Protein translocation in bacteria, eukaryotes- targeting signals- import, export systems: bacterial, ER, chloroplasts, peroxisomes, mitrochondria- nuclear import

10-1

Protein transport and translocationProtein transport and translocation

at least 40% of all cellular proteins are:

inserted into a membrane

translocated into an organelle, nucleus

exported outside the cell or to the periplasm

proteins must be kept in translocation-competent form (i.e., either partially or entirely unfolded

exception is peroxisomes, nucleus

proteins must be folded/assembled after translocation; molecular chaperones are usually involved

translocation is an energy dependent process

10-2

Overview of protein transportOverview of protein transportand translocationand translocation

IM, inner membraneIMS, inner membrane spaceP, periplasmOM, outer membraneTL, thylakoid lumenTM, thylakoid membrane

SecYEG, Sec61, TOM, TIM, TOC are proteinsubunits of the translocation systems

adapted from Schatz and Dobberstein,Science 271, 1519 (1996)

10-3Protein translocation systemsProtein translocation systems

e e

e

i

i

i

blue is hydrophilic (H-phil)

red is hydrophobic (H-phob)

curling lines are helical

zig-zags are turns

‘OH’ denotes hydroxylated residues

‘+’ denotes positively charged aa’s

most signals are at the N-terminus

can be cryptic

expo

rt s

igna

lsim

port

sig

nals

H-phobobic H-philic

H-phob

H-phil

H-phob

10-4

Targeting signalsTargeting signals

two major pathways for translocation in bacteria: Sec and SRP pathways

both converge at SecYEG translocon and use SecA, a peripherally-bound ATPase that supplies the energy for translocation

SecB binds to nascent chains containing a signal sequence and maintains the preprotein in translocation-competent form, then binds SecA; SRP docks with membrane receptor, FtsY (simpler homologues of eukaryotic SRP and SRP receptor)

archaea lack SecB, have SRP/FtsY but no SecA; what drives translocation?

archaeal SRP, FtsY, SecYEG more closely related to eukaryotic proteins (SecYEG)

10-5

Translocation in Translocation in bacteriabacteria

Structure and function of SecBStructure and function of SecB 10-6

SecB monomer; functionalcomplex assembles as atetramer (dimer of dimers)

shading of the hydrophobic subsites 1 and 2 in the assembled tetramer; the opposite surface contains the same groove with two separate subsites 1 and 2

Conserved residues shown to be important for the interaction of SecB with SecA:

Asp27, Glu31, Glu86 (green)

Ile 84(yellow)

PTB (phosphotyrosine Binding) domain

SecBmonomer

SecB has an

unexpected structural

similarity to the PTB domain

Sec61 is a hetero-trimeric complex composed of , , subunits related to SecYEG

SRP is a ribonucleoprotein complex composed of 7S RNA and numerous proteins

binding of signal sequence is modulated by NAC

SRP pathway is co-translational; SRP mediates arrest of elongation until it docks with SRP receptor; translocation then proceeds through Sec61

SRP is the major pathway used for import into ER

a post-translational translocation pathway that makes use of Sec61 also exists; preproteins are maintained in a translocation-competent form by Hsp70/Hsp40

10-7

Translocation into the Translocation into the ERER

10-9

Folding in the Endoplasmic reticulumFolding in the Endoplasmic reticulum

Toc components, mediate translocation (Toc75 is the translocon); it is unclear how preproteins are targeted to the channel; Hsp70/Hsp40 may be involved

Hsp70 in both the IMS and the stroma assist the threading of the preprotein into the chloroplast

an Hsp100 chaperone also called ClpC (AAA ATPase) also binds preproteins in the stroma

Hsp70/chaperonin (Cpn60) may assist folding/assembly of newly-imported protein

import into thylakoids (used for respiration) uses the SRP pathway

10-9

Translocation into Translocation into chloroplastschloroplasts

targeting of proteins is initiated post-translationally by Pex5/7 proteins, which bind the peroxisomal targeting signal (PTS)

translocon not well defined; possibility of vesicular budding?

gated pore that is regulated by membrane proteins?

first organelle demonstrated to import proteins without a PTS, by virtue of assembly with other proteins that contained a PTS

various protein oligomers are imported into peroxisomes

antibodies with PTS, and 9 nm gold particles could be imported

Other transport mechanisms likely involve folded proteins, including the twin-arginine (Tat) transport system of bacteria, and the cytoplasm-to-vacuole targeting pathway of yeast

10-10

Translocation into Translocation into peroxisomesperoxisomes

delivery of preproteins to mitochondria depends on either Hsp70/Hsp40 or MSF, mitochondrial import stimulation factor (MSF)

evidence now that Hsp90 is also involved

mtHsp70/Tim44/Mge (GrpE) is required for import; Tim44 contains J domain

Big debate:

brownian ratchet or pulling model for Hsp70 system-mediated import of proteins

protein folding following import depends on Hsp70, chaperonin (Hsp60)

10-11

Translocation into Translocation into mitochondriamitochondria

nuclear localization signal (NLS) is typically highly basic; e.g., the SV40 large tumor antigen (T ag) has the sequence PKKKRKV /1 importin hetero-dimer recognizes and binds the NLS (or importin alone) importin docks with NPC and mediates interaction with Ran (GDP form) directionality conferred by nature of guanine nucleotide bound to RanRan binding protein (RanBP) is required for importin binding to RanGDP; Ran GTPase activating protein (RanGAP) and nucleotide-exchange factor (RCC) are cytoplasmic and nuclear cytopl. RanGDP required for import; nuclear RanGTP required for release conversely, RanGTP binds substrate with NES in the export direction proteins to be imported can be in a native/near native form

10-12

Import into the Import into the nucleusnucleus

bar,50 nm

- RanGTP

+ RanGTP

Structure of the nuclear pore complexStructure of the nuclear pore complex

some nuclear pore proteins (nucleoporins) contain core FxFG repeats (yellow)

importin contains ‘heat’ repeats that bind the FxFG repeats (Heat repeats 5, 6, 7 are shown in red, green and blue)

the FxFG repeats interdigitate in grooves formed by the Heat repeats

interaction of importin with nucleoporins allows transport across the nuclear pore complex

Core FxFG repeats found in nucleoporins.Each repeat is separated by a ‘linker’ region:

Bayliss et al. (2000) Cell 102, 99-108.

10-14

Mechanism of import into nucleusMechanism of import into nucleus

15 heat repeats of protein phosphatase 2A

conservation is to one side of the repeat structureGroves et al. (1999) Cell 96, 99-110.

10-15

Heat repeat-containing proteinHeat repeat-containing protein