scholarly journals 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.

1996 ◽  
Vol 135 (1) ◽  
pp. 97-109 ◽  
Author(s):  
Y Elgersma ◽  
L Kwast ◽  
A Klein ◽  
T Voorn-Brouwer ◽  
M van den Berg ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Protein ◽  
Protein Import ◽  
Sh3 Domain ◽  
Type I ◽  
Peroxisomal Membrane ◽  
Peroxisomal Membrane Protein ◽  
Peroxisomal Targeting ◽  
Src Homology

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.

scholarly journals Saccharomyces cerevisiae PTS1 Receptor Pex5p Interacts with the SH3 Domain of the Peroxisomal Membrane Protein Pex13p in an Unconventional, Non-PXXP–related Manner

2000 ◽  
Vol 11 (11) ◽  
pp. 3963-3976 ◽  
Author(s):  
Gina Bottger ◽  
Phil Barnett ◽  
AndréT. J. Klein ◽  
Astrid Kragt ◽  
Henk F. Tabak ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Protein ◽  
Protein Import ◽  
Sh3 Domain ◽  
Site Directed Mutagenesis ◽  
Binding Motif ◽  
Binding Studies ◽  
Peroxisomal Membrane ◽  
Yeast Saccharomyces Cerevisiae

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 W204XXQF208, 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 pex5Δ 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.

scholarly journals Identification of Pex13p a peroxisomal membrane receptor for the PTS1 recognition factor.

1996 ◽  
Vol 135 (1) ◽  
pp. 111-121 ◽  
Author(s):  
R Erdmann ◽  
G Blobel
Keyword(s):  
Membrane Protein ◽  
Protein Import ◽  
Signal Sequence ◽  
Membrane Receptor ◽  
Matrix Proteins ◽  
Signal Recognition ◽  
Peroxisomal Membrane ◽  
Peroxisomal Membrane Protein ◽  
Src Homology 3 ◽  
Src Homology

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.

scholarly journals The peroxisomal protein import machinery displays a preference for monomeric substrates

Open Biology ◽  
2015 ◽  
Vol 5 (4) ◽  
pp. 140236 ◽  
Author(s):  
Marta O. Freitas ◽  
Tânia Francisco ◽  
Tony A. Rodrigues ◽  
Celien Lismont ◽  
Pedro Domingues ◽  
...  
Keyword(s):  
Protein Import ◽  
Urate Oxidase ◽  
Experimental Conditions ◽  
Peroxisomal Membrane ◽  
Oligomeric Proteins ◽  
Peroxisomal Protein ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Monomeric Proteins

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and transported by the shuttling receptor PEX5 to the peroxisomal membrane docking/translocation machinery, where they are translocated into the organelle matrix. Under certain experimental conditions this protein import machinery has the remarkable capacity to accept already oligomerized proteins, a property that has heavily influenced current models on the mechanism of peroxisomal protein import. However, whether or not oligomeric proteins are really the best and most frequent clients of this machinery remain unclear. In this work, we present three lines of evidence suggesting that the peroxisomal import machinery displays a preference for monomeric proteins. First, in agreement with previous findings on catalase, we show that PEX5 binds newly synthesized (monomeric) acyl-CoA oxidase 1 (ACOX1) and urate oxidase (UOX), potently inhibiting their oligomerization. Second, in vitro import experiments suggest that monomeric ACOX1 and UOX are better peroxisomal import substrates than the corresponding oligomeric forms. Finally, we provide data strongly suggesting that although ACOX1 lacking a peroxisomal targeting signal can be imported into peroxisomes when co-expressed with ACOX1 containing its targeting signal, this import pathway is inefficient.

scholarly journals Peroxisomal Membrane Protein Import Does Not Require Pex17p

2002 ◽  
Vol 277 (19) ◽  
pp. 16498-16504 ◽  
Author(s):  
Courtney C. Harper ◽  
Sarah T. South ◽  
J. Michael McCaffery ◽  
Stephen J. Gould
Keyword(s):  
Membrane Protein ◽  
Protein Import ◽  
Peroxisomal Membrane ◽  
Peroxisomal Membrane Protein

scholarly journals Involvement of Pex13p in Pex14p Localization and Peroxisomal Targeting Signal 2–dependent Protein Import into Peroxisomes

1999 ◽  
Vol 144 (6) ◽  
pp. 1151-1162 ◽  
Author(s):  
Wolfgang Girzalsky ◽  
Peter Rehling ◽  
Katharina Stein ◽  
Julia Kipper ◽  
Lars Blank ◽  
...  
Keyword(s):  
Binding Sites ◽  
Protein Import ◽  
Sh3 Domain ◽  
Loss Of Function ◽  
Peroxisomal Membrane ◽  
Peroxisomal Targeting ◽  
Dependent Protein ◽  
Targeting Signal ◽  
Docking Protein ◽  
Peroxisomal Targeting Signal

Pex13p is the putative docking protein for peroxisomal targeting signal 1 (PTS1)-dependent protein import into peroxisomes. Pex14p interacts with both the PTS1- and PTS2-receptor and may represent the point of convergence of the PTS1- and PTS2-dependent protein import pathways. We report the involvement of Pex13p in peroxisomal import of PTS2-containing proteins. Like Pex14p, Pex13p not only interacts with the PTS1-receptor Pex5p, but also with the PTS2-receptor Pex7p; however, this association may be direct or indirect. In support of distinct peroxisomal binding sites for Pex7p, the Pex7p/Pex13p and Pex7p/ Pex14p complexes can form independently. Genetic evidence for the interaction of Pex7p and Pex13p is provided by the observation that overexpression of Pex13p suppresses a loss of function mutant of Pex7p. Accordingly, we conclude that Pex7p and Pex13p functionally interact during PTS2-dependent protein import into peroxisomes. NH2-terminal regions of Pex13p are required for its interaction with the PTS2-receptor while the COOH-terminal SH3 domain alone is sufficient to mediate its interaction with the PTS1-receptor. Reinvestigation of the topology revealed both termini of Pex13p to be oriented towards the cytosol. We also found Pex13p to be required for peroxisomal association of Pex14p, yet the SH3 domain of Pex13p may not provide the only binding site for Pex14p at the peroxisomal membrane.

scholarly journals The importomer is a peroxisomal membrane protein translocase

2020 ◽  
Author(s):  
James S Martenson ◽  
Hanson Tam ◽  
Alexander J McQuown ◽  
Dvir Reif ◽  
Jing Zhou ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Matrix Protein ◽  
Protein Import ◽  
Protein Targeting ◽  
Fundamental Principle ◽  
Specific Protein ◽  
En Bloc ◽  
Peroxisomal Membrane ◽  
Peroxisomal Membrane Protein ◽  
Protein Biogenesis

ABSTRACTThree sites of membrane protein biogenesis (the endoplasmic reticulum, mitochondria and chloroplasts) receive unfolded substrates from organelle-specific protein targeting factors, then integrate them using separate translocation channels. Peroxisomes also receive membrane proteins from known targeting factors, but whether a separate translocase is needed for integration remains unknown. Here, using a novel genetic screening strategy, we reveal that the importomer–known for matrix protein import–integrates the peroxisomal membrane protein Pex14. In importomer mutants, Pex14 is arrested in a pre-integrated state on the peroxisome surface. To undergo integration, a Pex14 translocation signal binds the importomer’s substrate receptor Pex5 at a distinct site from matrix proteins. En bloc translocation of an engineered protein complex with Pex14’s luminal region argues that integration occurs without substrate unfolding. Our work shows that the handling of membrane protein targeting and integration by discrete machineries is a fundamental principle shared by diverse membrane protein biogenesis pathways.

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