targeting and processing of a chimeric protein with the n ... · in the chimeric proteins, the...

The Plant Cell, Vol. 8, 1601-1611, September 1996 O 1996 American Society of Plant Physiologists

Targeting and Processing of a Chimeric Protein with the N-Terminal Presequence of the Precursor to Glyoxysomal Citrate Synthase

Akira Kato, Makoto Hayashi, Maki Kondo, and Mikio Nishimura’

Department of Cell Biology, National lnstitute for Basic Biology, Okazaki 444, Japan

Glyoxysomal citrate synthase in pumpkin is synthesized as a precursor that has a cleavable presequence at its N-terminal end. To investigate the role of the presequence in the transport of the protein to the microbodies, we generated transgenic Arabidopsis plants that expressed f3-glucuronidase with the N-terminal presequence of the precursor to the glyoxysomal citrate synthase of pumpkin. lmmunogold labeling and cell fractionation studies showed that the chimeric protein was transported into microbodies and subsequently was processed. The chimeric protein was transported to functionally different microbodies, such as glyoxysomes, leaf peroxisomes, and unspecialized microbodies. These observations indicated that the transport of glyoxysomal citrate synthase is mediated by its N-terminal presequence and that the transport system is functional in all plant microbodies. Site-directed mutagenesis of the conserved amino acids in the presequence caused abnormal targeting and inhibition of processing of the chimeric protein, suggesting that the conserved amino acids in the presequence are required for recognition of the target or processing.

INTRODUCTION

Microbodies are ubiquitous organelles in eukaryotic cells; they are generally defined as organelles that contain catalase and at least one H202-producing oxidase. In higher plants, microbodies can be categorized into at least three classes that are distinguishable by function (Huang et al., 1983). (1) Glyoxy- somes, which are found in germinating cotyledons of oil seeds, contain some enzymes required for the B-oxidation of fatty acids and the glyoxylate cycle, which participates in the mobiliza- tion of lipids. (2) Leaf peroxisomes are found in green leaves and function together with mitochondria and chloroplasts in photorespiratory glycolate metabolism. (3) Unspecialized microbodies with undefined metabolic roles are present in other organs, such as roots and stems. Microbodies in cotyledons of fatty seedlings can undergo a change in metabolic function. Thus, glyoxysomes are transformed to leaf peroxisomes during the greening of cotyledons.

lmmunocytochemical studies have shown that glyoxysomes can change directly into leaf peroxisomes (Titus and Becker, 1985; Nishimura et al., 1986; Sautter et al., 1986). We have shown previously that the transition of microbodies from glyoxysomes to leaf peroxisomes is regulated both transcriptionally and post-transcriptionally (Mori and Nishimura, 1989; Mori et al., 1991; Tsugeki et ai., 1993; Katoet al., 1995,1996; Yamaguchi et al., 1995). The reverse conversion of microbodies was ob- served in senescent tissues. Leaf peroxisomes in senescing cotyledons were once again converted directly to glyoxysomes

’ To whom correspondence should be addressed.

(De Bellis and Nishimura, 1991; Nishimura et al., 1993; Kato et al., 1996).

Microbodies are surrounded by a single membrane, and their proteins are encoded in the nuclear genome. Microbody proteins are synthesized in the cytosol on free polysomes and are transported post-translationally into microbodies (Lazarrow and Fujiki, 1985). Most microbody proteins are synthesized as proteins that are the same size as their corresponding mature proteins. The mature proteins in this group have a signal for targeting their sequences to the microbodies. A tripeptide such as Ser-Lys-Leu at the C-terminal end has been identi- fied as a targeting signal, known as peroxisome targeting signal 1 (PTS1; reviewed in Subramani, 1992, 1993). Ser-Lys-Leu and related amino acid sequences have similar functions in mammals, insects, fungi, and plants (Gould et al., 1990). Glyox- ysomal enzymes, such as malate synthase (Comai et al., 1989a; Graham et al., 1989; Mori et al., 1991) and isocitrate lyase (Beeching and Northcote, 1987; Comai et al., 1989b; Turley et al., 1990), and leaf peroxisomal enzymes, such as glycolate oxidase (Volokita and Somerville, 1987; Tsugeki et al., 1993) and hydroxypyruvate reductase (Hayashi et al., 1996b), contain the PTSl signal at their C-terminal ends. In contrast, a small group of microbody proteins, such as 3-ketoacyl-COA thiolase (Hijikata et al., 1987; Preisig-Müllar and Kindl, 1993; Kato et al., 1996) and malate dehydrogenase (Gietl, 1990), are synthesized as precursor proteins with molecular masses greater than those of the mature proteins. They have a cleavable presequence at their N-terminal ends. Swinkels et

1602 The Plant Cell

al. (1991) showed that the N-terminal presequence of 3-ketoacyl-COA thiolase from rat liver functions as a targeting signal (PTS2) .

Glyoxysomal citrate synthase (gCS), which converts ox- aloacetate to citrate in the glyoxylate cycle, is synthesized as a longer precursor with an N-terminal presequence (Kato et al., 1995). The N-terminal region of gCS is highly homologous to those of other microbody proteins that are synthesized as precursors, and gCS has two conserved sequences in its N-terminal region (Kato et al., 1995). One such sequence is RL-X5-HL. It was first recognized by de Hoop and Ab (1992). This sequence seems to function for targeting to microbodies (Gietl et al., 1994; Glover et al., 1994; Tsukamoto et al., 1994). The other sequence is SXLXXAXCXA (where X stands for any amino acid; Kato et al., 1995, 1996), located at the cleavage site of the presequence, but its function has not been defined.

In this study, we focused on the roles of these two consensus sequences in the targeting of gCS to microbodies and in the processing of its N-terminal presequence. We generated transgenic Arabidopsis plants that expressed a chimeric protein composed of the N-terminal region of gCS and a bacterial protein, pglucuronidase (GUS). In addition, we constructed and expressed chimeric proteins with substituted amino acid residues in the two consensus sequences by site-directed mutagenesis. The localization of these chimeric proteins was determined by immunological and immunocytochemical anal- yses, and the processing of the chimeric proteins was examined. We discuss the functions of the two consensus sequences in the targeting and processing of gCS and also provide some details of protein transport, which is mediated by the N-terminal region of gCS, to various microbodies, namely, glyoxysomes, leaf peroxisomes, and unspecialized microbodies.

RESULTS

Expression of Chimeric Proteins in Transgenic Arabidopsis Plants

To investigate the function of the N-terminal region of gCS, we constructed a chimeric gene that encodes the N-terminal region of the enzyme from pumpkin and GUS. The gene encoding GUS originated from fscherichia co/i(uidA) and thus included no signal sequences for targeting to organelles in eukaryotic cells. Figure 1A shows the construction of chimeric proteins. A DNA fragment produced by polymerase chain reaction (PCR) encoded 47 amino acids; this included 43 residues of presequence and four residues of the N-terminal region of mature gCS. This fragment was conjugated to the N-terminal end of GUS, to yield a gene designated CS-GUS (Figure 16). Various amino acids, namely, Arg-16, Leu-17, Leu-24, Glu-30, and Cys-42 in the conserved sequences in the presequence, were replaced by glycine residues as described in Methods. In addition, Cys-42 was replaced by phenylalanine, which has a large and aromatic side chain, or it was eliminated from the presequence.

The chimeric genes were integrated downstream of the cauliflower mosaic virus 35s promoter in the binary vector pMAT037 (Figure 16; Matsuoka and Nakamura, 1991). The Ti plasmid vectors were transferred to Arabidopsis by Agro- bacterium-mediated transformation, and primary transformants were designated To plants. Among the kanamycin-resistant TI plants, transformants that produced the chimeric proteins were selected by immunoblotting, using specific antibodies raised against GUS. T2 seeds were germinated on kanamycin- containing medium, and kanamycin-resistant and homozygous

A Strain

CS-GUS

Rl6G

L17G

L24G

E30G

C42G

C42F

AC42

Gene

CS-GUS

R16G

LI 7G

L24G

E3OG

C42G

C42F

dC42

Amino acid sequence 6 1617 24 30 42 +

CS-GUS M P T D M E L S P S N L S A A S L E P P V M A S S L F A H C V S A Q T -

ME'TDMELSPSNVARHGLAVLAAHLSAASLEPPWSLEAHCVSAQT- 1 GUS H MPTDMELSPSNVARHRGAVLAAHLSAASLEPPVE.IASSLEAHCVSAQT- Tandem 35.9 6ad

M P T D M E L S P S N G S A A S L E P P W S L E A H C V S A Q T - GUS I ~ T D ~ ~ L S P S N V A R H R L A V L A A H L S A A S L P ~ ~ S L ~ ~ S A Q T - GUS

1 ~ T D ~ ~ L S P S N V A R H R L S M S L E P P ' J L . I A S S L E A H G V S A Q T - Tandem 3 5 9 6ad

1 " I P T D l ~ ~ L S P S N V ~ R ~ L I S A A ~ L E P P ~ ~ ~ " ~ I S S L ~ S ~ Q T ~

1 " I P T D P I E L S P S I . R ' h R H ~ ~ ~ L S ~ S L E P P ~ ~ " ~ S L ~ ~ ~ S A Q T -

Figure 1. Construction of Chimeric Genes.

In the chimeric proteins, the N-terminal sequence of gCS in pumpkin was conjugated with the N terminus of GUS. (A) N-terminal extra peptides of the chimeric proteins. Amino acid substitutions were introduced by PCR. The substituted amino acids are repre- sented in boldface letters. The arrow indicates the site of processing of the presequence. The designations at left represent transgenic Arabidopsis plants carrying the chimeric proteins; gene designations are italicized. (6) Construction of plasmids. The chimeric gene and the GUS gene were integrated into the Ti binary vector pMAT037 (see Methods). The stippled box indicates the N-terminal region of gCS. Tandem 35SP represents the tandemly duplicated 35s promoter of the cauliflower mosaic virus; 6ab' represents the transcription terminator region of T-DNA transcripts 6a and 6b.

Transport of Glyoxysomal Citrate Synthase 1603

Figure 2. Immunological Detection of Chimeric Proteins in CrudeExtracts of Green Seedlings of Transgenic Arabidopsis Seedlings.Crude extracts of green seedlings were subjected to SDS-PAGE (on6% acrylamide gels) and subsequently immunoblotted with GUS-specific antibodies. Ten micrograms of total protein was loaded in eachlane. Arrows indicate the sizes of the proteins.

T2 plants were isolated. The T2 seedlings that accumulatedthe largest amount of the product of each chimeric gene wereused for further analysis.

Figure 2 shows the accumulation of the chimeric proteinsin all of the transgenic plants. The transformant carrying Gl/S,which encoded native GUS (Figure 1B; Hayashi et al., 1996a),produced a single immunoreactive band of a protein with amolecular mass of 68 kD. In the case of other constructs, pro-teins with molecular masses of 72 kD were detected. Thedifference of 4 kD in the molecular masses corresponds tothe additional amino acids in the chimeric proteins. Some trans-genic plants expressed GUS proteins of both 68 and 72 kD.The 68-kD proteins detected in the transgenic plants wereCS-GUS, E30G, C42G, and C42F and may have been gener-ated by processing after translocation of the chimeric proteins.The accumulation levels of the products of the chimeric geneswere similar in each transgenic Arabidopsis plant.

quence, indicating that the N-terminal region of gCS functionsas a signal for targeting to glyoxysomes.

In higher plants, microbodies can be classified into threetypes, namely, glyoxysomes in etiolated cotyledons, leat perox-isomes in green leaves, and unspecialized microbodies in othertissues. Microbodies in dark-grown cotyledons from Arabidop-sis function as glyoxysomes (Hayashi et al., 1996a). BecausegCS is a glyoxysnme-specific protein in pumpkin cotyledons(Kato et al., 1995), the chimeric protein encoded by CS-GUSaccumulated in glyoxysomes of 7-day-old dark-growncotyledons (Figures 3 and 4A). Hayashi et al. (1996a) alsodemonstrated that a chimeric protein consisting of GUS plusthe C-terminal targeting signal of malate synthase is trans-ported to all types of microbodies, namely, glyoxysomes, leafperoxisomes, and unspecialized microbodies.

We emphasize that the chimeric protein containing theN-terminal targeting sequence also accumulated in all func-tionally differentiated microbodies in plants. The chimericprotein encoded by CS-GUS was localized in leaf peroxisomesin green cotyledons (Figure 4B) and in glyoxysomes in etio-lated cotyledons (Figure 4A). The chimeric protein was alsolocalized in leaf peroxisomes in mature leaves (Figure 4C) andin unspecialized microbodies in roots (Figure 4D). These resultsclearly indicate that the chimeric protein encoded by CS-GC/Swas transported to glyoxysomes, leaf peroxisomes, and un-specialized microbodies.

M

Localization of the Chimeric Protein Encodedby CS-GUS

To clarify the intracellular localization of the chimeric proteinencoded by CS-GUS, we performed immunocytochemicalanalysis using a transgenic plant expressing the chimeric pro-tein. Ultrathin sections were prepared from the cotyledon ofa 7-day-old dark-grown transgenic Arabidopsis plant carryingCS-GUS. Double staining was with antibodies raised againstGUS and catalase, which is a marker enzyme for microbod-ies. Figure 3 shows the results of double staining one of thesections. The larger gold particles and the smaller gold parti-cles represent the localization of GUS and catalase,respectively. The large gold particles are colocalized on anorganelle on which small gold particles are visible, but not onother organelles such as mitochondria and etioplasts. Thus,GUS was specifically localized in the microbodies. Native GUSis localized nonspecifically in the organelles of transgenicplants (Hayashi et al., 1996a). The specific translocation of GUSto glyoxysomes resulted from the additional N-terminal se-

Figure 3. Double-lmmunogold Labeling of a Section of a TransgenicArabidopsis Seedling.A transgenic Arabidopsis seedling carrying CS-GUS was grown for7 days in darkness. A thin section of the cotyledon was double-stainedwith specific antibodies raised against GUS and catalase. Large goldparticles (15 nm; large arrowhead) indicate the localization of the chi-meric protein that included GUS, and small gold particles (10 nm; smallarrowheads) indicate the localization of catalase. E, etioplast; G, glyox-ysome; M, mitochondrion. Bar = 1 nm.

1604 The Plant Cell

B

CP

D Urn

M

Figure 4. Localization of the Protein Encoded by CS-GUS in VariousMicrobodies.

Shown is immunoelectron microscopy of different tissues of transgenicArabidopsis plants carrying CS-GUS. The thin sections were stainedwith GUS-specific antibodies. Cp, chloroplast; E, etioplast; G, glyoxy-some; M, mitochondrion; R leaf peroxisome; Um, unspecializedmicrobody.(A) Glyoxysomes in the etiolated cotyledon of a seedling grown in dark-ness for 7 days.(B) Leaf peroxisomes in the green cotyledon of a seedling grown inthe light for 7 days.(C) Leaf peroxisomes in the leaf of a seedling grown in the light for17 days.(D) Unspecialized microbodies in the roots of a seedling grown in thelight for 17 days.Bar in (D) = 1 um for (A) to (D).

Localization of Chimeric Proteins with an AlteredN-Terminal Consensus Sequence

To investigate the function of the conserved sequences in theN-terminal region of gCS, we performed immunocytochemi-cal analysis with transgenic Arabidopsis plants expressingvarious chimeric proteins. Ultrathin sections were preparedfrom cotyledons of seedlings that had been grown in the lightfor 7 days and immunostained with antibodies raised againstGUS, and the sections were examined under an electron mi-croscope. The gold particles indicated the localization of thechimeric proteins that consisted of mutated N-terminal se-quences and GUS.

In the chimeric proteins encoded by R16G, L17G, and L24G,conserved arginine and leucine residues were replaced by gly-cine residues (Figure 1 A). The substitution of these conservedamino acids has been reported to increase the erroneous trans-location of a thiolase from Saccharomyces cerevisiae (Gloveret al., 1994), a thiolase from rat (Tsukamoto et al., 1994), anda malate dehydrogenase from watermelon (Gietl et al., 1994)to microbodies in mammalian cells or in yeast cells. Figures5D to 5F show the results of immunostaining of thin sectionsof Arabidopsis transformants that accumulated the mutatedchimeric proteins. Whereas the presence of the CS-GUS con-struct resulted in the labeling of GUS in the microbodies (Figure5B), the presence of the R16G, L17G, and L24G constructsresulted in no signals in any organelles, as was also observedin the case of the control section from a plant transformed withGUS (Figure 5A). We analyzed another transformant of R16Gand two others of L17G by using immunoelectron microscopy;two other transformants of L24G were analyzed by cell frac-tionation and immunoblotting. They did not show specificlocalization of the chimeric proteins to the microbodies (datanot shown), indicating that the erroneous localization of thechimeric proteins was not due to mutagenesis by transgeneinsertions into chromosomes. These results indicated that thechimeric proteins containing the mutation of Arg-16, Leu-17,and Leu-24 are not accumulated in microbodies of transgenicArabidopsis plants.

Localization of Chimeric Proteins with Alterations ofOther Amino Acids

In the chimeric protein encoded by E30G, Glu-30 was replacedby glycine. In the section from the corresponding Arabidopsisplant, the chimeric protein was found at the same location asthose in the CS-GUS—containing plant (Figures 5B and 5C),indicating that the chimeric protein encoded by E30G was trans-ported to microbodies.

The function of the second consensus sequence that is lo-cated at the cleavage site has not yet been defined. In thisconsensus sequence, a cysteine residue on the C-terminal sideof the presequence is conserved in all microbody proteins thatare synthesized as precursors (Kato et al., 1995). We inves-tigated the function of this cysteine residue, Cys-42 in thepresequence of pumpkin gCS, in the targeting of proteins tomicrobodies. Immunocytochemical analysis showed that thechimeric proteins encoded by C42G and C42F were localizedspecifically to microbodies in transgenic Arabidopsis plants(Figures 5G and 5H). In addition, the chimeric protein encodedby AC42, which was missing Cys-42, was also localized in themicrobodies (Figure 51). These results indicate that the chi-meric proteins encoded by C42G, C42F, and AC42 wereimported to microbodies. Also, the results suggest that N-ter-minal sequences containing the mutation of Cys-42 functionas targeting signals to microbodies in the same way as thenative sequence.


Processing of Chimeric Proteins That Have BeenImported into the Microbodies

The CS-GUS —containing plants expressed the 68- and 72-kD proteins that reacted immunologically with antibodies raisedagainst GUS (Figure 2). The 68-kD protein had the samemolecular mass as the product of GUS (Figure 2). Because

the native GUS protein was not imported into the microbod-ies, the 72-kD protein seemed to be processed by the removalof the N-terminal presequence in the microbodies and con-verted to the 68-kD mature protein. Miura et al. (1994)demonstrated that the precursor for 3-ketoacyl-CoA thiolasewas translocated and subsequently processed in rat liver perox-isomes in vitro. To confirm that the chimeric proteins were

Figure 5. Localization of the Chimeric Proteins in Transgenic Arabidopsis Seedlings.

harboring the chimeric proteins were grown for 7 days in the light. Thin sections of cotyledons were stained with GUS-specificchloroplast; M, mitochondrion; P, leaf peroxisome.

The seedlingsantibodies. Cp,(A) GUS.(B) CS-GUS.(C) E30G.(D) R16G.(E) L17G.(F) L24G.(G) C42G.(H) C42R(I) AC42.Bar in (I) = 1 urn for (A) to (I).

1606 The Plant Cell

processed in the microbodies, we performed subcellular frac-tionation with transgenic Arabidopsis plants by using sucrosedensity gradient centrifugation. The distribution of catalaseand GUS was determined by immunoblotting with specific anti-bodies. In GUS plants, the protein encoded by GUS was presentin the supernatant fractions, indicating that the 68-kD GUSprotein was not targeted to microbodies (Figure 6A). The chi-meric protein encoded by R16G was also detected in the topfractions of the density gradient but not in the microbodyfractions (Figure 6C). This result confirmed the immunocyto-chemical result for the plant carrying R16G (Figure 5D), namely,that the chimeric protein encoded by R76G did not accumu-late in microbodies. Similar fractionation experiments revealedthat localization of the chimeric proteins encoded by L17G andL24G was the same as that of the protein encoded by R16G(data not shown). Although the R16G, L17G, and L24G pro-teins contained a normal processing site in their N-terminalsequences, respectively, the detectable polypeptides in thegradients were only the 72-kD proteins. Because the chimericproteins encoded by R76G, L17G, and L24G were not targetedto microbodies and were not processed in microbodies, wecould not detect the 68-kD mature protein form.

In contrast with the plants carrying CS-GUS, both the 68-and 72-kD chimeric proteins were fractionated with catalase(Figure 6B), indicating that the immunoreactive polypeptideswere colocalized in the microbodies. This confirmed the re-sult of the immunoelectron microscopic analysis (Figure 5B).The distribution of the immunoreactive polypeptides mimickedthat of catalase, indicating that the immunoreactive poly-peptides in the supernatant had been released from themicrobodies during homogenization and subsequent centrifu-gation (Figure 6B). Furthermore, the ratio of the two proteinswas ~50% in the whole-cell extract and was the same as themicrobody fractions. These observations suggested that thechimeric protein encoded by CS-GUS that was synthesizedin the cytosol was immediately and exclusively transported intothe microbodies.

Densitometric analysis showed that the levels of the 68-kDproteins in the C42G and C42F constructs corresponded to~20 and 30% of the total immunoreactive proteins, respec-tively, whereas those in CS-GUS—containing seedlingscorresponded to ~50°/o (Figure 2). In addition, AC42, withoutCys-42, resulted in no accumulation of the 68-kD protein(Figure 2).

Cell fractionation assays showed that the distribution of thechimeric protein encoded by C42G coincided with that of cata-lase, but the extent of the accumulation of the 68-kD proteinin the microbody fractions was reduced (Figure 6D) as muchas the 68-kD protein in total homogenates of seedlings. Fur-thermore, in the microbody fractions of the C42F plants, theaccumulation of the 68-kD protein was also reduced (data notshown). These results indicated that the chimeric proteins en-coded by C42G and C42F were exclusively imported tomicrobodies post-translation ally, but the accumulation of the68-kD proteins, the mature form of the CS-GUS protein, was

reduced. In addition, the pattern of the distribution of the chi-meric proteins encoded by AC42 coincided with that of catalase(Figure 6E), indicating that the chimeric proteins were exclu-sively imported into the microbodies, as was the chimericprotein encoded by CS-GUS (Figure 6E). In microbody frac-tions of the AC42 plant, the 68-kD protein, the mature form,

GUS

GUS

GUS

1 3 5 7 9 11 13 15 17 19 21 23 25 27

1 3 5 7 9 11 13 15 17 19 21 23 25 27

GUS

Cat

GUS

Cat

5 7 11 13 15 17 19 21 23 25 27

F r a c t i o n n u m b e rFigure 6. Subcellular Fractionation of Transgenic Arabidopsis PlantsExpressing the Chimeric Proteins.Transgenic Arabidopsis plants harboring chimeric proteins were grownfor 4 to 6 weeks in the light. The whole plants without roots werehomogenized, and extracts were subjected to sucrose density gradientcentrifugation (30 to 60% [w/w]). The distribution of the chimeric pro-teins and catalase was determined by immunoblotting with specificantibodies raised against GUS and catalase (Cat).(A) GUS.(B) CS-GUS.(C) R16G.(D) C42G(E) AC42.


was not detected (Figure 6E). Thus, the chimeric protein encoded by AC42 with a molecular mass of 72 kD was imported into microbodies but did not seem to be processed there. These observations suggested that the substitution and elimination of Cys-42 in the N-terminal sequence of gCS caused the reduction and inhibition of precursor processing.

DlSCUSSlON

The N-Terminal Sequence of gCS Functions as a Targeting Signal

We established a stable system for analyzing the transport of a specific protein to microbodies, which are small and fragile organelles, by using transgenic plants. We constructed transgenic Arabidopsis plants expressing a GUS protein that included the N-terminal region of pumpkin gCS, and we determined the localization of the chimeric protein. The chimeric CS-GUS protein that included the N-terminal sequence of gCS was transported to glyoxysomes in etiolated cotyledons, to leaf peroxisomes, in green cotyledons and in mature leaves, and to unspecialized microbodies in roots (Figure 4). Therefore, the N-terminal presequence of gCS plays a role in targeting to plant microbodies. The PTS1 signal has been shown to function as a signal for targeting to glyoxysomes, leaf peroxisomes, and unspecialized microbodies in transgenic plants (Hayashi et al., 1996a). In this study, we showed that the PTS2 signal also functions as a signal for targeting to plant microbodies. These results suggest that all of the functionally different microbodies in higher plants are equipped with mechanisms for the import of both PTS1- and WS2-containing proteins.

Faber et al. (1994) reported that specific growth substrates induced the import pathway for PTSBcontaining proteins in the methylotrophic yeast Hansenula polymorpha. In other eu- karyotes, inducible protein transport pathways have not been reported. During greening of germinating cotyledons in oil seeds, the metabolic conditions in cells change dramatically, and the seedlings are transformed from heterotrophs to auto- trophs. We have shown that the levels of PTS2-containing proteins, such as citrate synthase and 3-ketoacyl-COA thiolase, decrease markedly at this transition stage in pumpkin cotyledons (Kato et al., 1995). It is possible that the pathway for transport of PTS2-containing proteins to microbodies might be repressed during greening of the cotyledons.

Roles of Conserved Amino Acids in the Targeting of Proteins to Microbodies

The targeting and the processing of the various chimeric proteins are summarized in Table l . Because thé chimeric proteins encoded by R16G, L17G, and L24G were not detected in the microbodies, the substitutions of Arg-16, Leu-17, and Leu-24

Table 1. Localization and Processing of the Chimeric Proteins

Efficiency of Strainl P roces i ng Protein Localization Processinga (O/O)~

CS-GUS R16G L I 7G L24G E30G C42G C42F AC42

Microbody Cytosol Cytosol Cytosol Microbody Microbody Microbody Microbody

+ 50 -

- + 40 + 20 + 30

a (+), 68-kD protein, the mature form, was detectable by immunoblotting; (-), not detectable.

68 kD/(68 kD + 72 kD) in Figure 2.

by glycine residues in the presequence of pumpkin gCS caused nonspecific localization of the chimeric proteins (Figures 5D to 5F and 6C). We concluded that the three amino acid residues Arg-16, Leu-17, and Leu-24, located in the first consensus sequence, RL-X5-HL, are essential for protein targeting to plant microbodies. It is difficult to detect the presence of a low concentration of proteins by immunogold labeling. However, the mutated proteins encoded by R16G, L17G, and L24G were not detected in microbody fractions by immunoblotting (Figure 6C and data not shown). Therefore, if the mutated proteins are imported into the microbodies, their con- tents in the microbodies are extremely low.

Where are the nontargeted chimeric proteins localized in transgenic Arabidopsis? In the R16G construct, the chimeric protein was not detected by immunoelectron microscopy in the cell (Figure 50). It was detected in the top fractions of density gradients and was not detectable in any other fractions of the gradients when immunoblotting was used (Figure 6C). This suggested that the chimeric protein encoded by R16G is not associated with any other organelles and remains in the cytosol. Although the products encoded by R16G, Ll7G, L24G, and GUS seemed to be localized in the cytosol, the concentration of the proteins in the cytosol was not sufficient to allow unequivocal detection by immunogold labeling. We could not provide direct evidence that the nontargeted chimeric proteins exist in the cytosol. Further analysis is necessary to detect the nontargeted chimeric proteins.

The consensus sequence is necessary for targeting of various PTSBcontaining proteins, such as mammalian and yeast thiolases to peroxisomes (Glover et al., 1994; Tsukamoto et al., 1994), yeast amine oxidase to peroxisomes (Faber et al., 1994), trypanosome aldolase to glycosomes (Blattner et al., 1995), and watermelon malate dehydrogenase to yeast peroxisomes (Gietl et al., 1994). Therefore, the consensus sequence seems generally to function in the targeting of proteins to microbodies in mammals, yeasts, trypanosomes, and plants.

1608 The Plant Cell

PTS2 appears to have been well conserved during evolution, as has F'TS1.

The accuracy and efficiency of protein transport to microbodies are influenced by the nature of the amino acids that replace the conserved amino acids (Glover et al., 1994; Tsukamoto et al., 1994). The replacement of the three amino acids in the gCS presequence by glycine residues caused no accumulation of the chimeric proteins in the microbodies. Gly- cine residues have no long side chains, and they allow greater flexibility in the conformation of polypeptides than do other amino acids. The substitutions of arginine and leucine residues by glycine residues might cause the loss of an electric charge and looseness of the rigid conformation of the presequence. The three original amino acids should endow the presequence with an electric charge and a precise conformation so that it can interact with the putative receptor on the microbody membrane.

The presequences of microbody proteins are rich in posi- tively charged amino acids. This feature is similar to those of mitochondrial enzymes, but a few negatively charged amino acids have been found in the presequences of the latter enzymes. Tsukamoto et al. (1994) demonstrated that substitution of the glutamic acid residue in the rat thiolase presequence, which is at a position homologous to that of Glu-30 in the gCS presequence, induced erroneous translocation of the chimeric proteins to mitochondria. They proposed that the negatively charged amino acid, which is not present in mitochondrial presequences, might be required to restrict the targeting activity of the presequence to peroxisomes and not to mitochondria. The substitution of Glu-30 by the noncharged amino acid glycine did not affect the targeting to microbodies in Arabidopsis (Figure 5C). We suggest that a negative charge at position 30 in the gCS presequence is not necessary in the recognition required for targeting to the microbodies.

Roles of Conserved Amino Acids in Processing of the Precursor

In the plant carrying CS-GUS, the molecular masses of the chimeric proteins were 68 and 72 kD. The 72-kD protein corresponds to the GUS-containing N-terminal region of gCS, and the 68-kD protein corresponds to the native GUS. The cell fractionation assay showed that the two GUS proteins were colocalized in the microbodies, indicating that the N-terminal presequence of CS-GUS was processed in the microbodies. If the processing of the chimeric protein occurred in another compartment, particularly in the cytosol, the chimeric proteins encoded by R76G, L17G, and L24G, which contained a normal processing site in the presequences, seemed to be processed. Nevertheless, we could not detect the 68-kD proteins, the mature form, in those constructs.

The processing of the chimeric protein encoded by CS-GUS was incomplete. In microbody fractions of CS-GUS construct, the 72-kD protein was detectable a ta leve1 of 50% of the total

(Figures 6B). It indicated that 50% of the chimeric protein accumulated as a precursor in the microbodies. Swinkels et al. (1991) failed to observe the processing of a chloramphenicol acetyltransferase fusion protein that included the presequence of rat thiolase in CHO cells. Tsukamoto et al. (1994) reported that an intact processing site, including the N-terminal side of the mature protein, is necessary for processing, and the efficiency of the processing depends on the length of the N-terminal region of the mature protein that is conjugated to the reporter protein. The N-terminal stretch of our GUS chimeric proteins included four of the N-terminal amino acids of the mature gCS. To increase the efficiency of the processing of the chimeric protein encoded by CS-GUS, it might be necessary to include a stretch of more N-terminal amino acids of the mature gCS.

The signals that determine the processing of precursor proteins are not known. A consensus sequence has been found around the cleavage sites of precursor proteins. In the consensus, a cysteine residue at the processing site is completely conserved. Yeast thiolase, which is not processed after translocation to the microbodies, does not have the second consensus or even the cysteine residue. The processing site of pumpkin gCS, determined by N-terminal sequencing of the mature protein, is on the C-terminal side of Val-43 (Kato et al., 1995), not on that of Cys-42. However, the processing sites of other precursor-type proteins from pumpkin, such as malate dehydrogenase (A. Kato, Y. Takeda-Yoshikama, and M. Nishimura, unpublished results) and thiolase (Kato et al., 1996), correspond to the cysteine residue. It is possible that the presequence of pumpkin gCS is cleaved on the C-terminal side of Cys-42 and that Val-43 might be removed after cleavage of the presequence. Here, we focused on the function of the cysteine residue in processing.

The chimeric proteins, in which Cys-42 was replaced by ei- ther a glycine or a phenylalanine residue, were imported into the microbodies (Figures 5G and 5H), but the extent of accumulation of the 68-kD proteins in the seedlings was reduced (Figure 2). There are two possible explanations for the reduced accumulations of the 68-kD proteins. One is that the import efficiencies of these proteins to the microbodies were reduced. The reduction in import efficiency resulted in the accumulation of nontargeted chimeric proteins; therefore, the relative content of the 68-kD protein declined. The second possibility is that the chimeric proteins were normally imported, but the processing reactions in the microbodies were inhibited. Be- cause microbodies are very fragile organelles, some of them were disrupted, and their matrix proteins were released into the supernatant during homogenization and subsequent centrifugation. In the CS-GUS plant, the distribution of the immunoreactive polypeptides mimicked that of catalase (Fig- ure 6B). In addition, the relative amounts of the 68- and 72-kD proteins were unchanged both in whole extracts (Figure 2) and in isolated microbodies (Figure 6B). These results support the latter possibility, namely, that the chimeric proteins encoded by CS-GUS, which have been synthesized in the cytosol, are immediately and exclusively transported into the microbodies.


The distribution of the chimeric proteins in C42G and C42F also coincided with that of catalase, indicating that the chimeric proteins encoded by C42G and C42f were also exclusively imported into the microbodies as the CS-GUS protein was. These observations suggested that replacement of Cys-42 did not affect the targeting to the microbodies but did affect the efficiency of processing of the chimeric proteins. It is possible that there are nontargeted chimeric proteins encoded by C42G, C426 and AC42 in other compartments. However, at least the ratio of the 68-kD protein in isolated microbodies in C42G- and C42F-containing plants was lower than that of CS-GUS-containing plants. This result strongly suggested that processing of the precursor in microbodies was reduced by the substitution of Cys-42. Furthermore, the mature form protein was not detected in isolated microbodies of plants carrying AC42. These results suggested that the chimeric proteins encoded by AC42 are transported into microbodies but that the processing is completely inhibited. The Cys-42 might play a role in recognition for the processing of the presequence.

The chimeric protein encoded by €30G, in which Glu-30 is replaced by a glycine residue, was less efficiently processed than was CS-GUS (Figure 2). The relative amount of the 68- kD protein in isolated microbodies was also reduced (data not shown). These results indicate that Glu-30, located upstream of the C-terminal consensus, participates in the recognition required for processing. A putative processing enzyme should recognize not only the cysteine residue but also the region around the cleavage site of the presequence. The secondary structure of the N-terminal region of the precursor might be important for recognition before processing.

METHODS

Mutagenesis and Construction of Plasmids

The plasmid containing part of a cDNA for the N-terminal portion of glyoxysome citrate synthase (gCS) from pumpkin (pGCS16A) in pBluescript SK- (Stratagene, La Jolla, CA) was described by Kato et al. (1995). The coding region of P-glucuronidase (GUS) originated from the plasmid pGUSN358-S (Clontech, Palo Alto, CA).

The DNA sequence corresponding to the 47 amino acids of the N-terminal region of gCS was amplified by polymerase chain reactions (PCR) with pGCS1GA as the template. The total volume of the reaction mixture for PCR was 30 pL, and it contained 100 ng of template DNA, 200 pM deoxynucleotide triphosphates, 1 FM oligonucleotide primers, supplied buffer (from Stratagene), and 0.75 units of Pfu DNA polymerase (Stratagene). Thirty cycles of reactions (94OC for 45 sec; 55OC for 30 sec; 74OC for 30 sec) were performed in a thermal cycler (Hybaid, Teddington, UK). We used the following primers: for CS-GUS, namely, the wild-type sequence, csl and cs2; for C42G, csl and cs3; for C42E csl and cs4; and for AC42, csl and cs5. To substitute amino acid residues, we used the double PCR method of Barik (1993).

The primers used for the first PCR are as follows: for R76G, csl and cs6; for L77G, cs1 and cs7; for L24G, csl and cs8; and for H O G , csl

and cs9. The primers used for the second PCR were the purified double- stranded products (used as sense primers) and cs2. Hindlll and Bglll restriction sites were ligated to the 5’ends of the amplified fragments. The resulting DNA fragments were digested with Hindlll and Ncol, the restriction site that was included in the sequence for gCS, and they were inserted between the Hindlll and Ncol sites of pGUSN358-S, whose Ncol site encompassed ATG and the second codon of the coding sequence for GUS.

The mutations in these plasmids were confirmed by direct sequence analysis (Sanger et al., 1977). The chimeric genes were prepared by digestion with Bglll and EcoRl and subsequently were cloned into the Ti binary vector pMAT037 (Matsuoka and Nakamura, 1991) that had been digested with BamHl and EcoRI. The Ti plasmids were transferred to Escherichia coli (strain DH5a) and purified on QlAprep spin columns (Qiagen, Hilden, Germany).

Sequences of the primers for PCR are as follows: csl (sense), 5’-ATCAAGCTTAGATCTATGCCCACCGACATGGAATT-3’; cs2 (antisense), 5’-CGCAACCATGGTCTGAGCTGA-3’; cs3 (antisense), 5’-CAACCATGGTCTGAGCTGACACGCCATGAGC-3’; cs4 (antisense), 5%AACCATGGTCTGAGCTGACACGAAATGAGC-3’; cs5 (antisense), 5-CAACCATGGTCTGAGCTGACACATGAGCCTCGA-3’; cs6 (antisense), 5’-ACGGCCAAGCCATGACGAGCA-3 cs7 (antisense),WACAACG- GCCCCGCGATGACGA-3‘; cs8 (antisense), 5‘-CAGCGCTCCCAT- GCGCTGC-3‘; and cs9 (antisense), 5-CACCGGCGGTCCCAAGGA- CGC-3’. The restriction sites are underlined.

Transformation of Plants

Ti plasmids were transferred to Agrobacterium tumefaciens (strain C58C1) by electroporation (Nagel et al., 1990). The constructs were introduced into Arabidopsis thaliana (ecotype C24) by the root transformation method (Valvekens et al., 1988). Primary transformants were designated To plants. TI seeds, harvested from To plants, were germinated on germination medium (Hayashi et al., 1996a) that contained 50 pglmL kanamycin (Meiji Seika Kaisha, Tokyo, Japan). A cotyledon was removed from a kanamycin-resistant plant that had been grown for 7 days in the light. The amount of the product of the chimeric protein in the cotyledon was analyzed by immunoblotting with a GUS-specific antibody (Molecular Probes, Eugene, OR). We selected severa1 transformants that accumulated large amounts of the product of the chimeric genes. TZ seeds were harvested from T, plants and grown on germination medium that contained 50 pglmL kanamycin. Kanamycin-resistant and homozygous TP plants were used for further analysis.

lmmunological Detection

SDS-PAGE was performed by the method of Laemmli (1970). Immu- noblot analysis was performed by the method of Towbin et al. (1979), with antibodies raised against GUS (Molecular Probes) and catalase (Yamaguchi and Nishimura, 1984). lmmunological reactions were detected by monitoring activities of horseradish peroxidase (Amersham Japan) with a chemiluminescence kit (ECL; Amersham).

lmmunoelectron Microscopy

Cotyledons, leaves, and roots were fixed, dehydrated, and embedded in LR white resin (London Resin Co., Basingstoke, UK), as described

1610 The Plant Cell

previously (Nishimura et al., 1993). Ultrathin sections were cut on a Reichert ultramicrotome (Leica, Nussloch, Germany) with a diamond knife and mounted on coated nickel grids. The protein A-gold labeling procedure was performed as described by Nishimura et al. (1993). Ultrathin sections were incubated overnight at room temperature with 1000- or 500-fold diluted antiserum raised against GUS (Molecular Probes) and then with a 50-fold diluted suspension of protein A-gold particles (15 nm; Amersham) at room temperature for 30 min. For double-staining, ultrathin sections were incubated overnight at room temperature with a 1000-fold diluted solution of antibodies raised against GUS and then with a 50-fold diluted suspension of protein A-gold particles (15 nm) at room temperature. Subsequently, the sections were incubated overnight at room temperature with a 2000-fold diluted antiserum raised against catalase and then with a50-fold diluted suspension of protein A-gold particles (10 nm; Amersham) at room temperature. The labeled sections were stained with 4% uranyl ace- tate and lead citrate and then examined with a transmission electron microscope (1200EX; JEOL, Tokyo, Japan).

Fractionation of Cells

T2 seeds were grown on germination medium at 22OC for 2 weeks under continuous illumination. The resulting seedlings were transferred to glass fiber blocks (Nitto Boseki, Chiba, Japan) and grown for 4 to 6 weeks under continuous illumination. One gram of green plants, ex- cluding roots, was chopped with a razor blade in a Petri dish with 2.5 mL of chopping buffer (150 mM Tricine-KOH, pH 7.5, 1 mM EDTA, 0.5 M sucrose) and filtrated with Miracloth (Calbiochem, La Jolla, CA). One milliliter of the homogenate was layered directly on top of a 16- mL linear sucrose density gradient (30 to 60% [w/w]) that contained 1 mM EDTA. Centrifugation was performed in an SW 28.1 rotor (Beck- man, Palo Alto, CA) at 21,000 rpm (60,OOOg) for 3 hr. Fractionation was then performed with a gradient fractionator (model 185; ISCO, Lin- coln, NE). The distribution of the chimeric protein and catalase in the various fractions was determined by immunoblotting.

Quantitation of Protein

T2 seedlings were homogenized with buffer (200 mM Tris-HCI, pH 7.5, 1 mM EDTA, 0.05% SDS, 1 mM phenylmethylsulfonyl fluoride) in microcentrifuge tubes, and supernatants were prepared by microcen- trifugation. Proteins in supernatants were quantitated by using a protein assay kit (Bio-Rad Japan, Tokyo), with BSA as the standard.

ACKNOWLEDGMENTS

We are grateful to Dr. Ken Matsuoka and Dr. Kenzo Nakamura (Nagoya University, Nagoya, Japan) for the Ti vector pMAT037. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (Nos. 04454017, 06248230, and 06261239) and the Nissan Science Foundation.

Received February 1, 1996; accepted July 22, -1996

REFERENCES

Barik, S. (1993). Site-directed mutagenesis by double polymerase chain reaction: Megaprimer method. In Methods in Molecular Biology, Vol. 15, B.A. White, ed (Clifton, NJ: Humana Press Inc.), pp. 277-286.

Beeching, J.R., and Northcote, D.H. (1987). Nucleic acid (cDNA) and amino acid sequences of isocitrate lyase from castor bean. Plant MOI. Biol. 8, 471-475.

Blattner, J., Dorsam, H., and Clayton, C.E. (1995). Function of N-terminal import signals in trypanosome microbodies. FEBS Lett.

Comai, L., Baden, C.S., and Harada, J.J. (1989a). Deduced sequence of a malate synthase polypeptide encoded by a subclass of the gene family. J. Biol. Chem. 264, 2778-2782.

Comai, L., Dietrich, R.A., Maslyar, D.J., Baden, C.S., and Harada, J.J. (1989b). Coordinate expression of transcriptionally regulated isocitrate lyase and malate synthase genes in Brassica napus. Plant Cell 1, 293-300.

De Bellis, L., and Nishimura, M. (1991). Development of enzymes of the glyoxylate cycle during senescence of pumpkin cotyledons. Plant Cell Physiol. 32, 555-561.

de Hoop, M.J., and Ab, G. (1992). lmport of proteins into peroxisomes and other microbodies. Biochem. J. 286, 657-669.

Faber, K.N., Haima, P., Gietl, C., Harder, W., Ab, G., and Veenhuis, M. (1994). The methylotrophic yeast Hansenula polymorpha con- tains an inducible import pathway for peroxisomal matrix proteins with an N-terminal targeting signal (PTS2 proteins). Proc. Natl. Acad. Sci. USA 91, 12985-12989.

Gietl, C. (1990). Glyoxysomal malate dehydrogenase from watermelon is synthesized with an amino-terminal transit peptide. Proc. Natl. Acad. Sci. USA 87, 5773-5777.

Gietl, C., Faber, K.N., van der Klei, I.J., and Veenhuis, M. (1994). Mutational analysis of the N-terminal topogenic signal of watermelon glyoxysomal malate dehydrogenase using the heterologous host Han- senula polymorpha. Proc. Natl. Acad. Sci. USA 91, 3151-3155.

Glover, J.R., Andrews, D.W., Subramani, S., and Rachubinski, R.A. (1994). Mutagenesis of the amino targeting signal of Sacchammyces cerevisiae 3-ketoacyl-COA thiolase reveals conserved amino acids required for import into peroxisomes in vivo. J. Biol. Chem. 269,

Gould, S.J., Keller, G.A., Schneider, M., Howell, S.H., Garrard, L.J., Goodman, J.M., Distel, B., Tabak, H., and Subramani, S. (1990). Peroxisomal protein import is conserved between yeast, plants, insects and mammals. EM60 J. 9, 85-90.

Graham, I.A., Smith, L.M., Brown, J.W.S., Leaver, C.J., and Smith, S.M. (1989). The malate synthase gene of cucumber. Plant MOI. Biol.

Hayashi, M., Aoki, M., Kato, A., Kondo, M., and Nishimura, M. (1996a). Transport of chimeric proteins that contain acarboxy-terminal targeting signal into plant microbodies. Plant J. 10, 225-234.

Hayashi, M., Tsugeki, R., Kondo, M., Mori, H., and Nishimura, M. (1996b). Pumpkin hydroxypyruvate reductases with and without a putative C-terminal signal for targeting to microbodies may be producing by alternative splicing. Plant MOI. Biol. 30, 183-189.

Hijikata, M., Ishii, N., Kagamiyama, H., Osumi, T., and Hashimoto, T. (1987). Structural analysis of cDNA for rat peroxisomal 3-ketoacyl- COA thiolase. J. Biol. Chem. 262, 8151-8158.

360, 310-314.

7558-7563.

13, 673-684.

Transpor? of Glyoxysomal Citrate Synthase 161 1

Huang, A.H.C., Trelease, R.N., and Moore, T.S. (1983). Plant Peroxi- somes. (New York: Academic Press).

Kato, A., Hayashi, M., Mori, H., and Nishimura, M. (1995). Molecu- lar characterization of a glyoxysomal citrate synthase that is synthesized as a precursor of higher molecular mass in pumpkin. Plant MOI. Biol. 27, 377-390.

Kato, A., Hayashi, M., Takeuchi, Y., and Nishimura, M. (1996). cDNA cloning and expression of a Qene for 3-ketoacYl-CoA thiolase in pumpkin cotyledons. Plant MOI. Biol., in press.

Laemmli, U.K. (1970). Cleavage of structural proteins during the as-

Lazarow, P.B., and Fujiki, Y. (1985). Biogenesis of peroxisomes. Annu. Rev. Cell Biol. 1, 489-530.

Matsuoka, K., and Nakamura, K. (1991). Propeptide of a precursor to a plant vacuolar protein required for vacuolar targeting. Proc. Natl. Acad. Sci. USA 88, 834-838.

Miura, S., Miyazawa, S., Osumi, T., Hashimoto, T., and Fujiki, Y.

Sautter, C. (1986). Microbody transition in greening watermelon cotyledons. Double immunocytochemical labeling of isocitrate lyase and hydroxypyruvate reductase. Planta 167, 491-503.

Subramani, S. (1992). Targeting of proteins into the peroxisomal matrix. J. Membr. Biol. 125, 99-106.

Subramani, S. (1993). Protein import into peroxisomes and biogenesis of the organelle. Annu. Rev. Cell Biol. 9, 445-478.

Swinkels, B.W., Gould, S.J., Bodnar, A.G., Rachubinski, R.A., and Subramani, S. (1991). A novel, cleavable peroxisomal targeting signal at the amino-terminal of the rat 3-ketoacyl-COA thiolase. EM60 J.

Titus, D.E., and Becker, W.E. (1985). lnvestigation of the glyoxysome- peroxisome transition in germinating cucumber cotyledons using double-label immunoelectron microscopy. J. Cell Biol. 101,1288-1299.

Towbin, H., Staehelin, T., and Gordon, J. (1979). Electrophoretic trans- fer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76,

Tsugeki, R., Hara-Nishimura, I., Mori, H., and Nishimura, M. (1993). Cloning and sequencing of cDNAfor glycolate oxidase from pumpkin cotyledons and Northern blot analysis. Plant Cell Physiol. 34, 51-57.

Tsukamoto, T., Hata, S., Yokota, S., Miura, S., Fujiki, Y., Hijikata, M., Miyazawa, S., Hashimoto, T., and Osumi, T. (1994). Charac-

peroxisomal 3-ketoacyl-COA thiolase precursor. J. Biol. Chem. 269,

Turley, R.B., Choe, S.M., and Trelease, R.N. (1990). Characteriza- tion of a cDNA clone encoding the complete amino acid sequence of cotton isocitrate lyase. Biochim. Biophys. Acta 1049, 223-226.

Valvekens, D., Van Montagu, M., and Van Lijsebettens, M. (1988). Agrobacterium tumefaciens-mediated transformation of Arabidop- sis thaliana root explants by using kanamycin selection. Proc. Natl. Acad. Sci. USA 85, 5536-5540.

sembly of the head of bacteriopharge T4. Nature 227, 680-685. 10, 3255-3262.

(1994). Post-translational import of 3-ketoacyl-COA thiolase into rat 4350-4354. liver peroxisomes in vitro. J. Biochem. 115, 1064-1068.

Mori, H., and Nishimura, M. (1989). Glyoxysomal malate synthetase is specifically degraded in microbodies during greening of pumpkin cotyledons. FEBS Lett. 244, 163-166.

Mori, H., Takeda-Yoshikawa, Y., Hara-Nishimura, I., and Nishimura, M. (1991). Pumpkin malate synthase: Cloning and sequencing of the cDNA and Northern blotanalysis, Eur, J, Biochem, 197, 331-336, terization Of the signal peptide at lhe amino terminLls Of the rat

Nagel, R., Elliott, A., Masel, A., Birch, R.G., and Manners, J.M. 6001-6010. (1990). Electroporation of binary Ti Plasmidvectorinto Agrobacterium tumefaciens and Agrobacterium rhizogenes. FEMS Microbiol. Lett. 67, 325-328.

Nishimura, M., Yamaguchi, J., Mori, H., Akazawa, T., and Yokota, S. (1986). lmmunocytochemical analysis shows that glyoxysomes are directly transformed to leaf peroxisomes during greening of pumpkin cotyledons. Plant Physiol. 81, 313-316.

Nishimura, M., Takeuchi, Y., De Bellis, L., and Hara-Nishimura, 1. (1993). Leaf peroxisomes are directly transformed to glyoxysomes during senescence of pumpkin cotyledons. Protoplasma 175, 131-1 37.

Preisig-Müller, R., and Kindl, H. (1993). Thiolase mRNA translated in vitro yields a peptide with a putative N-terminal presequence. Plant MOI. Biol. 22, 59-66.

Sanger, F., Nicklen, S., and Coulson, AR. (1977). DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467.

Volokita, M., and Somerville, C.R. (1987). The primary structure of spinach glycolate oxidase deduced from lhe DNA sequence of a cDNA clone. J. Biol. Chem. 262, 15825-15828.

Yamaguchi, J., and Nishimura, M. (1984). Purification of glyoxysomal catalase and immunochemical comparison of glyoxysomal and leaf peroxisomal catalase in germinating pumpkin cotyledons. Plant Phys- iol. 74, 261-267.

Yamaguchi, K., Takeuchi, Y., Mari, H., and Nishimura, M. (1995). Development of microbody membrane proteins during the transformation of glyoxysomes to leaf peroxisomes in pumpkin cotyledons. Plant Cell Physiol. 36, 455-464.

DOI 10.1105/tpc.8.9.1601 1996;8;1601-1611Plant Cell

A Kato, M Hayashi, M Kondo and M Nishimurato glyoxysomal citrate synthase.

Targeting and processing of a chimeric protein with the N-terminal presequence of the precursor

This information is current as of August 18, 2019

Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

eTOCs http://www.plantcell.org/cgi/alerts/ctmain

Sign up for eTOCs at:

CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain

Sign up for CiteTrack Alerts at:

Subscription Information http://www.aspb.org/publications/subscriptions.cfm

is available at:Plant Physiology and The Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists

https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

http://www.plantcell.org/cgi/alerts/ctmain

http://www.plantcell.org/cgi/alerts/ctmain

http://www.aspb.org/publications/subscriptions.cfm

targeting and processing of a chimeric protein with the n ... · in the chimeric proteins, the...

Documents