Moving proteins from the cytosol into mitochondria.

Mitochondria of the yeast Saccharomyces cerevisiae contain at least 750 different proteins, which perform diverse roles. Most of these proteins (approx. 99%) are translated on cytosolic ribosomes, and their import into mitochondria is essential for mitochondrial function. Proteinaceous machineries of great complexity, the so-called translocases, in the mitochondrial membranes mediate the import of these proteins.

Domain mapping of human PEX5 reveals functional and structural similarities to Saccharomyces cerevisiae Pex18p and Pex21p.

PEX5 functions as an import receptor for proteins with the type-1 peroxisomal targeting signal (PTS1). Although PEX5 is not involved in the import of PTS2-targeted proteins in yeast, it is essential for PTS2 protein import in mammalian cells. Human cells generate two isoforms of PEX5 through alternative splicing, PEX5S and PEX5L, and PEX5L contains an additional insert 37 amino acids long. Only one isoform, PEX5L, is involved in PTS2 protein import, and PEX5L physically interacts with PEX7, the import receptor for PTS2-containing proteins. In this report we map the regions of human PEX5L involved in PTS2 protein import, PEX7 interaction, and targeting to peroxisomes. These studies revealed that amino acids 1-230 of PEX5L are required for PTS2 protein import, amino acids 191-222 are sufficient for PEX7 interaction, and amino acids 1-214 are sufficient for targeting to peroxisomes. We also identified a 21-amino acid-long peptide motif of PEX5L, amino acids 209-229, that overlaps the regions sufficient for full PTS2 rescue activity and PEX7 interaction and is shared by Saccharomyces cerevisiae Pex18p and Pex21p, two yeast peroxins that act only in PTS2 protein import in yeast. A mutation in PEX5 that changes a conserved serine of this motif abrogates PTS2 protein import in mammalian cells and reduces the interaction of PEX5L and PEX7 in vitro. This peptide motif also lies within regions of Pex18p and Pex21p that interact with yeast PEX7. Based on these and other results, we propose that mammalian PEX5L may have acquired some of the functions that yeast Pex18p and/or Pex21p perform in PTS2 protein import. This hypothesis may explain the essential role of PEX5L in PTS2 protein import in mammalian cells and its lack of importance for PTS2 protein import in yeast.

The mitochondrial import machinery for preproteins.

Most mitochondrial proteins are transported from the cytosol into the organelle. Due to the division of mitochondria into an outer and inner membrane, an intermembrane space and a matrix, an elaborated system for recognition and transport of preproteins has evolved. The translocase of the outer mitochondrial membrane (TOM) and the translocases of the inner mitochondrial membrane (TIM) mediate these processes. Receptor proteins on the cytosolic face of mitochondria recognize the cargo proteins and transfer them to the general import pore (GIP) of the outer membrane. Following the passage of preproteins through the outer membrane they are transported with the aid of the TIM23 complex into either the matrix, inner membrane, or intermembrane space. Some preprotein families utilize the TIM22 complex for their insertion into the inner membrane. The identification of protein components, which are involved in these transport processes, as well as significant insights into the molecular function of some of them, has been achieved in recent years. Moreover, we are now approaching a new era in which elaborated techniques have already allowed and will enable us to gather information about the TOM and TIM complexes on an ultrastructural level.

Class C Vps protein complex regulates vacuolar SNARE pairing and is required for vesicle docking/fusion.

In yeast, the Class C Vps protein complex (C-Vps complex), composed of Vps11, Vps16, Vps18, and Vps33, functions in Golgi-to-vacuole protein transport. In this study, we characterized and purified this complex and identified its interaction with the syntaxin homolog Vam3. Vam3 pairs with the SNAP-25 homolog Vam7 and VAMP homolog Vti1 to form SNARE complexes during vesicle docking/fusion with the vacuole. The C-Vps complex does not bind to Vam3-Vti1-Vam7 paired SNARE complexes but instead binds to unpaired Vam3. Antibodies to a component of this complex inhibited in vitro vacuole-to-vacuole fusion. Furthermore, temperature-conditional mutations in the Class C VPS genes destabilized Vam3-Vti1-Vam7 pairing. Therefore, we propose that the C-Vps complex associates with unpaired (activated) Vam3 to mediate the assembly of trans-SNARE complexes during both vesicle docking/fusion and vacuole-to-vacuole fusion.

Pex8p, an intraperoxisomal peroxin of Saccharomyces cerevisiae required for protein transport into peroxisomes binds the PTS1 receptor pex5p.

We report the characterization of ScPex8p, which is essential for peroxisomal biogenesis in Saccharomyces cerevisiae. Cells lacking Pex8p are characterized by the presence of peroxisomal membrane ghosts and mislocalization of peroxisomal matrix proteins of the PTS1 and PTS2 variety to the cytosol. Pex8p is tightly associated with the lumenal face of the peroxisomal membrane. Consistent with its intraperoxisomal localization, Pex8p contains a peroxisomal targeting signal 1, and it interacts with the PTS1 receptor Pex5p. However, the Pex5p/Pex8p association is also observed upon deletion of the PTS1 of Pex8p, suggesting that Pex8p contains a second binding site for Pex5p. The pex8Delta mutant phenotype and the observed PTS1-independent interaction with the PTS1 receptor suggest that Pex8p is involved in protein import into the peroxisomal matrix. In pex8Delta cells, the PTS1 and PTS2 receptor still associate with membrane bound components of the protein import machinery, supporting the assumption that the Pex8p function in protein translocation follows the docking event.

Formation of AP-3 transport intermediates requires Vps41 function.

Transport of a subset of membrane proteins to the yeast vacuole requires the function of the AP-3 adaptor protein complex. To define the molecular requirements of vesicular transport in this pathway, we used a biochemical approach to analyse the formation and content of the AP-3 transport intermediate. A vam3tsf (vacuolar t-SNARE) mutant blocks vesicle docking and fusion with the vacuole and causes the accumulation of 50-130-nanometre membrane vesicles, which we isolated and showed by biochemical analysis and immunocytochemistry to contain both AP-3 adaptors and alkaline phosphatase (ALP) pathway cargoes. Inactivation of AP-3 or the protein Vps41 blocks formation of this vesicular intermediate. Vps41 binds to the AP-3 delta-adaptin subunit, suggesting that they function together in the formation of ALP pathway transport intermediates at the late Golgi.

Involvement of Pex13p in Pex14p localization and peroxisomal targeting signal 2-dependent protein import into peroxisomes.

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.

Pex17p of Saccharomyces cerevisiae is a novel peroxin and component of the peroxisomal protein translocation machinery.

The Saccharomyces cerevisiae pex17-1 mutant was isolated from a screen to identify mutants defective in peroxisome biogenesis. pex17-1 and pex17 null mutants fail to import matrix proteins into peroxisomes via both PTS1- and PTS2-dependent pathways. The PEX17 gene (formerly PAS9; Albertini, M., P. Rehling, R. Erdmann, W. Girzalsky, J.A.K.W. Kiel, M. Veenhuis, and W.-H Kunau. 1997. Cell. 89:83-92) encodes a polypeptide of 199 amino acids with one predicted membrane spanning region and two putative coiled-coil structures. However, localization studies demonstrate that Pex17p is a peripheral membrane protein located at the surface of peroxisomes. Particulate structures containing the peroxisomal integral membrane proteins Pex3p and Pex11p are evident in pex17 mutant cells, indicating the existence of peroxisomal remnants ("ghosts"). This finding suggests that pex17 null mutant cells are not impaired in peroxisomal membrane biogenesis. Two-hybrid studies showed that Pex17p directly binds to Pex14p, the recently proposed point of convergence for the two peroxisomal targeting signal (PTS)-dependent import pathways, and indirectly to Pex5p, the PTS1 receptor. The latter interaction requires Pex14p, indicating the potential of these three peroxins to form a trimeric complex. This conclusion is supported by immunoprecipitation experiments showing that Pex14p and Pex17p coprecipitate with both PTS receptors in the absence of Pex13p. From these and other studies we conclude that Pex17p, in addition to Pex13p and Pex14p, is the third identified component of the peroxisomal translocation machinery.

Pex14p, a peroxisomal membrane protein binding both receptors of the two PTS-dependent import pathways.

Pex14p, an S. cerevisiae peroxin, is attached to the outer face of the peroxisomal membrane and is a component of the protein import machinery. Pex14p interacts with both the PTS1 and PTS2 receptors. It is the only known peroxisomal membrane protein that binds the PTS2 receptor and might thus mediate the membrane docking event of PTS2-dependent protein import. These results suggest that the two import pathways overlap and, furthermore, that Pex14p represents the point of convergence. Pex14p also interacts with two other membrane-bound peroxins including Pex13p, another binding protein for the PTS1 receptor. The data presented here are consistent with the idea of a common translocation machinery for both PTS-dependent protein import pathways in the peroxisomal membrane.

The import receptor for the peroxisomal targeting signal 2 (PTS2) in Saccharomyces cerevisiae is encoded by the PAS7 gene.

The import of peroxisomal matrix proteins is dependent on one of two targeting signals, PTS1 and PTS2. We demonstrate in vivo that not only the import of thiolase but also that of a chimeric protein consisting of the thiolase PTS2 (amino acids 1-18) fused to the bacterial protein beta-lactamase is Pas7p dependent. In addition, using a combination of several independent approaches (two-hybrid system, co-immunoprecipitation, affinity chromatography and high copy suppression), we show that Pas7p specifically interacts with thiolase in vivo and in vitro. For this interaction, the N-terminal PTS2 of thiolase is both necessary and sufficient. The specific binding of Pas7p to thiolase does not require peroxisomes. Pas7p recognizes the PTS2 of thiolase even when this otherwise N-terminal targeting signal is fused to the C-terminus of other proteins, i.e. the activation domain of Gal4p or GST. These results demonstrate that Pas7p is the targeting signal-specific receptor of thiolase in Saccharomyces cerevisiae and, moreover, are consistent with the view that Pas7p is the general receptor of the PTS2. Our observation that Pas7p also interacts with the human peroxisomal thiolase suggests that in the human peroxisomal disorders characterized by an import defect for PTS2 proteins (classical rhizomelic chondrodysplasia punctata), a functional homologue of Pas7p may be impaired.

Functional expression and molecular characterization of AtUBC2-1, a novel ubiquitin-conjugating enzyme (E2) from Arabidopsis thaliana.

The first member of a novel subfamily of ubiquitin-conjugating E2-proteins was cloned from a cDNA library of Arabidopsis thaliana. Genomic blots indicate that this gene family (AtUBC2) consists of two members and is distinct from AtUBC1, the only other E2 enzyme known from this species to date (M.L. Sullivan and R.D. Vierstra, Proc. Natl. Acad. Sci. USA 86 (1989) 9861-9865). The cDNA sequence of AtUBC2-1 extends over 794 bp which would encode a protein of 161 amino acids and a calculated molecular mass of 18.25 kDa. The protein encoded by AtUBC2-1 is shown to accept 125I-ubiquitin from wheat E1 enzymes, when expressed from Escherichia coli hosts as fusion protein carrying N-terminal extensions. It is deubiquitinated in the presence of lysine and, by these criteria, is considered a functional E2 enzyme.