Pex15p of <i>Saccharomyces cerevisiae</i> Provides a Molecular Basis  for Recruitment of the AAA Peroxin Pex6p to Peroxisomal Membranes

Birschmann, Ingvíld; Stroobants, An K.; Berg, Marlene van den; Schäfer, Antje; Rosenkranz, Katja; Kunau, Wolf‐H.; Tabak, Henk F.

doi:10.1091/mbc.e02-11-0752

Cited by 126 publications

(149 citation statements)

References 46 publications

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“…In vivo experiments with Walker B mutants show that ATP hydrolysis at both sites is needed for cell viability (51,52). Thus, even the less important sites may hydrolyze ATP.…”

Section: Discussionmentioning

confidence: 99%

Unique double-ring structure of the peroxisomal Pex1/Pex6 ATPase complex revealed by cryo-electron microscopy

Blok

Tan

Wang

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Members of the AAA family of ATPases assemble into hexameric double rings and perform vital functions, yet their molecular mechanisms remain poorly understood. Here, we report structures of the Pex1/Pex6 complex; mutations in these proteins frequently cause peroxisomal diseases. The structures were determined in the presence of different nucleotides by cryo-electron microscopy. Models were generated using a computational approach that combines Monte Carlo placement of structurally homologous domains into density maps with energy minimization and refinement protocols. Pex1 and Pex6 alternate in an unprecedented hexameric double ring. Each protein has two N-terminal domains, N1 and N2, structurally related to the single N domains in p97 and N-ethylmaleimide sensitive factor (NSF); N1 of Pex1 is mobile, but the others are packed against the double ring. The N-terminal ATPase domains are inactive, forming a symmetric D1 ring, whereas the C-terminal domains are active, likely in different nucleotide states, and form an asymmetric D2 ring. These results suggest how subunit activity is coordinated and indicate striking similarities between Pex1/Pex6 and p97, supporting the hypothesis that the Pex1/Pex6 complex has a role in peroxisomal protein import analogous to p97 in ER-associated protein degradation.Pex1 | Pex6 | AAA ATPase | cryo-electron microscopy | peroxisome T he AAA (ATPases associated with diverse cellular activities) family of ATPases contains a large number of proteins that perform important functions in cells (1). Many members of this family form hexamers. These hexamers consist either of a single ring of ATPase domains or of stacked rings formed by tandem ATPase domains in a single polypeptide chain (type I and II ATPases, respectively). Both classes of AAA ATPases often use the energy of ATP hydrolysis to move macromolecules. Examples of single-ring ATPases include the F1 ATPase (2), which rotates a polypeptide inside its central pore, ClpX (3), which moves polypeptides into the proteolytic chamber of ClpP, and the helicase gp4 of T7 phage (4), which pulls a DNA strand through its center. Double-ring ATPases generally act on polypeptides. Prominent members of this family include N-ethylmaleimide sensitive factor (NSF), p97/VCP (valosin-containing protein), and Hsp104 in eukaryotes and ClpB in bacteria (5-7). NSF dissociates SNARE complexes generated during membrane fusion, p97/VCP plays a role in many processes, including ERassociated protein degradation (ERAD), and Hsp104 and ClpB disassemble protein aggregates.The molecular mechanism of many hexameric AAA ATPases is only poorly understood, particularly for those that move polypeptide chains. In addition, the mechanism appears to differ among the known examples. For the F1 ATPase, it has been established that ATPase domains hydrolyze ATP continuously in a strictly consecutive manner around the ring (2). In contrast, in another single-ring ATPase, ClpX, several ATPase domains hydrolyze ATP in sporadic bursts, either simultaneously or in short succession...

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“…In vivo experiments with Walker B mutants show that ATP hydrolysis at both sites is needed for cell viability (51,52). Thus, even the less important sites may hydrolyze ATP.…”

Section: Discussionmentioning

confidence: 99%

Unique double-ring structure of the peroxisomal Pex1/Pex6 ATPase complex revealed by cryo-electron microscopy

Blok

Tan

Wang

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…5). Among these five mutants, two groups can be distinguished based on the pattern of ubiquitinated Pex5p, and the peroxins deficient within a group are functionally related (18,22). The first group consists of the pex4⌬ and pex22⌬ mutants, whereas the second group comprises pex1⌬, pex6⌬, and pex15⌬.…”

Section: Discussionmentioning

confidence: 99%

“…Two peroxins, Pex1p and Pex6p, are known as ATPase associated with various cellular activities (AAA) ATPases (16,17). In Saccharomyces cerevisiae they partially associate with the membrane via the integral membrane protein Pex15p (18,19). AAA ATPases have been suggested to function in the dissociation of protein complexes and protein unfolding (for review, see Refs.…”

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confidence: 99%

The Saccharomyces cerevisiae Peroxisomal Import Receptor Pex5p Is Monoubiquitinated in Wild Type Cells

Kragt

Voorn-Brouwer

Berg

et al. 2005

Journal of Biological Chemistry

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Pex5p is a mobile receptor for peroxisomal targeting signal type I-containing proteins that cycles between the cytoplasm and the peroxisome. Here we show that Pex5p is a stable protein that is monoubiquitinated in wild type cells. By making use of mutants defective in vacuolar or proteasomal degradation we demonstrate that monoubiquitinated Pex5p is not a breakdown intermediate of either system. Monoubiquitinated Pex5p is localized to peroxisomes, and ubiquitination requires the presence of functional docking and RING finger complexes, which suggests that it is a late event in peroxisomal matrix protein import. In pex1, pex4, pex6, pex15, and pex22 mutants, all of which are blocked in the terminal steps of peroxisomal matrix protein import, polyubiquitinated forms of Pex5p accumulate, ubiquitination being dependent on the ubiquitin-conjugating enzyme Ubc4p. However, Ubc4p is not required for Pex5p ubiquitination in wild type cells, and cells lacking Ubc4p are not affected in peroxisome biogenesis. These results indicate that Pex5p monoubiquitination in wild type cells serves to regulate rather than to degrade Pex5p, which is supported by the observed stability of Pex5p. We propose that Pex5p monoubiquitination in wild type cells is required for the recycling of Pex5p from the peroxisome, whereas Ubc4p-mediated polyubiquitination of Pex5p in mutants blocked in the terminal steps of peroxisomal matrix protein import may function as a disposal mechanism for Pex5p when it gets stuck in the import pathway.

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“…Immunoblot analysis was performed according to Harlow and Lane (34) with polyclonal rabbit antibodies raised against Pex7p (35), Pex13p (36), Pex14p (9), Fox3p (37), Pcs60p (38), Gpd1p, Porin, and Kar2p (39), Fbp1p (40), protein A (Sigma, Munich, Germany), and GFP-GST (41). Primary antibodies were detected with an IRDye 800CW goat anti-rabbit IgG secondary antibody (LI-COR Bioscience, Bad Homburg, Germany).…”

Section: Methodsmentioning

confidence: 99%

Role of Pex21p for Piggyback Import of Gpd1p and Pnc1p into Peroxisomes of Saccharomyces cerevisiae

Effelsberg

Cruz-Zaragoza

Tonillo

et al. 2015

Journal of Biological Chemistry

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Background: PTS2 proteins Gpd1p and Pnc1p are imported into peroxisomes in a PTS2 receptor-dependent manner. Results: PTS2co-receptor Pex21p is required for peroxisomal piggyback import of Gpd1p and Pnc1p. Conclusion: PTS2 co-receptors Pex18p and Pex21p enable targeting of distinct cargo proteins under variable stress conditions. Significance: The life span-regulating Pnc1p is transported into peroxisomes via a novel import route.

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Pex15p of Saccharomyces cerevisiae Provides a Molecular Basis for Recruitment of the AAA Peroxin Pex6p to Peroxisomal Membranes

Cited by 126 publications

References 46 publications

Unique double-ring structure of the peroxisomal Pex1/Pex6 ATPase complex revealed by cryo-electron microscopy

Unique double-ring structure of the peroxisomal Pex1/Pex6 ATPase complex revealed by cryo-electron microscopy

The Saccharomyces cerevisiae Peroxisomal Import Receptor Pex5p Is Monoubiquitinated in Wild Type Cells

Role of Pex21p for Piggyback Import of Gpd1p and Pnc1p into Peroxisomes of Saccharomyces cerevisiae

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