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1.
The targeting of castor bean isocitrate lyase to peroxisomes was studied by expression in the heterologous host Saccharomyces cerevisae from which the endogenous ICL1 gene had been removed by gene disruption. Peroxisomal import of ICL was dependent upon the PTS1 receptor Pex5p and was lost by deletion of the last three amino acids, Ala-Arg-Met. However, removal of an additional 16 amino acids restored the ability of this truncated ICL to be targeted to peroxisomes and this import activity, like that of the full-length protein, was dependent upon Pex5p. The ability of peptides corresponding to the carboxyl terminal ends of wild-type and Delta 3 and Delta 19 mutants of ICL to interact with the PTS1-binding portion of Pex5p from humans, plants and yeast was determined using the yeast two-hybrid system. The peptide corresponding to wild-type ICL interacted with all three Pex5p proteins to differing extents, but neither mutant could interact with Pex5p from any species. Thus, ICL can be targeted to peroxisomes in a Pex5p-dependent but PTS1-independent fashion. These results help to clarify the contradictory published data about the requirement of the PTS1 signal for ICL targeting. 相似文献
2.
John A. Parkes Sigrid Langer Andreas Hartig Alison Baker 《Molecular membrane biology》2013,30(1):61-69
The targeting of castor bean isocitrate lyase to peroxisomes was studied by expression in the heterologous host Saccharomyces cerevisae from which the endogenous ICL1 gene had been removed by gene disruption. Peroxisomal import of ICL was dependent upon the PTS1 receptor Pex5p and was lost by deletion of the last three amino acids, Ala-Arg-Met. However, removal of an additional 16 amino acids restored the ability of this truncated ICL to be targeted to peroxisomes and this import activity, like that of the full-length protein, was dependent upon Pex5p. The ability of peptides corresponding to the carboxyl terminal ends of wild-type and Δ3 and Δ19 mutants of ICL to interact with the PTS1-binding portion of Pex5p from humans, plants and yeast was determined using the yeast two-hybrid system. The peptide corresponding to wild-type ICL interacted with all three Pex5p proteins to differing extents, but neither mutant could interact with Pex5p from any species. Thus, ICL can be targeted to peroxisomes in a Pex5p-dependent but PTS1-independent fashion. These results help to clarify the contradictory published data about the requirement of the PTS1 signal for ICL targeting. 相似文献
3.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2019,1866(2):199-213
Peroxisomal biogenesis depends on the correct import of matrix proteins into the lumen of the organelle. Most peroxisomal matrix proteins harbor the peroxisomal targeting-type 1 (PTS1), which is recognized by the soluble PTS1-receptor Pex5p in the cytosol. Pex5p ferries the PTS1-proteins to the peroxisomal membrane and releases them into the lumen. Finally, the PTS1-receptor is monoubiquitinated on the conserved cysteine 6 in Saccharomyces cerevisiae. The monoubiquitinated Pex5p is recognized by the peroxisomal export machinery and is retrotranslocated into the cytosol for further rounds of protein import. However, the functional relevance of deubiquitination has not yet been addressed.In this study, we have analyzed a Pex5p-truncation lacking Cys6 [(Δ6)Pex5p], a construct with a ubiquitin-moiety genetically fused to the truncation [Ub-(Δ6)Pex5p], as well as a construct with a reduced susceptibility to deubiquitination [Ub(G75/76A)-(Δ6)Pex5p]. While the (Δ6)Pex5p-truncation is not functional, the Ub-(Δ6)Pex5p chimeric protein can facilitate matrix protein import. In contrast, the Ub(G75/76A)-(Δ6)Pex5p chimera exhibits a complete PTS1-import defect. The data show for the first time that not only ubiquitination but also deubiquitination rates are tightly regulated and that efficient deubiquitination of Pex5p is essential for peroxisomal biogenesis. 相似文献
4.
Okumoto K Misono S Miyata N Matsumoto Y Mukai S Fujiki Y 《Traffic (Copenhagen, Denmark)》2011,12(8):1067-1083
Pex5p is the cytosolic receptor for peroxisome matrix proteins with peroxisome-targeting signal (PTS) type 1 and shuttles between the cytosol and peroxisomes. Here, we show that Pex5p is ubiquitinated at the conserved cysteine(11) in a manner sensitive to dithiothreitol, in a form associated with peroxisomes. Pex5p with a mutation of the cysteine(11) to alanine, termed Pex5p-C11A, abrogates peroxisomal import of PTS1 and PTS2 proteins in wild-type cells. Pex5p-C11A is imported into peroxisomes but not exported, resulting in its accumulation in peroxisomes. These results suggest an essential role of the cysteine residue in the export of Pex5p. Furthermore, domain mapping indicates that N-terminal 158-amino-acid region of Pex5p-C11A, termed 158-CA, is sufficient for such dominant-negative activity by binding to membrane peroxin Pex14p via its two pentapeptide WXXXF/Y motifs. Stable expression of either Pex5p-C11A or 158-CA likewise inhibits the wild-type Pex5p import into peroxisomes, strongly suggesting that Pex5p-C11A exerts the dominant-negative effect at the translocation step via Pex14p. Taken together, these findings show that the cysteine(11) of Pex5p is indispensable for two distinct steps, its import and export. The Pex5p-C11A would be a useful tool for gaining a mechanistic insight into the matrix protein import into peroxisomes. 相似文献
5.
Functional similarity between the peroxisomal PTS2 receptor binding protein Pex18p and the N-terminal half of the PTS1 receptor Pex5p 总被引:1,自引:0,他引:1 下载免费PDF全文
Schäfer A Kerssen D Veenhuis M Kunau WH Schliebs W 《Molecular and cellular biology》2004,24(20):8895-8906
Within the extended receptor cycle of peroxisomal matrix import, the function of the import receptor Pex5p comprises cargo recognition and transport. While the C-terminal half (Pex5p-C) is responsible for PTS1 binding, the contribution of the N-terminal half of Pex5p (Pex5p-N) to the receptor cycle has been less clear. Here we demonstrate, using different techniques, that in Saccharomyces cerevisiae Pex5p-N alone facilitates the import of the major matrix protein Fox1p. This finding suggests that Pex5p-N is sufficient for receptor docking and cargo transport into peroxisomes. Moreover, we found that Pex5p-N can be functionally replaced by Pex18p, one of two auxiliary proteins of the PTS2 import pathway. A chimeric protein consisting of Pex18p (without its Pex7p binding site) fused to Pex5p-C is able to partially restore PTS1 protein import in a PEX5 deletion strain. On the basis of these results, we propose that the auxiliary proteins of the PTS2 import pathway fulfill roles similar to those of the N-terminal half of Pex5p in the PTS1 import pathway. 相似文献
6.
Lee JR Park SC Kim MH Jung JH Shin MR Lee DH Cheon MG Park Y Hahm KS Lee SY 《Biochemical and biophysical research communications》2007,359(4):941-946
We have purified a novel antifungal protein from blast fungus (Magnaporthe grisea)-treated rice leaves using consecutive chromatographies on CM-Sepharose ion-change, Affi-gel blue, and HPLC gel filtration columns. We determined the N-terminal peptide sequence of the purified protein and subjected it to the NCBI/BLAST database and found the protein to be a partial fragment of the peroxisomal receptor protein in rice (OsPex5p). After cloning two cDNAs encoding OsPEX5L and OsPEX5S genes that are splice variants of OsPEX5 from a rice leaf cDNA library, we investigated their antifungal properties. The recombinant proteins were expressed in Escherichia coli and found to significantly inhibit cell growth of various pathogenic fungal strains. mRNA expression of the OsPEX5L gene was induced by diverse external stresses such as rice blast fungus, fungal elicitor, and other signaling molecules including H(2)O(2), abscisic acid, jasmonic acid, and salicylic acid. These results suggest that the peroxisomal receptor protein, OsPex5p, plays a critical role in the rice defense system against diverse external stresses including fungal pathogenic attack. 相似文献
7.
Kiel JA van den Berg M Bovenberg RA van der Klei IJ Veenhuis M 《Fungal genetics and biology : FG & B》2004,41(7):708-720
We have isolated the Penicillium chrysogenum pex5 gene encoding the receptor for microbody matrix proteins containing a type 1 peroxisomal targeting signal (PTS1). Pc-pex5 contains 2 introns and encodes a protein of approximately 75 kDa. P. chrysogenum pex5 disruptants appear to be highly unstable, show poor growth, and are unable to sporulate asexually. Furthermore, pex5 cells mislocalize a fluorescent PTS1 reporter protein to the cytosol. Pc-pex5 was expressed in a PEX5 null mutant of the yeast Hansenula polymorpha. Detailed analysis demonstrated that the PTS1 proteins dihydroxyacetone synthase and catalase were almost fully imported into microbodies. Surprisingly, alcohol oxidase, which also depends on Pex5p for import into microbodies, remained mainly in the cytosol. Thus, P. chrysogenum Pex5p has a different specificity of cargo recognition than its H. polymorpha counterpart. This was also suggested by the observation that Pc-Pex5p sorted a reporter protein fused to various functional PTS1 signals with different efficiencies. 相似文献
8.
In the present study, we investigated molecular mechanisms underlying the import of peroxisome-targeting signal type 2 (PTS2) proteins into peroxisomes. Purified Chinese hamster Pex7p that had been expressed in an Sf9/baculovirus system was biologically active in several assays such as those for PTS2 binding and assessing the restoration of the impaired PTS2 protein import in Chinese hamster ovary (CHO) pex7 mutant ZPG207. Pex7p was eluted as a monomer in gel filtration chromatography. Moreover, the mutation of the highly conserved cysteine residue suggested to be involved in the dimer formation did not affect the complementing activity in ZPG207 cells. Together, Pex7p more likely functions as a monomer. Together with PTS1 protein, the Pex7p-PTS2 protein complex was bound to Pex5pL, the longer form of Pex5p, which was prerequisite for the translocation of Pex7p-PTS2 protein complexes. Pex5pL-(Pex7p-PTS2 protein) complexes were detectable in wild-type CHO-K1 cells and were apparently more stable in pex14 CHO cells deficient in the entry site of the matrix proteins, whereas only the Pex7p-PTS2 protein complex was discernible in a Pex5pL-defective pex5 CHO mutant. Pex7p-PTS2 proteins bound to Pex14p via Pex5pL. In contrast, PTS2 protein-bound Pex7p as well as Pex7p directly and equally interacted with Pex13p, implying that the PTS2 cargo may be released at Pex13p. Furthermore, we detected the Pex13p complexes likewise formed with Pex5pL-bound Pex7p-PTS2 proteins. Thus, the Pex7p-mediated PTS2 protein import shares most of the steps with the Pex5p-dependent PTS1 import machinery but is likely distinct at the cargo-releasing stage. 相似文献
9.
Pex5p, a receptor for peroxisomal matrix proteins with a type 1 peroxisome targeting signal (PTS1), has been proposed to cycle from the cytoplasm to the peroxisomal membrane where it docks with Pex14p and Pex13p, the latter an SH3 domain-containing protein. Using in vitro binding assays we have demonstrated that binding of Pex5p to Pex14p is enhanced when Pex5p is loaded with a PTS1-containing peptide. In contrast, Pex5p binding to Pex13p, which involves only the SH3 domain, occurs at 20-40-fold lower levels and is reduced when Pex5p is preloaded with a PTS1 peptide. Pex14p was also shown to bind weakly to the Pex13p SH3 domain. Site-directed mutagenesis of the Pex13p SH3 domain attenuated binding to Pex5p and Pex14p, consistent with both of these proteins being binding partners for this domain. The SH3 binding site in Pex5p was determined to lie within a 114-residue peptide (Trp(100)-Glu(213)) in the amino-terminal region of the protein. The interaction between this peptide and the SH3 domain was competitively inhibited by Pex14p. We interpret these data as suggesting that docking of the Pex5p-PTS1 protein complex at the peroxisome membrane occurs at Pex14p and that the Pex13p SH3 domain functions as an associated component possibly involved in sequestering Pex5p after relinquishment of the PTS1 protein cargo to components of the translocation machinery. 相似文献
10.
Identification of Pex13p a peroxisomal membrane receptor for the PTS1 recognition factor 总被引:10,自引:3,他引:10 下载免费PDF全文
《The Journal of cell biology》1996,135(1):111-121
We have identified an S. cerevisiae integral peroxisomal membrane protein of M of 42,705 (Pex13p) that is a component of the peroxisomal protein import apparatus. Pex13p's most striking feature is an src homology 3 (SH3) domain that interacts directly with yeast Pex5p (former Pas10p), the recognition factor for the COOH-terminal tripeptide signal sequence (PTS1), but not with Pex7p (former Pas7p), the recognition factor for the NH2-terminal nonapeptide signal (PTS2) of peroxisomal matrix proteins. Hence, Pex13p serves as peroxisomal membrane receptor for at least one of the two peroxisomal signal recognition factors. Cells deficient in Pex13p are unable to import peroxisomal matrix proteins containing PTS1 and, surprisingly, also those containing PTS2. Pex13p deficient cells retain membranes containing the peroxisomal membrane protein Pex11p (former Pmp27p), consistent with the existence of independent pathways for the integration of peroxisomal membrane proteins and for the translocation of peroxisomal matrix proteins. 相似文献
11.
Klein AT Barnett P Bottger G Konings D Tabak HF Distel B 《The Journal of biological chemistry》2001,276(18):15034-15041
We have studied how Pex5p recognizes peroxisomal targeting signal type 1 (PTS1)-containing proteins. A randomly mutagenized pex5 library was screened in a two-hybrid setup for mutations that disrupted the interaction with the PTS1 protein Mdh3p or for suppressor mutations that could restore the interaction with Mdh3p containing a mutation in its PTS1. All mutations localized in the tetratricopeptide repeat (TPR) domain of Pex5p. The Pex5p TPR domain was modeled based on the crystal structure of a related TPR protein. Mapping of the mutations on this structural model revealed that some of the loss-of-interaction mutations consisted of substitutions in alpha-helices of TPRs with bulky amino acids, probably resulting in local misfolding and thereby indirectly preventing binding of PTS1 proteins. The other loss-of-interaction mutations and most suppressor mutations localized in short, exposed, intra-repeat loops of TPR2, TPR3, and TPR6, which are predicted to mediate direct interaction with PTS1 amino acids. Additional site-directed mutants at conserved positions in intra-repeat loops underscored the importance of the loops of TPR2 and TPR3 for PTS1 interaction. Based on the mutational analysis and the structural model, we put forward a model as to how PTS1 proteins are selected by Pex5p. 相似文献
12.
Saccharomyces cerevisiae PTS1 receptor Pex5p interacts with the SH3 domain of the peroxisomal membrane protein Pex13p in an unconventional, non-PXXP-related manner 下载免费PDF全文
Bottger G Barnett P Klein AT Kragt A Tabak HF Distel B 《Molecular biology of the cell》2000,11(11):3963-3976
A number of peroxisome-associated proteins have been described that are involved in the import of proteins into peroxisomes, among which is the receptor for peroxisomal targeting signal 1 (PTS1) proteins Pex5p, the integral membrane protein Pex13p, which contains an Src homology 3 (SH3) domain, and the peripheral membrane protein Pex14p. In the yeast Saccharomyces cerevisiae, both Pex5p and Pex14p are able to bind Pex13p via its SH3 domain. Pex14p contains the classical SH3 binding motif PXXP, whereas this sequence is absent in Pex5p. Mutation of the conserved tryptophan in the PXXP binding pocket of Pex13-SH3 abolished interaction with Pex14p, but did not affect interaction with Pex5p, suggesting that Pex14p is the classical SH3 domain ligand and that Pex5p binds the SH3 domain in an alternative way. To identify the SH3 binding site in Pex5p, we screened a randomly mutagenized PEX5 library for loss of interaction with Pex13-SH3. Such mutations were all located in a small region in the N-terminal half of Pex5p. One of the altered residues (F208) was part of the sequence W(204)XXQF(208), that is conserved between Pex5 proteins of different species. Site-directed mutagenesis of Trp204 confirmed the essential role of this motif in recognition of the SH3 domain. The Pex5p mutants could only partially restore PTS1-protein import in pex5Delta cells in vivo. In vitro binding studies showed that these Pex5p mutants failed to interact with Pex13-SH3 in the absence of Pex14p, but regained their ability to bind in the presence of Pex14p, suggesting the formation of a heterotrimeric complex consisting of Pex5p, Pex14p, and Pex13-SH3. In vivo, these Pex5p mutants, like wild-type Pex5p, were still found to be associated with peroxisomes. Taken together, this indicates that in the absence of Pex13-SH3 interaction, other protein(s) is able to bind Pex5p at the peroxisome; Pex14p is a likely candidate for this function. 相似文献
13.
The majority of proteins targeted to the peroxisomal lumen contain a C-terminal peroxisomal targeting signal-1 (PTS1) that is bound by the peroxin Pex5p. The PTS1 is generally regarded as a C-terminal tripeptide that adheres to the consensus (S/A/C)(K/R/H)(L/M). Previously, we studied the binding affinity of peptides of the form YQX(-3)X(-2)X(-1) to the peptide-binding domain of human Pex5p (referred to as Pex5p-C). Optimal affinity was found for YQSKL, which bound with an affinity of 200 +/- 40 nM. To extend this work, we investigated the properties of a peptide containing the last 9 residues of acyl-CoA oxidase (RHYLKPLQSKL) and discovered that it binds to Pex5p-C with a dissociation constant of 1.4 +/- 0.4 nM, 180 times tighter than YQSKL. Further analysis revealed that the enhanced affinity is primarily due to the presence of leucine in the (-5) position. In addition, a peptide corresponding to the luciferase C-terminus (YKGGKSKL) was found to bind Pex5p-C about 20 times tighter than YQSKL. The majority of this effect results from having lysine in position (-4). Catalase contains a noncanonical PTS1 (-AREKANL). The affinity of YQANL was found to be 3600 +/- 400 nM. This relatively weak binding is consistent with previous unsuccessful attempts to direct chloramphenicol acetyltransferase to the peroxisome by fusing -ANL to its C-terminus (-GGA-ANL). The peptides YKANL, YEKANL, YREKANL, and YAREKANL all bound Pex5p-C with higher affinities than did YQANL, but the affinities are still lower than peptides that correspond to functional targeting signals in other contexts. Because both catalase and Pex5p are tetramers (as opposed to the monomeric Pex5p-C and the peptides used in our studies), multidentate effects on binding affinity between Pex5p and other oligomeric proteins should be considered. Our study provides direct thermodynamic data revealing that peptide binding to Pex5p-C binding is favored by lysine in the (-4) position and leucine in the (-5) position. Our results suggest that peptides or proteins with optimized residues in the (-4) and/or (-5) positions can bind to Pex5p with affinities that are at least two orders of magnitude greater than that of YQSKL, and that this stabilization can compensates for otherwise weakly binding PTS1s. 相似文献
14.
The SH3 domain of the Saccharomyces cerevisiae peroxisomal membrane protein Pex13p functions as a docking site for Pex5p, a mobile receptor for the import PTS1-containing proteins 总被引:2,自引:3,他引:2 下载免费PDF全文
《The Journal of cell biology》1996,135(1):97-109
We identified a Saccharomyces cerevisiae peroxisomal membrane protein, Pex13p, that is essential for protein import. A point mutation in the COOH-terminal Src homology 3 (SH3) domain of Pex13p inactivated the protein but did not affect its membrane targeting. A two-hybrid screen with the SH3 domain of Pex13p identified Pex5p, a receptor for proteins with a type I peroxisomal targeting signal (PTS1), as its ligand. Pex13p SH3 interacted specifically with Pex5p in vitro. We determined, furthermore, that Pex5p was mainly present in the cytosol and only a small fraction was associated with peroxisomes. We therefore propose that Pex13p is a component of the peroxisomal protein import machinery onto which the mobile Pex5p receptor docks for the delivery of the selected PTS1 protein. 相似文献
15.
Yarrowia lipolytica Pex20p, Saccharomyces cerevisiae Pex18p/Pex21p and mammalian Pex5pL fulfil a common function in the early steps of the peroxisomal PTS2 import pathway 下载免费PDF全文
Import of peroxisomal matrix proteins is essential for peroxisome biogenesis. Genetic and biochemical studies using a variety of different model systems have led to the discovery of 23 PEX genes required for this process. Although it is generally believed that, in contrast to mitochondria and chloroplasts, translocation of proteins into peroxisomes involves a receptor cycle, there are reported differences of an evolutionary conservation of this cycle either with respect to the components or the steps involved in different organisms. We show here that the early steps of protein import into peroxisomes exhibit a greater similarity than was thought previously to be the case. Pex20p of Yarrowia lipolytica, Pex18p and Pex21p of Saccharomyces cerevisiae and mammalian Pex5pL fulfil a common function in the PTS2 pathway of their respective organisms. These non-orthologous proteins possess a conserved sequence region that most likely represents a common PTS2-receptor binding site and di-aromatic pentapeptide motifs that could be involved in binding of the putative docking proteins. We propose that not necessarily the same proteins but functional modules of them are conserved in the early steps of peroxisomal protein import. 相似文献
16.
We earlier isolated peroxisome biogenesis-defective Chinese hamster ovary (CHO) cell mutants, ZPEG241, by the 9-(1'-pyrene)nonanol/ultraviolet selection method, from TKaEG2, the wild-type CHO-K1 cells transformed with two cDNAs encoding rat Pex2p and peroxisome targeting signal type 2 (PTS2)-tagged enhanced green fluorescent protein (EGFP). Peroxisomal localization of PTS2-EGFP was specifically impaired in ZPEG241 due to the failure of Pex5pL expression. Analysis of partial genomic sequence of PEX5 revealed one-point nucleotide-mutation from G to A in the 3'-acceptor splice site located at 1 nt upstream of exon 7 encoding Pex5pL specific 37-amino acid insertion, thereby generating 21-nt deleted mRNA of PEX5L in ZPEG241. When ZPEG241-derived Pex5pL was ectopically expressed in ZPEG241, PTS2 import was not restored because of no interaction with Pex7p. Together, we confirm the pivotal role of Pex5pL in PTS2 import, showing that the N-terminal 7-amino acid residues in the 37-amino acid insertion of Pex5pL are essential for the binding to Pex7p. 相似文献
17.
The majority of peroxisomal matrix proteins are recognized by the import receptor Pex5p. The receptor is dynamic in terms of its overall architecture and association with the peroxisomal membrane. It participates in different protein complexes during the translocation of cargos from the cytosol to the peroxisomal matrix. Its sequence comprises two structurally and functionally autonomous parts. The N-terminal segment interacts with several peroxins that assemble into distinct protein complexes during cargo translocation. Despite evidence for alpha-helical binding motifs for some of these components (Pex13p, Pex14p) its overall appearance is that of a molten globule and folding/unfolding transitions may play a critical role in its function. In contrast, most of the C-terminal part of the receptor folds into a ring-like alpha-helical structure and binds folded and functionally intact peroxisomal targets that bear a C-terminal peroxisomal targeting signal type-1. Some of these targets also bind to secondary binding sites of the receptor. 相似文献
18.
Interactions of Pex7p and Pex18p/Pex21p with the peroxisomal docking machinery: implications for the first steps in PTS2 protein import 下载免费PDF全文
Stein K Schell-Steven A Erdmann R Rottensteiner H 《Molecular and cellular biology》2002,22(17):6056-6069
Peroxisomal PTS2-dependent matrix protein import starts with the recognition of the PTS2 targeting signal by the import receptor Pex7p. Subsequently, the formed Pex7p/cargo complex is transported from the cytosol to the peroxisomal docking complex, consisting of Pex13p and Pex14p. In Saccharomyces cerevisiae, the latter event is thought to require the redundant Pex18p and Pex21p. Here we mapped the Pex7p interaction domain of Pex13p to its N-terminal 100 amino acids. Pex18p and Pex21p also interacted with this region, albeit only in the presence of Pex7p. Expression of an N-terminally deleted version of Pex13p in a pex13delta mutant failed to restore growth on fatty acids due to a specific defect in the import of PTS2-containing proteins. We further show by yeast two-hybrid analysis, coimmunoprecipitation, and in vitro binding assays that Pex7p can bind Pex13p and Pex14p in the absence of Pex18p/Pex21p. The PTS2 protein thiolase was shown to interact with Pex14p but not with Pex13p in a Pex7p- and Pex18p/Pex21p-dependent manner, suggesting that only Pex14p binds cargo-loaded PTS2 receptor. We also found that the cytosolic Pex7p/thiolase-containing complex includes Pex18p. This complex accumulated in docking mutants but was absent in cells lacking Pex18p/Pex21p, indicating that Pex18p/Pex21p are required already before the docking event. 相似文献
19.
Characterization of peroxisomal Pex5p from rat liver. Pex5p in the Pex5p-Pex14p membrane complex is a transmembrane protein 总被引:1,自引:0,他引:1
Gouveia AM Reguenga C Oliveira ME Sa-Miranda C Azevedo JE 《The Journal of biological chemistry》2000,275(42):32444-32451
Pex5p is the receptor for the vast majority of peroxisomal matrix proteins. Here, we show that about 15% of rat liver Pex5p is found in the peroxisomal fraction representing 0.06% of total peroxisomal protein. This population of Pex5p displays all the characteristics of an intrinsic membrane protein. Protease protection assays indicate that this pool of Pex5p has domains exposed on both sides of the peroxisomal membrane. The strong interaction of Pex5p with the membrane of the organelle is not affected by mild protease treatment of intact organelles, conditions that result in the partial degradation of Pex13p. Cytosolic Pex5p is a monomeric protein. In contrast, virtually all peroxisomal Pex5p was found to be part of a stable 250-kDa protein assembly. This complex was isolated and shown to comprise just two subunits, Pex5p and Pex14p. 相似文献
20.
The recognition of the conserved ATP-binding domains of Pex1p, p97 and NSF led to the discovery of the family of AAA-type ATPases. The biogenesis of peroxisomes critically depends on the function of two AAA-type ATPases, namely Pex1p and Pex6p, which provide the energy for import of peroxisomal matrix proteins. Peroxisomal matrix proteins are synthesized on free ribosomes in the cytosol and guided to the peroxisomal membrane by specific soluble receptors. At the membrane, the cargo-loaded receptors bind to a docking complex and the receptor-docking complex assembly is thought to form a dynamic pore which enables the transition of the cargo into the organellar lumen. The import cycle is completed by ubiquitination- and ATP-dependent dislocation of the receptor from the membrane to the cytosol, which is performed by the AAA-peroxins. Receptor ubiquitination and dislocation are the only energy-dependent steps in peroxisomal protein import. The export-driven import model suggests that the AAA-peroxins might function as motor proteins in peroxisomal import by coupling ATP-dependent removal of the peroxisomal import receptor and cargo translocation into the organelle. 相似文献