MEMBRANE-INTERACTIONS OF GPI-PLC IN VITRO AND IN TRYPANOSOMA

BRUCEI

by

CLYDE FRANK HARDIN JR

(Under the direction of Kojo Mensa-Wilmot)

ABSTRACT

Trypanosoma brucei is the causative agent of human African sleeping

sickness. T. brucei is covered with a surface coat that primarily consists of

variant surface glycoprotein (VSG). A glycosyl phosphatidylinositol (GPI) anchors

the VSG protein to the extracellular surface of the plasma membrane.

T. brucei contains a GPI-specific phospholipase C (GPI-PLCp) that is

capable of cleaving GPI-anchors and GPI-intermediates. GPI-PLCp is an

integral membrane protein however, how the protein binds to membranes, and

where it localizes in the cell, is not known.

The purpose of this study is to identify regions of GPI-PLCp that bind to

membranes and to identify where the enzyme localizes in vivo. The enzyme

does not contain any transmembrane domains or sequences homologous to

characterized membrane-binding domains. Therefore, in an effort to identify the

membrane-binding regions of GPI-PLCp, amino and carboxyl-terminal

truncations of the enzyme were made. Constructs were assayed for their ability

to target a soluble reporter protein, green fluorescent protein (GFP), to T. brucei

microsomes in vitro. Amino acids 60-120 of GPI-PLCp targeted GFP to

microsomes.

In vivo experiments indicate that GPI-PLCp colocalizes with the

glycosomal protein, hypoxanthine-guanine phosphoribosyltransferase (HGPRT).

To further study where the enzyme localizes in vivo, T. brucei glycosomes were

isolated by 15-60 % sucrose-gradient sedimentation of microbodies isolated by

differential centrifugation. Fractions that contained GPI-PLCp activity, also

contained the glycosomal protein, aldolase. These results indicate that GPI-

PLCp localizes to the glycosome in T. brucei.

We propose that amino acids 60-120 contribute to the association of GPI-

PLCp with glycosomes.

INDEX WORDS: Trypanosoma brucei, African sleeping sickness, Variantsurface glycoprotein, Glycosylphosphatidylinositol, GPI-PLC,GFP, HGPRT, Microsome, Glycosome, Density-gradientcentrifugation, Membrane-binding domain, Transmembranedomain.

MEMBRANE-INTERACTIONS OF GPI-PLC IN VITRO AND IN TRYPANOSOMA

BRUCEI

by

CLYDE FRANK HARDIN JR

B.S., The University of Georgia, 1999

A Thesis Submitted to the Graduate Faculty of The University of Georgia in

Partial Fulfillment of the Requirements for the Degree

MASTER OF SCIENCE

ATHENS, GEORGIA

2003

© 2003

Clyde Frank Hardin Jr

All Rights Reserved

MEMBRANE-INTERACTIONS OF GPI-PLC IN VITRO AND IN TRYPANOSOMA

BRUCEI

by

CLYDE FRANK HARDIN JR

Major Professor: Kojo Mensa-Wilmot

Committee: Marcus Fechheimer Kelley Moremen

Electronic Version Approved:

Maureen GrassoDean of Graduate SchoolThe University of GeorgiaDecember 2003

iv

DEDICATION

I dedicate this thesis to my family for their unconditional love, support and

encouragement. I will love you always.

v

ACKNOWLEDGEMENTS

I am grateful to have the opportunity to formerly thank those who helped

make this possible. First and foremost, I would like to express my sincere

gratitude to my major professor, Dr. Kojo Mensa-Wilmot for all his help and

support over the last few years. I am privledged to have had the opportunity to

work with such a fine teacher, mentor and scientist.

I thank the members of my committee, Dr. Marcus Fechheimer and Dr.

Kelley Moremen, for their support and encouragement. I appreciate the

generosity of Drs. Michael Terns and Claiborne Glover for use of their

equipment. I thank Dr. Charlie Keith, Dr. Jacek Gaertig and Dr. Jason Brown for

sparking my interest in research and encouraging me to pursue a post-graduate

education.

I am thankful to have worked with the past and present members of the

lab. I learned a tremendous amout from these individuals, they are not only

excellent collegues but wonderful friends as well.

Finally, I am blessed to have such a fine family and network of friends.

They have unconditionally loved and supported me in every aspect of my life. I

would have never have accomplished so much had it not been for them, and for

them, I thank God.

vi

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ............................................................................................... v

CHAPTER I – INTRODUCTION AND LITERATURE REVIEW....................................... 1

CHAPTER II – MEMBRANE INTERACTIONS OF GPI-PLC IN VITRO AND IN

TRYPANOSOMA BRUCEI ...................................................................................... 34

CHAPTER III – DISCUSSION....................................................................................... 89

1

CHAPTER I

INTRODUCTION AND LITERATURE REVIEW

TRYPANOSOMA BRUCEI1 Trypanosomes

1.1 Trypanosomiasis

Trypanosoma brucei is a protozoan parasite that causes human African

trypanosomiasis (sleeping sickness). The parasite is transmitted to the

mammalian bloodstream through the bite of an infected tsetse fly from the genus

Glossina. The disease is thereby limited to the ecology of the insect vector, only

found in intertropical regions of Africa. Among the many species of

trypanosomes, only two, belonging to the group brucei, are infectious to humans.

T.b. gambiense is primarily found in West and Central Africa and causes a

chronic form of the disease. T. b. rhodesiense, found in East and Central Africa,

causes a more acute and virulent condition (for review [1, 2]).

Upon receiving a bite from a tsetse fly, a small nodule, called the

trypanosomal chancre, forms at the site. It is here that initial parasite

multiplication occurs. Anywhere from weeks to months after the onset of the

chancre, the trypanosomes invade the host's bloodstream leading to the

systemic or hemolymphatic stage of the disease [3, 4]. This stage is often

considered the early stage of the disease, defined as the period prior to

proliferation into the central nervous system (CNS). It is marked by general signs

2

of infection, including: moderate fever, intense itching, headache, swelling, and

ocular disturbances. The late stage or meningo-encephalitic phase is

characterized by the passing of the parasite across the blood-brain barrier, into

the CNS. At this stage, the infected individual may suffer from disturbances of

consciousness and circadian rhythm of sleep, sensory disturbances or

physiological symptoms such as memory loss, dementia, depression, agitation or

mania. Although the amount of time spent sleeping does not differ significantly

from that of an uninfected individual, the disease is named sleeping sickness due

to alterations in sleep patterns and profound lethargy seen in patients in the late

stage of the disease. Most of the infected individuals are not aware of their

disease until the onset of symptoms associated with the late-stage of the

infection [1].

1.2 Treatment

African sleeping sickness affects more than 300,000 people and

approximately 60 million people in 36 countries of sub-Saharan Africa are at risk.

Today, chemotherapy is the only method of treatment available. If the disease is

diagnosed early, the chances of cure are high. Treatment depends on the phase

of infection: early or late. The chance of cure in the latter phase depends on the

drugs ability to cross the blood-brain barrier in order to target the parasite. First

phase treatments include suramin and pentamidine; late phase treatments

include melarsoprol and eflornithine (the only currently available drug used to

treat the late phase of T.b. gambiense). In general, these drugs are toxic, difficult

to administer in poor, underdeveloped areas (due to financial and/or

3

geographical constraint), and not always successful. These considerations

aside, new strains of the parasite are beginning to emerge that exhibit resistance

to the drugs. Therefore, in order to develop new, safer, more effective drugs and

ultimately, eradicate the disease, research must be done to further understand

the biological and biochemical processes of the parasite (W.H.O.).

1.3 Life Cycle

Trypanosomes live in the circulatory systems of infected individuals and

are completely dependent on glycolysis for ATP production [5]. Bloodstream

form (BSF) trypanosomes are covered by a dense layer of variant surface

glycoproteins (VSG) that canopies underlying membrane proteins and prevents

lysis by host-mediated defenses [6-8]. Metacyclics that have just been

introduced into the mammalian bloodstream begin to differentiate into long

slender trypomastigotes and divide. At peak parasitemia, a proportion of the

parasites differentiate into short stumpies, pre-adapted for transmission into the

tsetse fly. After a tsetse fly ingests a blood meal, the short stumpy

trypomastigotes begin to differentiate into procyclics. At this stage, synthesis of

the VSG surface coat is suppressed [9], the coat is shed and replaced by an

invariant coat composed of procyclin [10, 11]. During differentiation, the

parasites begin their migration to the mid-gut of the fly. Here, they begin to

transform into epimastigotes and divide by longitudinal binary fission in the lumen

of the mid-gut. Upon differentiating into epimastigotes, the parasites continue

their migration to the salivary glands where they become attached to the

epithelium and develop into infective metacyclic trypomastigotes. At this stage,

4

procyclin expression is lost in the metacyclic forms and a specific subset of VSG

is expressed in preparation for infection in the mammalian host [6]. The entire

cycle of development in the fly takes approximately 3 weeks. Once infective, the

fly remains as such, due to the continual reproduction of metacyclics in the

salivary glands. The fly introduces trypanosomes into humans by injecting saliva

into the puncture wound produced during feeding [12] and the cycle continues

(Fig.1).

2 Antigenic Variation

Trypanosoma brucei is capable of establishing and maintaining an

infection in humans and animals. The parasite survives the host’s antibody-

mediated defenses by a process knows as “antigenic variation” [13, 14]. Each

bloodstream trypanosome expresses a single VSG gene from a repertoire of an

estimated 1000 genes [14, 15]. Upon establishing infection, the host develops

antibodies against the parasite's VSG coat and kills a large proportion of the

population. However, a small amount (10-5/generation, [14]) of the population

express an antigenically different VSG. This small population of trypanosomes

goes undetected by the hosts immune system, survives the immune response,

and proliferates [15, 16].

3 Glycosomes

When living in the mammalian bloodstream, Trypanosoma brucei is

completely dependent on glycolysis for ATP generation [5]. Glycosomes are

intracellular, membrane-bound, peroxisome-related microbodies found in all

kinetoplastids; they house the first 7-9 enzymes of the glycolytic pathway [17,

5

18]. The presence of these glycolytic enzymes for the conversion of glucose

into 3-phosphoglycerate is the hallmark that distinguishes glycosomes from

peroxisomes [18]. Glycosomes are essential for the survival of the parasite within

its mammalian host [17].

There is no net ATP production in the glycosome. Rather,

3-phosphoglycerate generated in the organelle is further metabolized in the

cytosol generating ATP via substrate-level phosphorylation. Under these

conditions, 2 molecules of ATP and 2 pyruvate are generated from each

molecule of glucose. Nicotinamide adenosine dinucleotide (NADH), produced in

the oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate, is

reoxidized by conversion of dihydroxyacetone phosphate to a-glycerophosphate

(a-GP), in the glycosome. a-GP is then reoxidized by an a-glycerophosphate

oxidase system localized in the mitochondrion [19].

Glycosomes belong to the microbody family of organelles. They are

surrounded by a single phospholipid bilayer and do not contain DNA. This family

includes peroxisomes and glyoxysomes.

Glycosomes are related to peroxisomes of other eukaryotes. In

comparison to peroxisomes, the protein content is very high in glycosomes.

However, both organelles contain enzymes involved in fatty acid b-oxidation,

ether-lipid biosynthesis and pyrimidine biosynthesis. Catalase, the hallmark

enzyme of peroxisomes is not found in kinetoplastid glycosomes, but it is present

in glycosomes of related organisms Crithidia deanei and Phytomonas serpens

[20-23].

6

Biogenesis and protein import is similar. Most of their matrix proteins are

synthesized on free ribosomes in the cytosol and imported into or inserted into

the organelle membranes [20, 24-26]. It has also been shown that similar

sequences are used in routing polypeptides to these organelles [20, 27, 28].

Together, these studies suggest that both organelles may have been derived

from a single ancestral peroxisome-like organelle [29].

Peroxisomes, PEX genes and their products (peroxins) have been studied

much more extensively than have glycosomes. So far, approximately 25 PEX

genes, and their products have been identified and characterized (Fig. 2).

Because little is known about glycosome biogenesis, researchers typically use

peroxisomes as a model for studying the glycosome in trypanosomatids.

Therefore, peroxisomes, will be referenced extensively throughout this review.

3.1 Glycosomal Targeting Sequences

In order for proteins to become localized to glycosomes they must contain

a glycosomal targeting sequence. The nature of the targeting sequence dictates

whether the protein is imported into the glycosome lumen (matrix proteins) or

inserted into the glycosomal membrane (membrane proteins).

There are two types of peroxisome targeting signals (PTS) that direct

proteins into the lumen of peroxisomes, and there is evidence that a third exists.

Two of these signals, PTS1 and PTS2 function in T. brucei [27, 28, 30].

The most common of the three signals is PTS1, a C-terminal tripeptide: -

SKL (single letter amino acid code) or a variation thereof. The second and less

common type of signal sequence is PTS2. PTS2 is an N-terminal nonapeptide.

7

In peroxisomes, it has a loosely conserved sequence motif of RLXXXXX(Q/H)L

where x may be any amino acid (for review [19]). So far, the PTS2 signal has

only been observed in two glycosomal proteins, aldolase and hexokinase [30,

31].

The existence of a third type of glycosomal targeting sequence has been

postulated. Acyl-CoA oxidase in both Saccharomyces cerevisiae and Candida

tropicalis does not contain either PTS1 or PTS2 signal, yet it is imported into

peroxisomes. The enzyme contains two 120 amino acid segments that may

facilitate import of the protein into peroxisomes in vitro [32]. The peroxisomal

catalase A of S. cerevisiae and phosphoglycerate kinase A of T. brucei also

contain an ill-defined “internal signal sequence” [19, 33]. These data suggest

that there must be a third branch of matrix import in yeast and trypanosomes. It

has also been shown that some proteins may enter glycosomes by associating

with proteins that contain a PTS. This method is referred to as “piggyback

import” [34].

Membrane proteins are found in peroxisomes and glycosomes These

peroxisomal membrane proteins (PMPs) localize to the organelles independent

of PTS1 or PTS2 targeting sequences. Membrane peroxisomal targeting

sequences (mPTS) have been identified in several integral PMPs. The first

mPTS was identified as a hydrophilic loop in PMP47 of Candida boidinii [35].

The loop contains a central group of positively charged amino acids followed by a

group of diverse residues. A similar sequence was also found in PMP70 and

PEX3p that led to a consensus mPTS sequence of XX(K/R)(K/R)3-

8

7X(T/S)XX(D/E)X [36] (using a single letter amino acid code, where x is any

amino acid). Human PMP34 contains a similar mPTS but has the opposite

topology, both N and C-termini face the cytosol [37]. In Pichia pastoris, humans

and rat, the first 40 amino acids of PEX3p and the first 54 of Saccharomyces

cerevisiae PEX3p sufficiently target a soluble membrane polypeptide (GFP) to

peroxisomal membranes [38-41]. Each domain contains a conserved block of

positively charged amino acids (that resembles the center of the PMP47 loop)

followed by a stretch of hydrophobic amino acids that may serve as a membrane

anchor [34]. A comparison of yeast and mammalian PEX3p sequences led to a

consensus of RX(K/R)XK [42] (single amino acid code, x is any amino acid).

The mPTS of rat PMP22 is contained within the first 37 amino acids.

Orthologs of PMP22 have been characterized in mouse, Saccharomyces

cerevisiae and Arabidopsis thaliana. Alignment of their N-terminal sequences

revealed a conserved mPTS of YXXXLXXXPXXX(K/N) [43] (single amino acid

code, x is any amino acid). The three-dimensional structure of the N-termini is a-

helical as predicted by the secondary structure program GOR IV [43, 44].

3.2 Glycosomal Targeting Sequence Receptor Proteins

The mechanism of import of both peroxisomal and glycosomal proteins

share common features; glycosomal import is therefore modeled after

peroxisomal import. This hypothesis is strengthened by the identification of PTS-

2 in two glycosomal matrix proteins and of glycosomal proteins homologous to

PEX proteins.

9

Approximately 24 peroxins (PEX) have been identified in yeasts and

mammalian cells that are involved in peroxisomal biogenesis (for review see [34,

45]) (Fig. 2). Peroxisomal membrane and matrix proteins are synthesized on

free ribosomes in the cytosol and either inserted or imported into peroxisomes.

Peroxisomal proteins are recognized by one of three cytosolic receptors, PEX5p,

PEX7p or PEX19p.

PEX5p is the receptor for peroxisomal proteins bearing PTS1 and is

essential for the import of all proteins bearing this class of PTS. After binding,

the PEX5p/cargo complex migrates to the peroxisomes where cargo is imported

and PEX5p recycles to the cytosol for additional rounds of import. Studies using

human peroxisomes also show that PEX5p enters the peroxisome during normal

function and then reemerges to the cytosol to carry out additional rounds of

import [46]. These studies suggests the receptor-protein complex is imported

prior to dissociation, after which, PEX5p is exported out of the peroxisome and

recycled to the cytosol.

PEX7p is the import receptor for peroxisomal proteins that contain a

PTS2. PEX7p is required for the import of proteins bearing this class of targeting

sequence. Subcellular localization studies indicate that PEX7p is found in both

the cytosol and peroxisomes [34], suggesting that the receptor-protein complex is

imported, similar to PEX5p. PEX7p requires a species-specific auxiliary protein

for PTS2 import: PEX18p or PEX21p in S. cerevisiae [47] , PEX20p in Yarrowia

lipolytica [48] and Neurospora crassa [49] or the longer of two splice isoforms of

PEX5p of mammals [50].

10

Finally, PEX19p is thought to bind newly synthesized PMPs and facilitate

their insertion into the peroxisomal membrane [51, 52]. The molecular machinery

required for inserting PMPs is different from that which is required for import of

PTS1 or PTS2 proteins. Very little is know about this process. PEX19p has

been shown to interact with various PMPs and peroxins, including the docking

protein, PEX17p, [51, 53]. However, the mechanism by which it facilitates

insertion is relatively unknown.

3.3 Glycosomal Import and Insertion

The receptors and their cargo dock at the peroxisomal membrane to a

complex consisting of PEX13p, PEX14p and PEX17p and are subsequently

transported into or inserted into the membrane of the peroxisome. After docking,

PMPs interact with PEX3p and PEX16p. Both of these peroxins are integral

membrane proteins and are thought to facilitate the insertion of PMPs into the

membrane. The mechanism of insertion has yet to be discovered. The

molecular details of translocation and receptor recycling are also unknown.

However, several PEX proteins have been shown to be required for import (Fig.

2.1).

PEX2p is thought to work downstream of PEX10p/PEX12p [54] and is

required for PTS1 and PTS2 import. PEX4p belongs to a family of E2 ubiquitin-

conjugating enzymes and is anchored to the peroxisomal membrane by the

integral membrane protein PEX22p. Both of these proteins are required for

import and are thought to be involved in PEX5p recycling [55]. PEX1p and

PEX6p are ATPases that belong to the functionally diverse family of “ATPases

11

associated with various cellular activities” (AAA). ATP is required for

PEX1p/PEX6p interaction, however their function is unknown.

Other peroxins that may function in import, but may not be required, have

been identified. PEX10p and PEX12p are integral peroxisomal membrane

proteins that may be associated with translocation machinery [56]. PEX8 is

required for import, it localizes to the cytosolic side of peroxisomes and interacts

with PEX5p and PEX20p [57, 58]. PEX9p and PEX23p are integral membrane

proteins found only in Y. lipolytica [59]. PEX15p is a phosphorylated integral

membrane protein [53].

4 Trypanosomal Peroxins and Their Functions

Several glycosomal proteins have been found in Trypanosmes that are

homologous to peroxisomal proteins. PEX5p, the cytosolic receptor for proteins

bearing a PTS1 receptor was found in T. brucei [20]. T. brucei glycosomes

contain two predominant integral membrane proteins. One of them, a 24 kDa

protein, was found to be the functional homologue to PEX11. PEX11p is

required for peroxisome division in Saccharomyces cerevisiae and Candida

boidinii [60, 61]. Over-expression of PEX11 in T. brucei leads to growth arrest

and deformation of glycosomes; reduced expression leads to fewer, but larger

glycosomes [62]. A PEX14 homologue has also been found in T. brucei. In

peroxisomes, PEX14p is part of the peroxisomal docking complex. In T. brucei,

PEX14p interacts specifically with PEX5p and is essential in both bloodstream

form (BSF) and procyclics. RNAi of PEX14 resulted in the release of part of the

glycosomal proteins, proposed to be due to the inability to import newly

12

synthesized proteins [63]. Finally, the PEX2 gene has been found in T. brucei.

PEX2p is essential for the import of proteins into glycosomes [17]

5 Gycosyl phosphatidylinositol (GPI)

Glycosyl phosphatidylinositols (GPIs) contain a conserved structural

Mana1-4GlcNa1-6myo-phosphatidylinositol (PI) [64]. Protein-linked GPIs (type-

1 GPIs) are based on the Mana1-6Mana1-4GlcNa1-6PI motif and can have a

wide variety of modifications [65]. GPIs may also function to anchor

polysaccharides to the plasma membrane; these GPIs contain the motif Mana1-

3Mana1-4GlcNa1-6PI and are called type-2 GPIs [66]. The third type of GPI is

free GPIs or hybrid GPIs. These GPIs are based on the branched glycan

Mana1-6(Mana1-3)Mana1-4GlcNa1-6PI [67] (for review see [66]).

The cell surface of T. brucei is covered with GPI-anchored proteins during

all stages of their life cycle. Bloodstream form parasites are covered with 107

molecules of GPI-anchored VSG [68]. The VSG GPI was the first GPI structure

to be solved [69]. This glycolipid consists of EtN-P-Mana1-2Mana1-6Mana1-

4GlcNa1-6myo-Ins-P. This core motif is decorated at the first mannose from the

lipid end with galactose (Gal) residues in either a1-2, a1-3 or a1-6 linkages [69].

5.1 GPI Biosynthesis

GPI biosynthesis begins on the cytoplasmic face of the endoplasmic

reticulum [70]. It is a sequential process in which sugars and an EtN are added

to PI to generate a GPI precursor to which a protein can be added. Synthesis

initiates with the transfer of GlcNAc from UDP-GlcNAc to PI [71] to yield GlcNAc-

PI. This molecule is then deacetylated to give GlcN-PI. Following deacetylation,

13

mannose residues from the donor dolichol-phosphoryl-mannose (Dol-P-Man) are

added [72]. Following the addition of mannose residues, a phospho-EtN is

donated from phosphatidylethanolamine to Man3-GlcN-PI to yield glycolipid A’

(EtN-P-Man3-GlcN-Ins-P-diacylglycerol) [73]. T. brucei GPIs undergo fatty acid

remodeling; the fatty acids on glycolipid A’ are replaced with myristate to yield

glycolipid A, a T. brucei VSGp GPI anchor precursor.

Concomitant with the formation of glycolipid A, T. brucei also produces

glycolipid C, in which the inositol group of glycolipid A is palmitoylated [74].

While both glycolipid A and C have been shown to be competent for transfer to

VSG protein in a cell-free system [75], there is no evidence of VSG transfer to

glycolipid C in vivo [74]. Thus the role of glycolipid C in T. brucei is unknown.

The transfer of glycolipid A GPI precursor to VSG polypeptide is thought to

occur in the lumen of the ER [76, 77]. Two signals in the polypeptide sequence

are required in order for a GPI anchor to be added. The amino (N)-terminal

signal directs the nascent protein to the lumen of the ER [78]. The second signal

resides in the carboxy (C)-terminus. GPI transfer requires a hydrophobic

sequence of 10-20 amino acids at the C-terminus. It is the hydrophobicity, rather

than the specific sequence of this site that is important for GPI transfer [79].

There must also be a cleavage/attachment site (w), 5-12 residues (hydrophilic

spacer region) N-terminal to the hydrophobic region. A transamidation reaction

occurs between the w-site of the newly synthesized VSG and the amino group of

ethanolamine, yielding GPI-anchored VSG [80] (figure 3).

14

6 GPI-specific Phospholipase C (GPI-PLCp)

Trypanosoma brucei express a glycosyl phosphatidylinositol-specific

phospholipase C (GPI-PLCp) [81, 82] that is highly specific for GPIs [83, 84] and

requires a glucosaminylinositol for efficient substrate recognition [85]. GPI-PLCp

is an integral membrane protein [81-84, 86] that may associate with glycosomes

(Z. Zheng, unpublished). How the protein binds to membranes, its localization,

three-dimensional structure and function have yet to be solved. However, the

enzyme is a virulence factor. GPI-PLCp influences parasitemia in mice [87], and

may also regulate endocytosis (S. Subramanya, unpublished).

There are two potential substrates of GPI-PLCp in vivo: (i) GPI-anchored

proteins and (ii) GPI-intermediates. VSG is anchored to the exoplasmic leaflet of

the plasma membrane, whereas GPI-intermediates are synthesized on the

cytoplasmic side of the endoplasmic reticulum [70, 88]. GPI-PLCp is thought to

localize to the “cytoplasmic side of intracellular vesicles” [89] . Thus, the most

likely substrates of GPI-PLCp in intact cells are GPI-intermediates.

6.1 Regulation of GPI-PLCp: Lessons From a Leishmania Model

Leishmania major (L. major) is related to T. brucei but does not contain an

endogenous GPI-PLCp. It is, therefore commonly used to study in vivo

regulation of GPI-PLCp. In L. major cells that have been stably transfected with

a T. brucei GPI-PLC gene, the cells become GPI-deficient and constitutively

secrete gp63p, the major GPI-linked protein on the plasma membrane into the

culture medium [90]. If GPI-PLCp cleaved GPI-intermediates in T. brucei, one

would expect to see a GPI-deficiency and secretion of VSG into the culture

15

medium. However, this is not the case; little or no VSG is released from long

slender T. brucei [91]. Thus, GPI-PLCp does not easily cleave GPI-

intermediates. Given the topological constraint between GPI-anchored VSG and

GPI-PLCp, one could infer that GPI-PLCp does not cleave membrane-form VSG

(mfVSG) either. Therefore, the function of GPI-PLCp in vivo remains to be

defined. These data also indicate that T. brucei may have a way of suppressing

GPI-PLCp activity in vivo in order to avoid the deleterious effects that enzyme

activity could have on the GPI biosynthetic pathway. It is estimated that there

are approximately 2.4 x 104 molecules of glycolipid A [70] and 3.5 x 104

molecules GPI-PLCp per cell [92]. It is theorized that GPI-PLCp could cleave all

the glycolipid A within the cell in a matter of seconds [93]. However, VSG is still

added to GPI-anchors, implying that access of GPI-PLCp to GPIs in vivo is

limited.

There are two ways in which T. brucei may regulate GPI-PLCp activity: (i)

by suppression of enzyme activity or (ii) localizing the enzyme away from the GPI

biosynthetic pathway on the ER. It has been shown that the first possibility does

occur in T. brucei. GPI-PLCp is primarily monomeric. However, in L. major,

when GPI-PLCp is expressed and gp63 is constitutively secreted, it exists

predominantly as a tetramer [93]. This data suggests that oligomerization may

activate GPI-PLCp activity [93]. In parallel, GPI-PLCp is also reversibly

myristoylated in vivo [94]. In vitro studies indicate that deacylation decreases

enzyme activity up to 30-fold, indicating that myristoylation may also regulate the

activity of GPI-PLCp [94].

16

7 Primary Objectives

The biological function of GPI-PLCp in T. brucei is unknown.

Biochemically, GPI-PLCp is an integral membrane protein. However, how the

enzyme associates with membranes and where it localizes in the cell is

unknown. Therefore, the purpose of this study is to determine how GPI-PLCp

binds membranes and to identify where the enzyme localizes in the cell; in an

effort to better understand the function of GPI-PLCp in vivo.

17

Figure 1: Trypanosoma brucei life cycle (from [95]). During a blood meal, the

tsetse fly inoculates the mammalian host with metacyclic parasites. These

quickly differentiate into long slender bloodstream forms (BSF). At peak

parasitemia the slender BSF differentiate into the short stumpy non-dividing form,

pre-adapted for survival in the insect vector. Once the tsetse fly ingests the short

stumpies, they differentiate into the procyclic form and begin to divide and

migrate to the fly midgut. Here they begin to differentiate into epimastigotes,

divide and continue migration to the salivary glands. Finally, the epimastigotes

differentiate into the non-dividing infective metacyclic form, pre-adapted for

survival in the mammalian host. The life cycle is complete when the metacyclics

are reintroduced back into a mammalian host.

18

Figure 1

19

Figure 2: Role of peroxins in peroxisomal import, biogenesis and division

(from [52]). (a) Peroxisomal proteins are synthesized on free ribosomes in the

cytosol. PTS1 proteins are chaperoned to the peroxisome by PEX5p, and PTS2

proteins by PEX7p, PEX18p, PEX20p and PEX21p. (b) These proteins are then

imported into the peroxisome. (c) Those of which have an mPTS reach the

peroxisome by PEX19p and are inserted into the peroxisomal membrane. (d-h)

Identify peroxins that are thought to play a role in peroxisomal

division/proliferation (e), biogenesis (c, g, h) and maintenance of peroxisomes (d,

f).

20

Figure 2

21

Figure 2.1: Proposed peroxisomal matrix-protein import: (from [45]) (a)

Proteins with a PTS-1 bind the chaperone PEX5p. (b) PEX5p shuttles the matrix

protein to the peroxisome surface. (c) The matrix-protein then docks with

membrane proteins like PEX14p via protein-protein interactions. (d) The matrix-

protein translocates through the proteinaceous pore thought to consist of PEX8p,

PEX10p and PEX12p. (e) The PEX5p receptor is then recycled back into the

cytosol, which may involve PEX4p and PEX22p.

22

Figure 2.1

23

Figure 3: Biosynthesis of GPIs (from [96]). Biosynthesis takes place on the

cytosolic side of the ER, beginning with the addition of GlcNAc to

phosphatidylinositol ¨. GlcNAc-PI is deacetylated ! to GlcN-PI followed by the

addition of three mannose residues " - # and the donation of a phospho-EtN by

PE ¥. Once synthesized, glycolipid A is thought to be flipped into the lumen of

the ER where it is decorated with Gal residues and added to VSG or a

carbohydrate moiety (not shown). It is then targeted to the extracellular surface

of the parasite.

24

Figure 3

25

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34

CHAPTER II

MEMBRANE INTERACTIONS OF GPI-PLC IN VITRO AND IN

TRYPANOSOMA BRUCEI1

1 Hardin, Clyde, Kimberly Butler, Robyn Howard, Zhifeng Zheng andKojo Mensa-Wilmot. To be submitted to Journal of Biological Chemistry

35

ABSTRACT

Trypanosoma brucei expresses an endogenous glycosyl

phosphatidylinositol-specific phospholipase C (GPI-PLCp). Biochemical

evidence suggests the enzyme is an integral membrane protein. However,

methods used to predict membrane-binding domains do not identify any such

domains in GPI-PLCp. In an attempt to clarify the mechanism by which GPI-

PLCp binds to membranes, amino and carboxy terminal truncations of the protein

were fused to a soluble reporter protein, green fluorescent protein (GFP) and

assayed for their ability to bind T. brucei membranes post-translationally. These

experiments indirectly identified two potential regions of GPI-PLCp that may

target GFP to the membranes. In this study, these domains, membrane binding

regions (MBR1and MBR2, amino acids 60-120 and 238-298, respectively), were

fused to GFP and assayed for their ability to bind T. brucei membranes. Only

MBR1 sufficiently targeted GFP to T. brucei membranes. Helical wheel analysis

of MBR1 region suggests that it could contain a stretch of amino acids that may

form an amphipathtic a-helix. We propose that GPI-PLCp interacts with

membranes via the hydrophobic face of MBR1 and one leaflet a lipid bilayer.

GPI-PLCp is therefore a membrane associated protein without a transmembrane

domain.

In vivo, immunofluorescence data reveals that GPI-PLCp colocalizes with

hypoxanthine-guanine phosphribosyltransferase (HGPRT), a glycosomal protein.

This data suggests that the enzyme localizes to glycosomes in T. brucei. In a

36

second approach, T. brucei glycosomes were purified by velocity sedimentation

and sucrose density-gradient fractionation. The fractions were assayed for GPI-

PLCp activity and for the presence of a glycosomal enzyme, aldolase. GPI-PLCp

enzyme activity and aldolase cosedimented on sucrose gradients. These data

support the immunofluorescence data, confirming the localization of GPI-PLCp to

glycosomes in T. brucei.

We determined the topological orientation of GPI-PLCp on glycosomes.

Purified glycosomes were subjected to proteinase K digestion in the presence or

absence of NP-40 detergent. After treatment, the integrity of GPI-PLCp was

determined using enzyme assays. Glycosomal GPI-PLCp is fully active after

treatment with proteinase K. However, during protease treatment in detergent,

GPI-PLCp is digested. These data indicate that glycosomes protect GPI-PLCp

from proteolysis by proteinase K. We infer that GPI-PLCp is imported into the

lumen of the glycosomes.

We conclude that (i) GPI-PLCp is a glycosomal protein, (ii) GPI-PLCp

resides inside glycosomes, and that (iii) MBR1 may direct the association of GPI-

PLCp with membranes inside glycosomes.

37

INTRODUCTION

Glycosyl phosphatidylinositol-specific phospholipase C (GPI-PLCp) from

T. brucei is an integral membrane protein [1-5]. GPI-PLCp associates with the

pellet of a hypotonic cell lysate and cannot be extracted by reagents known to

solubilize peripheral membrane proteins (1M NaCl and 20 mM EDTA) or by low

concentrations of detergent (0.1% nonidet P-40). High concentrations of

detergent (1% NP-40), however, can solubilize the protein. The purified enzyme

is also found in the detergent fraction after Triton X-114 phase partitioning [4].

How GPI-PLCp binds to membranes is unknown. Kyte-Doolittle analysis

[6] and Pfam (Protein families database of alignment and hidden Markov models)

[7] analysis, are used to identify hydrophobic domains or peptide sequences

homologous to lipid-binding domains of other proteins, respectively. These

computational analyses do not provide clues about how GPI-PLCp associates

with membranes.

GPI-PLCp may associate with membranes by: (i) interaction with other

proteins; (ii) by lipid modification; or (iii) lipid-binding domains. Membrane

binding by the enzyme is not due to eukaryotic-specific modifications [4] or

protein-protein interactions [2]. Therefore, we investigated the possibility of

membrane binding via lipid-binding domains in T.brucei.

Conceding that GPI-PLCp does not have a transmembrane domain, a

search for short hydrophobic segments with a shorter window was performed

using Kyte-Doolittle analysis [6]. Five ‘domains’ of GPI-PLCp were predicted to

be hydrophobic enough to bind membranes (K. Butler, M.S. Thesis, UGA).

38

Protein truncations containing these domains were constructed and fused to

green fluorescent protein (GFP), a soluble reporter polypeptide. The fusion

proteins were assayed for their ability to target the reporter to T. brucei

membranes in vitro. From these experiments, two domains within GPI-PLCp,

amino acids 60-120 and 238-298 were proposed to be capable of targeting GFP

to microsomes and were named membrane-binding region (MBR)1 and MBR2,

respectively (K. Butler, M.S. Thesis, UGA). In this work we determined if these

domains are sufficient to target GFP to T. brucei microsomes. We found that

only MBR1 of GPI-PLCp could target GFP to microsomes, suggesting that this

region plays a role in membrane-binding by GPI-PLCp.

Attempts have been made in an effort to elucidate the intracellular location

of GPI-PLCp in T. brucei [8, 9]. The most recent attempt suggests that the

protein associates with the “cytoplasmic side of intracellular vesicles” and that the

function of these vesicles was not known [9]. Recently, however, it was shown

that GPI-PLCp colocalizes with a glycosomal protein, hypoxanthine-guanine

phosphoribosyltransferase (HGPRT) (Z. Zheng, unpublished).

To determine if GPI-PLCp localizes with glycosomes in vivo, we purified

glycosomes from T. brucei and assayed them for GPI-PLCp activity and for the

presence of aldolase (a glycosome marker). We show that GPI-PLCp and

aldolase cosedimented on sucrose gradients and conclude that GPI-PLCp binds

to T. brucei glycosomes in vivo. We also determined the topological orientation of

GPI-PLCp in glycosomes, by using protease protection assays. It was found that

glycosomes protected GPI-PLCp from digestion. Only after solubilization with

39

detergent was GPI-PLCp susceptible to digestion. These results indicate that

GPI-PLCp is imported into the lumen of glycosomes. We speculate on how GPI-

PLCp is targeted to glycosomes and how membrane binding regions may

mediate binding of GPI-PLCp to these membranes.

MATERIALS AND METHODS

Materials

Restriction enzymes Bam HI, Hind III and Xba I were from New England

Biolabs (Beverly, MA); nonidet P-40 was from Calbiochem (San Diego, CA);

sodium dodecyl sulfate (SDS), 5-bromo-4-chloro-indoyl phosphate (BCIP) and p-

nitroblue tetrazolium chloride (NBT) were from BioRad (Melville, NY); leupeptin

and DNase I were from Roche (Indianapolis, IN); butanol was from Fisher

Scientific (Norcross, GA); Immobilon P membrane was purchased from Millipore

(Bedford, MA); Redivue Promix was from Amersham Biosciences (Piscataway,

NJ); DE52 was from Whatman (Hillsboro, OR); Amplitaq DNA polymerase was

from Perkin Elmer (Boston, MA); T4 DNA ligase was from Promega (Madison,

WI); phenol/chloroform/isoamyl/alcohol and methanol were from Fisher

(Pittsburgh, PA); Ampliscribe T7 kit was from Epicentre (Madison, WI);

bicinchoninic acid assay kit was from Pierce (Rockford, IL); anti-aldolase

antibody was a gift from Dr. Christine Clayton (ZMBH, Heidelberg, Germany); all

other reagents were from Sigma (St. Louis, MO).

40

Cell Types / Strain

Monomorphic T. brucei strain 427 bloodstream form was used in this

work. Parasites were grown in rodents and harvested by chromatography on

DE52 [10].

Plasmids

The plasmids pCO64 and pCO59 were from Dr. George Cross

(Rockefeller University): they contain NH2- and COOH-terminal fusions,

respectively, of GPI-PLC to GFP. These vectors were used as templates to

produce various GPI-PLC:GFP fusions.

Generation of GPI-PLC and GFP DNA

Polymerase chain reaction (PCR) was used to amplify GPI-PLC and GFP

genes from pCO59 using forward and reverse primers. All forward primers

include a T7 promoter [11] for RNA synthesis in vitro and a short 5’ untranslated

region (UTR) to enhance translation [12]. All reverse primers introduced a stop

codon downstream of the coding region, unless noted otherwise.

PCR reactions were set up in Innis buffer (10 mM Tris-HCl pH 8, 2.5 mM

MgCl2, 0.05% Tween-20, 0.05% nonidet P-40, 50 mM KCl). To this buffer, 30 ng

pCO59 template, 0.25 mM of each deoxynucleotide, 20 mM of the indicated

primers, and 5U of Taq DNA polymerase were added [13]. Cycling conditions

were (i) denaturation at 95°C for 90 seconds, (ii) annealing at 56°C for 90

seconds, and, (iii) extension at 74°C for 2 min. Twenty-five cycles were

performed. Amplified products were purified using the Wizard PCR DNA

purification system (Promega) and resuspended in T10E1 (10 mM Tris-HCl pH 8,

41

1 mM EDTA) to a final concentration of 1 mg/ml as determined by OD260. Aliquots

(500 ng) were electrophoresed on a 1% agarose gel to verify product size and

concentration.

Generation of MBR1/MBR2 DNA by PCR

Production of MBR1:GFP and MBR2:GFP DNA required two PCR

reactions. The first reaction was used to synthesize the “megaprimer” [14], a

sense strand of 264 nucleotides containing (5’-3’) T7 promoter, Hind III restriction

site, 5’ untranslated region, and a segment of nucleotides encoding amino acids

60-120 (MBR1) or 238-298 (MBR2) of GPI-PLCp joined to a segment of

nucleotides that encodes amino acids 2-11 of GFP. The second reaction uses

the forward “megaprimer” and a reverse GFP primer to fuse the MBR1 or MBR2

coding region of GPI-PLC to GFP. The final product is flanked by 5’ Hind III and

3’ Bam HI restriction sites, generated from forward and reverse primers.

The first round of PCR was used to construct “megaprimers” that encoded

amino acids 60-120 or 238-358 of GPI-PLCp. To amplify the fragment encoding

residues 60-120 joined to amino acids 2-11 of GFP, forward primer fT7H3MBR1,

5’GGGTAATACGACTCACTATAGGGAGAAAGCTTGTAACACAGGAGGCAGAT

CATGCCATATGTGGACCTTTCTTTTA3’ and reverse primer rGFP-MBR1,

5’CCCGGTGAACAGCTCCTCGCCCTTGCTCACTGGAACAGAAATATGGAAAT

G3’ (restriction sites underlined) were used. To amplify the fragment encoding

residues 238-298 of GPI-PLCp joined to amino acids 2-11 of GFP, forward

primer fT7H3MBR2, 5’GGGTAATACGACTCACTATAGGGAGAAAGCTTGTA-

ACACAGGAGGCAGATCATGTTGGAAGATGTCAGTATTGGC3’ and reverse

42

primer 5’CCCGGTGAACAGCTCCTCGCCCTTGCTCACTAAATCATA-

AAACCACTTCAA 3’ were used. PCR was carried out as described using 50 ng

GPI-PLC amplified from pCO59 (K. Butler, M.S. Thesis, UGA) as the template.

Cycling conditions were (i) denaturation at 95°C for 90 seconds, (ii) annealing at

45°C for 90 seconds, and, (iii) extension at 74°C for 2 min. Twenty-five cycles

were performed. The second round of PCR contained using 50 ng GFP

template, 1.9 mg of the above “megaprimers” (forward primer), the reverse primer

(rGFP), 5’TGGCAGGATCCTATTTAACACCCGGGGTACAGCTCGTC3’ (Hind III)

restriction site underlined), and 45°C annealing temperature were used. The

rGFP reverse primer introduces a Bam HI restriction site N-terminal of the stop

codon.

PCR products were separated from templates by agarose gel

electrophoresis, and isolated from the agarose with homemade spin columns.

Columns were made by (i) piercing a hole in the bottom of a 0.8 ml Eppendorf

tube, (ii) stuffing the 0.8 ml tube with glass wool, and (iii) placing the 0.8 ml tube

in a 1.5 ml Eppendorf tube. Gel slices containing PCR products were placed in

the 0.8 ml tube and centrifuged twice for 15 min (8,000 x g, 25°C). DNA was

extracted from the eluate with phenol/chloroform/isoamyl alcohol (P/CIA) and

ethanol precipitation [13].

The second round of PCR produced a relatively low yield of the desired

1030 nucleotide fragment. After purification, the DNA was cloned into pBSKII

and transforming for amplification in Escherichia coli XL-10 cells as indicated

below.

43

Restriction Digest and Transformation

MBR-GFP DNA and pBSKII were digested with Bam HI and Hind III (NEB)

then ligated using 50 ng pBSKII and 25 ng MBR-GFP [15]. Five ml of each

ligation reaction was transformed into E. coli XL-10 cells [15]. Plasmid DNA was

extracted by mini alkaline lysis preparation [16]. The correct orientation of the

insert in the vector was determined by PCR using fT7H3MBR1/2 and rGFP

primers.

In Vitro Transcription of DNA

Plasmid DNA containing MBR1/2-GFP was linearized with Xba I and

concentrated to 1 mg/ml using ethanol precipitation [13]. Purified DNA fragments

were transcribed using the AmpliscribeTM T7 Kit (Epicentre Technologies). One

ml (1 MBU) of RNase-Free DNase I was added to the reaction and incubated for

15 min at 37°C. Twenty ml of 5 M ammonium acetate was added to the mixture

and incubated for 15 min. on ice. Precipitated RNA was pelleted (14, 000 x g, 15

min, 4°C) and washed in 70% ethanol. Purified RNA was resuspended in 10 ml

T10E1 (10 mM Tris, 1 mM EDTA pH 6.8) and concentration was determined by

absorbance at OD260. Samples were diluted to a concentration of 1 mg/ml with

T10E1. RNA concentration was verified by electrophoresing 1 mg on a 1%

agarose gel [16].

In Vitro Protein Synthesis

Reagents for in vitro translation reaction were added in the following order

on ice: 2 mg mRNA, 1 mM methionine/cysteine-free amino acid mixture

(Promega), 1.5 ml (21 mCi) [35S] Redivue Promix (Amersham Biosciences), 10.5

44

ml nuclease-free water, 15 ml rabbit reticulocyte lysate (Promega). Translation

was mixed thoroughly by pipetting and incubated at 30°C for 2 h. Translation

products were separated by SDS-PAGE (14% mini gel), and radiolabeled

proteins were visualized by phosphorimaging (Personal Molecular Imager FX,

BioRad).

Preparation of T. brucei Microsomes

A pellet of 2 x 109 bloodstream form T. brucei (BSF) was washed in PBS

and resuspended in 1 ml of homogenization buffer (HB) (250 mM sucrose, 50

mM KOAc, 6 mM Mg(OAc)2, 1 mM EDTA, 1 mM DTT) cells were lysed by

sonciation at 4°C (Vibra Cell Ultrasonicator model #VC-501: amplitude 20 for 4

min, cells were in a 1.5 ml microfuge tube and floated in an ice bath). The

mixture was centrifuged (2000 x g, 10 min., 4°C) to sediment large cellular

debris. The supernatant was centrifuged (12, 000 x g, 20 min., 4°C) and the

pellet was resuspended in 25 ml of rough microsome buffer (RMB) (250 mM

sucrose, 50 mM HEPES-KOH, pH 7.6, 50 mM KOAc, 1 mM DTT) supplemented

with1mg/ml TLCK, 5 mg/ml leupeptin. The OD260 of a 1:50 dilution of the

resuspended membranes in 0.5% SDS was determined. The

microbody/microsome stock was diluted with RMB to a final OD260 of 30,

aliquoted into 25 ml portions, quick frozen in liquid nitrogen, and stored at -80°C

(B. Patham, unpublished). Seven ml of these microsomes were used in binding

experiments (see below).

45

Post-Translational Binding of Proteins to T. brucei Membranes

Twenty microliters of an in vitro translation reaction was mixed with 37 ml

of RMB either with or without T. brucei microsomes (210 OD260 units). These

samples were incubated at 30°C for 10 min. to allow the protein to bind the

membranes. Next, 100 ml of dilution buffer (DB) (100 mM KOAc, 20 mM HEPES-

KOH pH 7.2, 2.5 mM Mg(OAc)2, 20 mM EDTA) was added and the samples were

incubated on ice for 15 min. This mixture was layered on top of a 70 ml sucrose

cushion (500 mM sucrose, 20 mM HEPES-KOH, pH 7.2) and centrifuged at

100,000 x g for 30 min. at 4°C (52,000 rpm in a TLA 100.3 rotor, Beckman

OptimaTM TL Ultracentrifuge). The supernatant was transferred to a fresh

microfuge tube. The pellet, containing membrane-bound protein was solubilized

by resuspending in 70 ml of pellet resuspension buffer (PRB) (500 mM Tris base,

5% SDS, 10 mM DTT) and heating at 55°C for 15 min [17]. Equivalent volumes

of each fraction (2 ml of the translation reaction, 22 ml of supernatant and 7 ml of

pellet) were separated by SDS-PAGE (14% mini gel), and radiolabeled proteins

were visualized by phosphorimaging (Personal Molecular Imager FX, BioRad).

QuantityOne software (BioRad) was used to detect the amount of signal in each

band (K. Butler, M.S. Thesis, UGA).

Purification of T. brucei Glycosomes

Isolation of glycosomes as follows was modified from [18]. T. brucei

(1010) were resuspended in 1 ml of HEDS buffer (50 mM HEPES-KOH pH 7.8, 1

mM EDTA, 1 mM DTT, 250 mM sucrose) plus 50 mM KOAc, 5% glucose)

supplemented with leupeptin (1 mg/ml). Cells were disrupted using 10 strokes

46

with a Dounce homogenizer (Kontes, 15 ml homogenizer, low clearance pestle),

and centrifuged at 1500 x g for 15 min at 4°C. Supernatant was collected into a

new microfuge tube. The pellet was resuspended in 1 ml HEDS buffer and

centrifuged as before, supernatant was collected. This process was repeated

again and all three supernatants were pooled and centrifuged at 33,000 x g

(25,000 rpm with Beckman TLA 100.3 rotor) for 30 min. The pellet (crude

microbodies) was resuspended in 3 ml HEDS buffer with 50 mM KOAc and 5 mM

MgCl2, and 0.25 mg/ml DNase I [18] (Roche) was added. The mixture was

incubated on ice for 30 min. One ml of the sample was added to a 12 ml,15-60%

continuous sucrose gradient (sucrose was dissolved in HEDS buffer with 1 mg/ml

leupeptin). The gradient was centrifuged overnight (36,000 rpm, 4°C) in an

SW41 Ti rotor. To fractionate the gradient, the bottom of the tube (Beckman

Ultra-Clear Tube #44059) was punctured by a Tube Piercer (ISCO) and 62%

sucrose was pumped into the bottom at 40% speed (1ml/min) (Tris-Pump,

ISCO). Eighteen 750 ml fractions were collected from the top of the gradient

using the Foxy Jr. Fraction Collector (ISCO). Total protein was determined with

a bicinchoninic acid assay kit (Pierce).

Cosedimentation Assays

Four ml of an in vitro translated reaction mixture was added to 3 ml (84

mg/ml) of enriched glycosomes. Briefly, glycosomes were purified as above

except they were layered on a 1-2 M (34-68%)sucrose gradient. The fractions

with peak GPI-PLCp activity were pooled (4 ml), diluted to 25 ml with HEDS

buffer and centrifuged at 22,000 x g for 30 min. at 4°C (Beckman 50.2 Ti rotor,

47

L7-55 centrifuge). Pellets were resuspended in 25 ml HEDS buffer with 50 mM

KOAc, 5 mM MgCl2. The reaction was incubated for 30°C for 30 min. to allow for

binding then cooled on ice for 1 hour. The mixture was centrifuged at 33,000 x g

for 30 min at 4°C (25,000 rpm TLA 100.3 rotor). Pellets were resuspended in 25

ml SDS-sample buffer. Ten ml of supernatant and pellet fraction were analyzed

by SDS-PAGE (14% mini gel, BioRad) as previously described.

TCA Precipitation

Microbodies were precipitated with TCA prior to Western analysis. Each

750 ml sample (from above) was raised to 1 ml with water, 500 ml were saved

prior to precipitation for GPI-PLCp activity assays (see below). The other 500 ml

was used as follows: to 500 ml (5 x 107 cell equivalents), 40 ml of water was

added followed by one ml 1% (w/v) sodium deoxycholate and for a final

concentration of 0.0015% [19]. The mixture was incubated on ice for 30 minutes.

Sixty ml trichloroacetic acid (TCA) was added yielding a final concentration of

10% TCA, and the reaction was mixed by vortexing. Fractions were incubated

on ice for 1 h and centrifuged for 10 min. at 14,000 x g, 4°C. Supernatant was

removed and the pellet was gently washed with 500 ml ice-cold acetone. Pellets

were allowed to air dry for 10 minutes, followed by exposure to ammonium

hydroxide (28-30%) vapor for 30 seconds per tube to neutralize the pH of the

pellet. Pellets were resuspended in 30 ml 5X SDS sample buffer, 5 ml was

heated for 5 min, and separated by SDS-PAGE (14% mini gel) (BioRad) [16].

Proteins were transferred to Immobilon P with a Trans-Blot semi-dry cell

(BioRad) [20].

48

Western Blots

The Immobilon P membrane was first blocked in PBS pH 7.5 solution

containing 3% (w/v) Carnation powdered milk, 1% (v/v) Tween 20) for 1 h at

room temperature. Aldolase was detected by Western blotting with anti-aldolase

antiserum (1:000) [20].

Assay of GPI-PLCp Enzyme Activity

All eighteen fractions (from above) were assayed for GPI-PLCp activity.

One ml of each fraction was added to 19 ml GPI-PLC assay buffer (50 mM Tris-

HCl, pH 8.0, 5 mM EDTA, 1.0% NP-40) and 2 mg [3H]myristate-labeled

membrane form variant surface glycoprotein dissolved in 10 ml GPI-PLC assay

buffer (30 µl final volume) [5]. The reaction was incubated at 37°C for 20 min,

and terminated by addition of 500 ml water-saturated n-butanol. Four hundred

microliters of the upper organic phase were retrieved for liquid scintillation

counting [5].

Proteinase Protection Assays

Fraction 14, the fraction that contained the highest GPI-PLCp activity, was

used for proteinase protection assays (fraction 14, Fig. 7). One microliter (28

mg/ml) of this fraction, or 1 ml (1.4U) purified GPI-PLCp [5], was added to 8 ml

HEDS buffer with or without proteinase K (300 ng/ml). The reaction was

incubated overnight at 15°C and quenched with 1 ml (20 mM) phenyl-methyl-

sulfonyl-fluoride (PMSF). When specified, HEDS buffer may be supplemented

to: 1% (v/v) nonidet P-40 detergent. After protease digestion, 5 ml of the reaction

was assayed for GPI-PLCp activity [5].

49

RESULTS

GPI-PLCp Is Not Predicted to Have Membrane-Spanning a-Helices

The biochemical properties of GPI-PLCp indicate that it is an integral

membrane protein. It cannot be extracted with high ionic solutions or by low

concentrations of detergents. High concentrations of detergent (1% NP-40)

solubilize GPI-PLCp. The purified enzyme is also found in the detergent fraction

after Triton X-114 phase partitioning [4].

To determine if GPI-PLCp contained transmembrane regions, Kyte-

Doolittle hydrophobicity analysis [6] was used, with a window of 19 amino acids,

the length required to span a lipid bilayer as an a-helix [6]. Typically, a value

greater than 1.6 correlates to a transmembrane domain. Figure 1 shows the

hydrophobicity plots of GPI-PLCp and Sec61p. Sec61p has 10 trans-membrane

domains which have been verified experimentally [21], as predicted by Kyte-

Doolittle hydrophobicity plots (Fig. 1B). GPI-PLCp does not contain a peak with

a hydrophobicity index of 1.6 (Fig. 1A). Computational analysis of GPI-PLCp

with Pfam [7] also yielded negative results for sequences homologous to known

membrane-binding domains (data not shown).

To resolve the discrepancy between the biochemical data [1-5] and the

predictions from the computational analysis, fragments of GPI-PLCp were fused

to GFP and assayed for their ability to target GFP to T. brucei microsomes

(K. Butler, M.S. Thesis, UGA).

50

GPI-PLCp Targets GFP to T. brucei Microsomes

To determine if GFP:GPI-PLCp could target GFP to microsomes, the

fusion protein and GFP were assayed by cosedimentation in the presence or

absence of T. brucei microsomes (Fig. 2). GFP is a soluble protein [21] and

does not pellet in either the presence or absence of T. brucei microsomes (Fig.

2A). GFP fused to the N-terminus of GPI-PLCp was also predominantly found in

the soluble fraction in the absence of membranes (Fig. 2A). To quantitate the

differences in the effectiveness of protein binding to microsomes, the amount of

signal in the pellet was compared to the total signal (sum of signal in supernatant

and pellet). The ratios obtained were plotted for each protein tested (indicated in

figures) in the presence or absence of T. brucei microsomes (Fig. 2B).

Approximately 2% of the total signal (translated protein) was found in the

pellet fraction in absence of microsomes. The amount of signal in the pellet

fraction was not affected upon the addition of microsomes (data not shown). For

GFP:GPI-PLC, there was an increase of signal found in the pellet fraction after

the addition of microsomes (Fig. 2B). Roughly 10% of the total signal was found

in the pellet fraction without microsomes. Addition of microsomes increased the

pelletable signal 4.6 fold (Fig. 2B).

These results indicate that GPI-PLCp can target a soluble reporter

polypeptide, GFP, to T. brucei microsomes.

Amino Acids 60-120 and 238-358 May Contribute to Membrane-Binding ofGPI-PLCp

To identify membrane binding regions in GPI-PLCp, truncations of the

protein were fused to GFP and assayed for the ability to bind T. brucei

51

microsomes using a cosedimentation assay (K. Butler, M.S. Thesis, UGA) (Fig.

3). Both full-length proteins with N- and C-terminal fusions of GFP to GPI-PLCp

showed increased binding to microsomes as compared to assays without

microsomes (Fig. 3A, E). Signal in the pellet fraction for N- and C-terminal

fusions were 27% and 23% (respectively) without microsomes. In the presence

of microsomes 63% of the proteins pelleted.

To identify membrane binding regions in the N-terminal half of GPI-PLCp,

truncations of residues 1-179 of the protein (1-60, 1-120 and 1-179) were fused

to the C-terminus of GFP (Fig. 3I) and assayed for the ability to bind T. brucei

microsomes using a cosedimentation assay (Fig. 3). Residues 1-120 and 1-179

of GPI-PLCp fused to GFP showed an increase in binding to microsomes as

compared to assays without microsomes (Fig. 3B, C). Signal in the pellet

fraction for residues 1-179 and 1-120 were approximately 38% without

microsomes. Addition of microsomes increased the amount of pelletable protein

to 76% (residues 1-179) and 88% (residues 1-120) (Fig. 3B, C). However, when

amino acids 1-60 were fused to GFP and assayed, there was no increase in the

amount of pelletable protein upon the addition of microsomes (Fig. 3D).

Therefore, amino acids 1-60 are not sufficient to target the reporter protein (GFP)

to microsomes.

Next, to identify membrane binding regions in the C-terminal half of GPI-

PLCp, truncations of residues 179-358 of GPI-PLCp (179-358, 238-358, 298-

358) were fused to the N-terminus of GFP, these constructs were assayed as for

binding as previously described. Residues 179-358 and 238-358 fused to GFP

52

showed an increase in pelleted fractions in the presence of microsomes as

compared to assays without microsomes (Fig. 3F, G). Approximately 35% of

signal (residues 179-358 and 238-358) was found in pellet fraction without

microsomes. Addition of microsomes increased these signals to 89% (179-358)

and 95% (238-358) (Fig. 3F, G). The presence of multiple bands in figure 3F

may be due to internal initiation of synthesis or perhaps due to proteolysis,

regardless, all signal was quantitated collectively. However, the last sixty amino

acids (residues 298-358) of GPI-PLCp do not significantly increase the fraction of

GFP that is pelleted upon the addition of T. brucei microsomes (Fig. 3H). Only a

10% increase in signal (from 32% to 42%) in the pellet fractions was observed,

(the top band was alone was quantitated in these experiments because the full-

length polypeptide signal was easily distinguished by size). Therefore, amino

acids 298-358 of GPI-PLCp are not sufficient to target the reporter protein (GFP)

to microsomes (Fig. 3H).

In summary, these results indirectly indicate residues 60-120 and 238-

298, termed membrane binding regions (MBR)1 and 2, respectively, may be

sufficient for binding GPI-PLCp to microsomes (Fig. 3I) (K. Butler, M.S. Thesis,

UGA).

Amino Acids 60-120 Is Sufficient to Target GFP to Microsomes

To determine if amino acids 60-120 (membrane binding region 1, MBR1)

or 238-358 (MBR2) from GPI-PLCp are capable of targeting GFP to T. brucei

microsomes, GFP was fused to the N-terminus of each region (Fig. 4A). Fusion

proteins were assayed for binding to microsomes from T. brucei. Without

53

microsomes, approximately 9% of the total signal of MBR1-GFP was found in the

pellet fraction (Fig. 4B, C). However, 25% of MBR1-GFP is found in the pellet

fraction after the addition of microsomes (Fig. 4B, C). Therefore, residues 60-120

is capable of directing GFP to T. brucei microsomes. In contrast, very little fusion

protein is found in the pellet fraction when MBR2-GFP was cosedimented with

microsomes (Fig 4B). Quantitation of the data indicates that with or without

microsomes, less than 5% of the fusion protein is pelleted (Fig. 4C). These data

indicate amino acids 238-358 (MBR2) of GPI-PLCp are not sufficient to target

GFP to T. brucei microsomes (Fig. 4B, C)

Together, these data suggest that residues MBR1 is capable of binding to

T. brucei microsomes. When comparing binding of MBR1-GFP to GPI-PLC:GFP

to T. brucei microsomes, there was a 3-fold increase and a 4.6-fold increase (Fig

2C, 4C). Therefore, the binding of MBR1:GFP was almost as efficient as GPI-

PLC:GFP, indicating that MBR1 may contribute to most of the membrane binding

activity of GPI-PLCp.

GPI-PLCp Binds To Glycosomes In T. brucei

Until recently, GPI-PLCp was thought to associate with the “cytoplasmic

side of intracellular vesicles” [9]; the identity of these vesicles was not known. In

order to better understand the function of GPI-PLCp in T. brucei, it was

imperative that the identity of these vesicles was resolved. In order to determine

the location of GPI-PLCp in T. brucei two approaches were used:

immunofluorescence and subcellular fractionation.

54

Bloodstream form T. brucei (Fig. 5A) were fixed, permeabilized and

incubated with anti-GPI-PLCp or anti-HGPRT (a glycosome marker [22])

antibodies. Anti-HGPRT was detected by Alexa Fluor 488 conjugated goat anti-

rabbit IgG (Fig. 5B, green). Anti-GPI-PLCp was detected using Alexa Fluor 594

goat anti-mouse IgG conjugate (Fig. 5C, red). The merge of images B and C is

shown in D. Colocalization of GPI-PLCp and glycosomes is evident (Z. Zheng,

unpublished).

In a second approach, intracellular location of GPI-PLCp was determined

by subcellular fractionation. T. brucei glycosomes were purified by velocity

sedimentation and sucrose density-gradient fractionation from homogenized

cells. Crude microbodies obtained by velocity sedimentation were separated

using sucrose density-gradient centrifugation [23]. The gradient was fractionated

into eighteen portions, each 0.75 ml portion was assayed for the presence of

GPI-PLCp by an enzyme assay and for aldolase (a marker of glycosomes [24])

by Western blotting (Fig. 6). Approximately 70% of GPI-PLCp activity was found

in samples 13-16 (Fig. 6). A small peak is also present in less dense fractions,

this may be newly synthesized, cytosolic GPI-PLCp not yet associated with

membranes. Aldolase was primarily found in samples 13-16 by Western

analysis; a small peak was also detected in fractions 3-5 (Fig. 6). The presence

of both GPI-PLCp and aldolase in fractions 13-16 indicate that the two

cosedimented on the sucrose gradient. These observations confirm the

immunofluorescence data that GPI-PLCp is a glycosome protein in vivo.

55

In the aforementioned experiments, as negative controls, we assayed

each fraction for the presence of the endoplasmic reticulum (ER) proteins, BiP

and Sec61 [25, 26] and lysosomal protein p67 [27]. The results of these

experiments were inconclusive, perhaps due to inappropriate assay conditions

(data not shown).

In vitro studies were performed to test whether GPI-PLCp binds to

glycosomes purified from T. brucei. For this purpose, GPI-PLCp was translated

in vitro and incubated with or without glycosomes (see Post-translational binding

of proteins to glycosomes and cosedimentation analysis). Samples were

separated into supernatant and pellet fractions and were analyzed by SDS-

PAGE/phosphorimaging. Figure 7A shows the distribution of GPI-PLCp in

supernatant and pellet fractions in the presence or absence of glycosomes (Fig.

7A). To quantitate the differences in the effectiveness of protein binding to

microsomes, the amount of signal in each pellet was compared to the total signal

(sum of signal in supernatant and pellet). The ratios obtained were plotted for

each protein tested in the presence or absence of T. brucei microsomes (Fig.

7B). In the absence of glycosomes, twenty one percent of GPI-PLCp pelleted.

Upon adding glycosomes, the protein signal in the pellet fraction increased to

51% of GPI-PLCp cosedimented with the organelle (Fig. 7B). This 2-fold

increase in sedimentation of GPI-PLCp in the presence of glycosomes indicates

that GPI-PLCp associates with glycosomes.

Titration analysis was also performed to determine if the amount of

glycosomes affected the binding of GPI-PLCp. To test this, in vitro translated

56

GPI-PLCp was incubated with different amounts of glycosomes (1, 3 and 5 ml);

the mixture was then sedimented and analyzed as above. From these

experiments we found 3 ml of glycosomes to be sufficient and there was no

difference in binding of GPI-PLCp when incubated with 5 ml of glycosomes (not

shown).

As shown (Fig. 7B), not all of the in vitro translated GPI-PLCp associates

with glycosomes. There are several reasons as to why this may occur. First,

endogenous GPI-PLCp may have saturated the T. brucei glycosomes.

Therefore, only a limited amount of the in vitro translated protein can bind.

Second, if GPI-PLCp is imported into glycosomes, the molecular machinery

required for import may not be present in the in vitro system. Third, there may be

an excess of in vitro translated GPI-PLCp as compared to glycosomes and not all

may be able to bind. Last, other proteins in the lysate used to translate GPI-PLC

mRNA may compete with GPI-PLCp in binding.

GPI-PLCp Is A Lumenal Protein of Glycosomes

Having identified glycosomes as the organelle that GPI-PLCp binds in T.

brucei, we wanted to determine the topological orientation of GPI-PLCp on

glycosomes since GPI-PLCp is a membrane protein [1-5]. To determine whether

GPI-PLCp associated with cytoplasmic membrane or lumenal membranes of

glycosomes, we used protease protection assays. In control studies, purified

GPI-PLCp [5] was incubated overnight with proteinase K; the phospholipase C

was digested by the protease (Fig. 8A).

57

Next, to determine whether glycosomal GPI-PLCp was susceptible to

protease digestion, purified glycosomes from T. brucei were incubated in the

presence or absence of proteinase K and/or NP-40 (a non-ionic detergent) (Fig.

8B). Purified glycosomes retain GPI-PLCp activity in the presence of proteinase

K or NP-40 alone. However, in the presence of both proteinase K and NP-40,

there is a dramatic loss of GPI-PLCp activity (Fig. 8B). These data indicate that

(i) glycosomes protect GPI-PLCp from proteinase K digestion, and (ii) detergent

permeabilization of glycosome membranes exposes GPI-PLCp to proteinase K

digestion. Based on these data we infer that GPI-PLCp is imported into

glycosomes of T. brucei.

DISCUSSION

Membrane Binding of GPI-PLCp

Biochemically, GPI-PLCp behaves in a mannar similar to an integral

membrane protein [1-5]. Integral membrane proteins are proteins that are not

released from the membrane by relatively gentle extraction procedures, such as

exposure to solutions of very high ionic strength or of extreme pH. Trans-

membrane proteins, and many proteins that are bound to the bilayer by lipids,

are integral membrane proteins [28]. Kyte-Doolittle analysis and Pfam analysis,

are used to identify hydrophobic domains or lipid-binding domains, respectively.

These computational approaches failed to recognize any such domains in GPI-

PLCp (Fig. 1A). Membrane binding of GPI-PLCp is not due to eukaryotic-specific

modifications [4] or protein-protein interactions [2, 29].

58

Cosedimentaton analysis of segments of GPI-PLCp fused to a soluble

reporter protein (GFP) indirectly revealed two potential membrane-binding

regions (MBRs) (Fig. 3), amino acids 60-120 (MBR1) and residues 238-298

(MBR2). These MBRs were fused to GFP in an effort to directly test a

hypothesis that either MBR1 or MBR2 was sufficient for membrane binding by

GPI-PLCp. Only MBR1 was capable of targeting GFP to microbodies (Fig. 4).

MBR1 of GPI-PLCp May Contain An Amphipathic a-Helix

MBR 1 does not have a 19 amino acid stretch that might span a lipid

bilayer (Fig. 1A). However, biochemical evidence indicates that GPI-PLCp is an

integral membrane. We therefore speculate that MBR1 had a hydrophobic patch

in its secondary structure that facilitated binding to membranes. To test this idea,

we analyzed GPI-PLCp by hydropathy analysis using an 8 amino acid window

(not shown). Alanine is the least aliphatic of the non-polar amino acids, a stretch

of 8 alanines has a hydrophobicity index of 1.8. Therefore, a value of 1.8 (Kyte-

Doolittle scale [6]) was used to identify potential hydrophobic regions within GPI-

PLCp. With these parameters, 5 peaks of at least 1.8 were found. Amino acids

60-74 of MBR1 contain a peak with a hydrophobicity index of 2.0 (Fig. 9A).

PSIPRED Protein Structure Prediction Server [30] predicted that these residues

have the potential to form an a-helix. Helical wheel analysis [31] of these amino

acids generated a possible amphipathic a-helix. Hydrophobic amino acids

(valine, leucine and phenylalanine) are on one side of the proposed a-helix in

MBR1 (Fig. 9B, black arch), forming a hydrophobic patch that may interact with a

59

lipid bilayer. Therefore, we propose that GPI-PLCp interacts with membranes by

a hydrophobic patch in MBR1 that has the potential to form an amphipathic a-

helix.

Amphipathic a-helices are used by other proteins to associate with

membranes. MinD binds to phospholipid vesicles with a single, short strech of

10 amino acids which have the potential to form an amphipathic helix [32].

Similarily, membrane binding of prostaglandin H2 synthase [33, 34] and

mammalian PI-PLCd [35] depend on amphipathic a-helices. Figure 9C illustrates

a model of prostaglandin H2 synthase associating with one leaflet of the plasma

membrane via 4 amphipathic a-helices.

Experimental data suggest that GPI-PLCp does not associate with

membranes by protein modification [4]. GPI-PLCp expressed in E. coli exhibits

the same tight association to membranes as it does in T. brucei [4]. However,

recombinant GPI-PLCp is extracted by 8M Urea whereas the native enzyme in T.

brucei cannot (R. Howard, unpublished). GPI-PLCp is myristoylated in T. brucei

[36]. This modification may contribute to the effectiveness of binding and explain

why native enzyme cannot be extracted by the chaotropic agent. Further

expermentation may be done to validate this hypothesis.

Glycosome Interactions

Glycosomes are peroxisome-like microbodies found in all kinetoplastids;

they contain the first 7-9 enzymes of glycolysis [24, 37]. Recent experiments

suggest GPI-PLCp localizes to glycosomes in vivo (Z. Zheng, unpublished). To

investigate this result further, T. brucei organelles were resolved by velocity

60

sedimentation and sucrose density-gradient centrifugation. The gradient was

fractionated into 18 portions, each of which was assayed for GPI-PLCp enzyme

activity and for the presence of aldolase (a glycosome marker) by Western

analysis. Aldolase and GPI-PLCp cosedimented on the sucrose gradient (Fig.

6), indicating that endogenous GPI-PLCp localizes to glycosomes in vivo. In

support of this result, we found that GPI-PLCp bound to purified glycosomes from

T. brucei (Fig. 7).

The topological orientation of GPI-PLCp on glycosomes was studied.

Here, glycosomes were subjected to proteinase K digestion in the presence or

absence of NP-40. Glycosomal GPI-PLCp is not susceptible to proteinase K

(Fig. 8B). However, when NP-40 detergent is added to the digestion reaction,

GPI-PLCp is digested by proteinase K digestion. Based on these results, we

surmise that GPI-PLCp resides within glycosomes of T. brucei. This result is

however, inconsistent with data that detected GPI-PLCp on the cytoplasmic side

of vesicles [9]. In this study, only a few gold particles (indicitative of GPI-PLCp)

were detected on vesicles, resulting in data that is relatively difficult to interpret.

We believe that this result was due to a loss of epitope signal that may have

been due to fixation of T. brucei cells prior to electron microscopy analysis [9].

Biochemical properties strongly suggest that GPI-PLCp is associated with

the glycosomal membranes. GPI-PLCp binds to T. brucei microbodies (Fig. 2)

and glycosomes (Fig. 8). The recombinant protein, expressed in Escherichia

coli, which lacks glycosomes, also strongly associates with membranes [4]. The

purified protein binds detergent micelles during Triton X-114 phase partitioning

61

[2]. Based on these observations, we propose that GPI-PLCp binds to

membranes non-specifically. This inference raises two questions: (i) how is GPI-

PLCp prevented from binding to local membranes after its synthesis in the

cytosol,and (ii) how does GPI-PLCp get specifically to glycosomal membranes?

Proposed Model for GPI-PLCp – Glycosome Interactions

We offer a hypothetical model based on peroxisomal import of matrix

proteins (Fig. 10). In this scheme, GPI-PLCp, synthesized in the cytosol, binds to

a cytosolic chaperone protein. Alternatively, GPI-PLCp may associate with a

protein containing a peroxisomal targeting sequence (PTS) that binds a PTS

receptor protein (“piggy-backing”). This initial complex formation could inhibit

non-specific interactions between GPI-PLCp and nearby membranes after

synthesis. We propose that the peroxin complex would then transport GPI-PLCp

to docking proteins. After docking, the complex could enter the glycosome.

Following import, the complex dissociates and the cytosolic chaperone is

recycled back to the cytosol. Released from the chaperone, GPI-PLCp

associates (non-specifically) with the lumenal leaflet of the glycosome inner

membrane [38, 39].

62

Figure 1: Kyte-Doolittle Hydropathy Analysis of GPI-PLCp and Sec61p. A)

Hydropathy analysis of GPI-PLCp from T. brucei using a 19 amino acid window.

B) Hydropathy analysis of Sec61p from S. cerevisiae using a 19 amino acid

window. The number ruler above each plot indicates amino acid number in each

peptide. Relative hydrophobicity is indicated by the scale to the left of each plot.

A hydrophobicity of 1.6 or greater predicts a potential transmembrane domain.

Nineteen is the minimum number of amino acids required to span a membrane

[16].

63

Figure 1

AGPI-PLCp (T. brucei)

Kyte-Doolittle Hydrophobicity Plot19 Amino Acid Window

BSec61p (S. cerevisiae)

Kyte-Doolittle Hydrophobicity Plot(19 Amino Acid Window)

64

Figure 2: GPI-PLCp targets a soluble reporter protein to T. brucei

microsomes. A) GFP and GFP:GPI-PLCp were translated in vitro as

described. Twenty ml of each reaction was incubated with (+) or without (-) T.

brucei rough microsomes (TbRM) (7 ml, 210U) in RMB. Reactions were

incubated for 10 min. at 30°C then 3-fold diluted with dilution buffer. Samples

were incubated on ice for 15 min, then layered on a sucrose cushion and

centrifuged at 52,000 rpm for 30 min. Supernatant was removed and pellet was

solubilized with 70 ml PRB. Five percent total volume of both supernatant (S) and

pellet (P) were analyzed by 14% SDS-PAGE and phosphorimaging. B) Graphs

indicate the ratio of signal in pellet to total signal in absence (-) or presence (+) of

T. brucei microsomes.

65

Rat

io o

f S

ign

al in

Pel

let

to T

ota

l Sig

nal

Figure 2

A

S P S P

- +GFP

S P S P

- +

GFP:GPI-PLC

TbRM

B

- +

GFP

- +

GFP:GPI-PLC

TbRM

66

Figure 3: Effect of NH2-terminal and COOH-terminal fragments of GPI-PLCp

on targeting a soluble reporter protein to T. brucei microsomes (K. Butler,

unpublished). Indicated fusion constructs were translated in vitro as described.

Twenty ml of Radiolabeled proteins were incubated in the absence (-) or

presence (+) of TbRM (210U) for 10 min. at 30°C. Twenty ml of each sample was

diluted 5-fold in DB and incubated for 10 min. on ice. Each fraction was layered

on a 500 mM sucrose cushion and centrifuged at 52,000 rpm for 30 min at 4°C.

Equivalent volumes of supernatant (s) and pellet (p) fractions were analyzed by

14% SDS-PAGE and phosphorimaging. Images were quantitated using

QuantityOne software (BioRad). (A-D) NH2-terminal fragments. (E-H) COOH-

terminal fragments. (I) is an illustration of each construct analyzed and indicates

which polypeptides were (3) or were not (7) capable of targeting GFP to T.

brucei rough microsomes.

67

GFP:GPI-PLC1-60

S P S P

- +GFP:GPI-PLC1-120

S P S P

- +

GFP:GPI-PLC1-179

S P S P

- +GFP:GPI-PLC1-358

S P S P

- +TbRM

TbRM

A B

C D

A

rbitr

ary

Phos

phor

imag

er U

nits

A

rbitr

ary

Phos

phor

imag

er U

nits

Arb

itrar

y Ph

osph

orim

ager

Uni

ts

A

rbitr

ary

Phos

phor

imag

er U

nits

Figure 3

68

GPI-PLC1-358:GFP

S P S P

- +GPI-PLC179-358:GFP

S P S P

- +

GPI-PLC238-358:GFP

S P S P

- +GPI-PLC298-358:GFP

S P S P

- +

TbRM

TbRM

E F

G H

A

rbitr

ary

Phos

phor

imag

er U

nits

Arb

itrar

y Ph

osph

orim

ager

Uni

ts

Arb

itrar

y Ph

osph

orim

ager

Uni

ts

A

rbitr

ary

Phos

phor

imag

er U

nits

Figure 3

69

Figure 3

I

70

Figure 4: Effect of putative membrane binding regions (MBR) of GPI-PLCp

on targeting a soluble reporter protein to T. brucei microsomes. A)

Membrane binding regions (MBR1), amino acids 60-120, and MBR2, amino

acids 238-298 of GPI-PLCp were fused to the soluble reporter polypeptide, green

fluorescent protein (GFP). Constructs were linked to GFP by megaprimer PCR

as described. DNA was transcribed and RNA translated, in vitro, as described.

B) Twenty ml of each radiolabeled polypeptide was added to RMB with (+) or

without (-) T. brucei rough microsomes (7 ml, 210U) and incubated at 30°C for 10

min. Each sample was diluted 3-fold and layered on a 500 mM sucrose cushion.

Samples were centrifuged at 52,000 rpm for 30 min at 4°C. Supernatant was

removed and the pellet fraction was solubilized in pellet resuspension buffer

(PRB). Equivalent volumes of supernatant (S) and pellet (P) fractions were

analyzed by 14% SDS-PAGE and phosphorimaging. QuantityOne software

(BioRad) was used to quantitate each fraction. C) Graphs indicate the ratio of

signal in pellet to the total signal in the absence (-) or presence (+) of

microsomes.

71

R

atio

of S

igna

l in

Pelle

t to

Tota

l Sig

nal

Figure 4

A

B

S P S P

- +

MBR1GPI-PLC:GFP

S P S P

- +

MBR2GPI-PLC:GFP

TbRM

C

- +MBR1GPI-PLC:GFP

- +

MBR2GPI-PLC:GFP

TbRM

72

Figure 5: GPI-PLCp colocalizes with a glycosomal protein, HGPRT, in T.

brucei. (A-D) T. brucei was fixed, permeablilized and incubated with anti-GPI-

PLCp and anti-HGPRT antibody. Anti-HGPRT was detected by Alexa Fluor 488

goat anti-rabbit IgG conjugate (B, green). Anti-GPI-PLCp was detected using

Alexa Fluor 594 goat anti-mouse IgG conjugate (C, red). The merge of images B

and C is shown in D [Z. Zheng, unpublished].

73

HGPRT

GPI-PLCp Merge

Figure 5

74

Figure 6: GPI-PLCp colocalizes with a glycosomal protein on a sucrose

gradient. T. brucei glycosomes were isolated by sucrose-gradient

sedimentation of microbodies obtained by differential centrifugation. The sucrose

gradient was fractionated into 18, 750 ml samples as described. One ml of each

sample was assayed for GPI-PLCp activity. The y-axis indicates the amount of

[3H] dimyristoylglycerol (DMG) released by GPI-PLCp. The x-axis denotes the

fraction number. Each of the 18 samples was precipitated using TCA. Pellets

were resuspended in 30 ml 5X SDS-sample buffer. Five ml of each sample was

analyzed by 14% SDS-PAGE followed by Western analysis using an anti-

aldolase antibody.

75

Figure 6

A

76

Figure 7: GPI-PLCp binds to glycosomes in vitro. GPI-PLC was translated in

vitro as described. Four ml of the translation mixture was incubated with (+) or

without (-) 3 ml purified glycosomes for 30 min. at 30°C then on ice for 1 hour.

Samples were centrifuged at 25,000 rpm for 30 min. Supernatants were

removed and pellets were resuspended in 25 ml 2.5X SDS-sample buffer. Ten ml

of supernatant (S) and pellet (P) were analyzed by 12% SDS-PAGE and

phosphorimaging. Quantitation was done using QuantityOne software (BioRad).

B) Graphs indicate the ratio of signal in pellet to total signal in arbitrary units.

77

Figure 7

A

S P S P

+-Glycosomes

GPI-PLCp

B

+-GPI-PLCp

Glycosomes

78

Figure 8: Glycosomal GPI-PLCp is protected from digestion by proteinase

K A) Effect of proteinase K on activity of purified GPI-PLCp. One ml (1.4U) of

purified GPI-PLCp was incubated with or without proteinase K (300 ng/ml)

overnight at 15°C. Reactions were quenched with 2mM PMSF and assayed for

GPI-PLCp activity as described. Released [3H]dimyristoylglycerol was quantified

by scintillation counting. B) Purified glycosomes (1 ml) containing endogenous

GPI-PLCp were incubated in the presence (+) or absence (-) of proteinase K

(300 ng/ml), 1% NP-40, or both, overnight at 15°C. Reactions were quenched

with 2mM PMSF and analyzed for GP-PLCp activity as described.

79

Figure 8

A

B

80

Figure 9: Hydropathy analysis of MBR1 and helical wheel analysis of

amino acids 60-74 of GPI-PLCp. A) Hydropathy plot of MBR1 of GPI-PLCp

using an 8 amino acid window. Numbers above the plot indicate amino acid

number of the polypeptide. Relative hydrophobicity is indicated to the left of the

plot. A hydrophobic patch is found within amino acids 60-74. B) Helical wheel

analysis predicts an amphipathic alpha-helix. Hydrophobic patch is indicated by

a solid line. C) Prostaglandin H2 Synthase binds membranes via four

amphipathic alpha-helices. Prostaglandin H2 Synthase is a peripheral-

membrane protein that binds to membranes by direct protein-lipid interactions.

The protein contains 4 alpha helices that have a series of hydrophobic residues

that are postulated to insert into the bilayer.

81

Figure 9

A GPI-PLCp (60-120)

Kyte-Doolittle Hydropathy Plot8 Amino Acid Window

B

82

Figure 9

CProstaglandin H2 Synthase

Plasma Membrane

Adapted from Theoretical Biophysics Group, University of Illinois at Urbana-Champaign

83

Figure 10: Hypothetical model of GPI-PLCp association with glycosomes.

GPI-PLCp is synthesized on free ribosomes in the cytosol. (a) After synthesis, it

may bind to a peroxin (PEX, green); (b) transport of the enzyme-receptor

complex to the glycosome surface; (c) the complex docks to a receptor complex

possibly including PEX17p (yellow) PEX14p (grey) and perhaps PEX13p (red).

GPI-PLCp-receptor complex enters through a proteinaceous pore that may

contain PEX8p, PEX10p and PEX12p (all shown in purple). After translocation,

the complex dissociates (e) and the receptor is recycled back to the cytosol

which may involve PEX4p and PEX22p (orange). Once GPI-PLCp is inside the

glycosome and has dissociated from the receptor, MBR1 may direct GPI-PLCp to

associate with the inner leaflet of the glycosome membrane. PEX, peroxin [27,

28, Figure is adapted from, 28].

84

Figure 10

85

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29. Morris, J. C., Ping-Sheng, L., Zhai, H.-X., Shen, T. Y. & Mensa-Wilmot, K.(1996) Phosphatidylinositol Phospholipase C is Activated Allosterically By TheAminoglycoside G418. 2-Deoxy-2-Fluoro-Scyllo-Inositol-1-O-Dodecylphosphonate and its Analogs Inhibit Glycosyl PhosphatidylinositolPhospholipase C, J.Biol.Chem. 271, 15468-15477.

30. McGuffin, L. J., Bryson, K. & Jones, D. T. (2000) The PSIPRED proteinstructure prediction server, Bioinformatics. 16, 404-5.

31. Schiffer, M. & Edmundson, A. B. (1967) Use of helical wheels to representthe structures of proteins and to identify segments with helical potential., BiophysJ. 7, 121-35.

32. Zhou, H. & Lutkenhaus, J. (2003) Membrane binding by MinD involvesinsertion of hydrophobic residues within the C-terminal amphipathic helix into thebilayer, J Bacteriol. 185, 4326-35.

33. Picot, D., Loll, P. J. & Garavito, M. (1994) The X-ray crystal structure of themembrane protein prostaglandin H2 synthase-1, Nature. 367, 243-249.

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35. Essen, L. O., Perisic, O., Cheung, R., Katan, M. & Williams, R. L. (1996)Crystal structure of a mammalian phosphoinositide-specific phospholipase Cdelta, Nature. 380, 595-602.

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36. Ochatt, C. M., Butikofer, P., Navarro, M., Wirtz, E., Boschung, M., Armah, D.& Cross, G. A. (1999) Conditional expression of glycosylphosphatidylinositolphospholipase C in Trypanosoma brucei, Mol Biochem Parasitol. 103, 35-48.

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CHAPTER III

DISCUSSION

Trypanosoma brucei is a protozoan parasite that causes African sleeping

sickness in humans. It is covered with a surface coat that consists of variant

surface glycoprotein (VSG) [1, 2]. This coat plays an important role in the

parasite’s ability to evade a host’s immune response through “antigenic variation”

[3]. The VSG protein is anchored to the extracellular surface of the plasma

membrane by a glycosyl phosphatidylinositol (GPI) anchor [1].

The trypanosome also express GPI-specific phospholipase C (GPI-PLCp)

that is highly specific for GPIs [4, 5]. GPI-PLCp is an integral membrane protein

however, how the protein binds membranes, and where it binds in vivo, is

unknown. Under normal conditions GPI-PLCp does not cleave GPI-anchored

proteins or intermediates [6]; therefore, the parasite remains viable in its host.

The enzyme is a virulence factor. It influences parasitemia in mice [7] and

may also regulate endocytosis (S. Subramanya, unpublished). It is because of

this that we wished to further characterize this enzyme in T. brucei. In an effort

to understand the function of GPI-PLCp in vivo, it was necessary to determine

how the enzyme binds to membranes and where it localizes in the cell.

In this work we found one region of GPI-PLCp that is sufficient in targeting

a soluble reporter polypeptide (GFP) to T. brucei microsomes. This region is

between amino acids 60-120. Kyte-Doolittle analysis of this region identified a

90

hydrophobic patch in residues 60-74. Further analysis predicts that these

residues may form an amphipathic a-helix. We propose GPI-PLCp binds

membranes by at least one amphipathic a-helix in residues 60-74.

In addition, we also found that GPI-PLCp cosedimented with aldolase on a

sucrose gradient. This, along with immunofluorescence experiments (Z. Zheng,

unpublished) suggests that GPI-PLCp localizes to glycosomes in vivo.

Endogenous GPI-PLCp associated with glycosomes is resistant to protease

digestion. Only after the addition of detergent is GPI-PLCp susceptiable to

digestion. Therefore, we propose that GPI-PLCp is imported into glycosomes

and resides in the lumen of the organelle.

GPI-PLCp does not contain any sequence homologous to known

peroxisomal targeting sequences (PTS). How GPI-PLCp is targeted to

glycosomes and imported is of interest. We believe the enzyme may contain a

novel PTS that has yet to be discovered or it may bind to another protein that

contains a PTS and “piggy-backs” its way into glycosomes.

In conlusion, GPI-PLCp is an integral membrane protein. It binds

membranes by at least one amphipathic a-helix. We believe that association of

GPI-PLCp with membranes is non-specific. GPI-PLCp is synthesized in the

cytosol and immediately after synthesis, could potentially bind to surrounding

membranes. However, GPI-PLCp localizes to glycosomes in vivo. Thus, binding

may be regulated in some way. We hypothisize, that after synthesis, GPI-PLCp

immediately associates with cytosolic factors that target the enzyme to

glycosomes, thereby sequestering non-specific binding. Only after GPI-PLCp

91

enters the glycosome and dissociates from the receptor protein, is it able to bind

membranes, specifically glycosomal membranes.

Further experimentation could be done to characterize the different stages

of our model in an effort to better understand the mechanisms by which GPI-

PLCp is imported into glycosomes. Also, immunofluorescene studies using the

truncated fusion constructs may be done to elucidate which region of GPI-PLCp

contains a glycosome targeting sequence. Furthermore, mutational anaylsis of

this region could be perfomed to identify the specific sequence of the targeting

signal.

92

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2. Mensa-Wilmot, K., Morris, J. C., Al-Qahtani, A. & Englund, P. T. (1995)Purification and use of recombinant glycosylphosphatidylinositol phospholipaseC, Methods Enzymol. 250, 641-655.

3. Turner, C. M. (1999) Antigenic variation in Trypanosoma brucei infections: anholistic view., J Cell Sci. 112, 3187-3192.

4. Bulow, R. & Overath, P. (1986) Purification and characterization of themembrane-form variant surface glycoprotein hydrolase of Trypanosoma brucei, JBiol Chem. 261, 11918-23.

5. Hereld, D., Krakow, J. L., Bangs, J. D., Hart, G. W. & Englund, P. T. (1986) Aphospholipase C from Trypanosoma brucei which selectively cleaves theglycolipid on the variant surface glycoprotein, J.Biol.Chem. 261, 13813-13819.

6. Black, S. J., Hewett, R. S. & Sendashonga, C. N. (1982) Trypanosoma bruceivariable surface antigen is released by degenerating parasites but not by activelydividing parasites, Parasite.Immunol. 4, 233-244.

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