Wolfgang Schliebs

Proteomics characterization of mouse kidney peroxisomes by tandem mass spectrometry and protein correlation profiling.

The peroxisome represents a ubiquitous single membrane-bound key organelle that executes various metabolic pathways such as fatty acid degradation by α- and β-oxidation, ether-phospholipid biosynthesis, metabolism of reactive oxygen species, and detoxification of glyoxylate in mammals. To fulfil this vast array of metabolic functions, peroxisomes accommodate ∼50 different enzymes at least as identified until now. Interest in peroxisomes has been fueled by the discovery of a group of genetic diseases in humans, which are caused by either a defect in peroxisome biogenesis or the deficient activity of a distinct peroxisomal enzyme or transporter. Although this research has greatly improved our understanding of peroxisomes and their role in mammalian metabolism, deeper insight into biochemistry and functions of peroxisomes is required to expand our knowledge of this low abundance but vital organelle. In this work, we used classical subcellular fractionation in combination with MS-based proteomics methodologies to characterize the proteome of mouse kidney peroxisomes. We could identify virtually all known components involved in peroxisomal metabolism and biogenesis. Moreover through protein localization studies by using a quantitative MS screen combined with statistical analyses, we identified 15 new peroxisomal candidates. Of these, we further investigated five candidates by immunocytochemistry, which confirmed their localization in peroxisomes. As a result of this joint effort, we believe to have compiled the so far most comprehensive protein catalogue of mammalian peroxisomes.

Peroxisomal matrix protein import: the transient pore model

Peroxisomes import folded, even oligomeric, proteins, which distinguishes the peroxisomal translocation machinery from the well-characterized translocons of other organelles. How proteins are transported across the peroxisomal membrane is unclear. Here, we propose a mechanistic model that conceptually divides the import process into three consecutive steps: the formation of a translocation pore by the import receptor, the ubiquitylation of the import receptors, and pore disassembly/receptor recycling.

Recombinant Human Peroxisomal Targeting Signal Receptor PEX5 STRUCTURAL BASIS FOR INTERACTION OF PEX5 WITH PEX14

Import of matrix proteins into peroxisomes requires two targeting signal-specific import receptors, Pex5p and Pex7p, and their binding partners at the peroxisomal membrane, Pex13p and Pex14p. Several constructs of human PEX5 have been overexpressed and purified by affinity chromatography in order to determine functionally important interactions and provide initial structural information. Sizing chromatography and electron microscopy suggest that the two isoforms of the human PTS1 receptor, PEX5L and PEX5S, form homotetramers. Surface plasmon resonance analysis indicates that PEX5 binds to the N-terminal fragment of PEX14-(1–78) with a very high affinity in the low nanomolar range. Stable complexes between recombinant PEX14-(1–78) and both the full-length and truncated versions of PEX5 were formed in vitro. Analysis of these complexes revealed that PEX5 possesses multiple binding sites for PEX14, which appear to be distributed throughout its N-terminal half. Coincidentally, this part of the molecule is also responsible for oligomerization, whereas the C-terminal half with its seven tetratricopeptide repeats has been reported to bind PTS1-proteins. A pentapeptide motif that is reiterated seven times in PEX5 is proposed as a determinant for the interaction with PEX14.

MINOS1 is a conserved component of mitofilin complexes and required for mitochondrial function and cristae organization

MINOS1/Mio10, a conserved mitochondrial protein, is required for mitochondrial inner membrane organization and cristae morphology. MINOS1/Mio10 is a novel constituent of the mitofilin/Fcj1 complex of the inner membrane, linking the morphology phenotype of the mutant to the activity of the mitochondrial inner membrane organizing complex.

Recognition of a Functional Peroxisome Type 1 Target by the Dynamic Import Receptor Pex5p

Peroxisomes require the translocation of folded and functional target proteins of various sizes across the peroxisomal membrane. We have investigated the structure and function of the principal import receptor Pex5p, which recognizes targets bearing a C-terminal peroxisomal targeting signal type 1. Crystal structures of the receptor in the presence and absence of a peroxisomal target, sterol carrier protein 2, reveal major structural changes from an open, snail-like conformation into a closed, circular conformation. These changes are caused by a long loop C terminal to the 7-fold tetratricopeptide repeat segments. Mutations in residues of this loop lead to defects in peroxisomal import in human fibroblasts. The structure of the receptor/cargo complex demonstrates that the primary receptor-binding site of the cargo is structurally and topologically autonomous, enabling the cargo to retain its native structure and function.

Peroxisomal protein import and ERAD: variations on a common theme

Despite their distinct biological functions, there is a surprising similarity between the composition of the machinery that imports proteins into peroxisomes and the machinery that degrades endoplasmic reticulum (ER)-associated proteins. The basis of this similarity lies in the fact that both machineries make use of the same basic mechanistic principle: the tagging of a substrate by monoubiquitylation or polyubiquitylation and its subsequent recognition and ATP-dependent removal from a membrane by ATPases of the ATPases associated with diverse cellular activities (AAA) family of proteins. We propose that the ER-associated protein degradation (ERAD)-like removal of the peroxisomal import receptor is mechanically coupled to protein translocation into the organelle, giving rise to a new concept of export-driven import.

Yarrowia lipolytica Pex20p, Saccharomyces cerevisiae Pex18p/Pex21p and mammalian Pex5pL fulfil a common function in the early steps of the peroxisomal PTS2 import pathway

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.

Functional Similarity between the Peroxisomal PTS2 Receptor Binding Protein Pex18p and the N-Terminal Half of the PTS1 Receptor Pex5p

ABSTRACT 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.

Membrane Association of the Cycling Peroxisome Import Receptor Pex5p

Peroxisomal proteins carrying a peroxisome targeting signal type 1 (PTS1) are recognized in the cytosol by the cycling import receptor Pex5p. The receptor-cargo complex docks at the peroxisomal membrane where it associates with multimeric protein complexes, referred to as the docking and RING finger complexes. Here we have identified regions within the Saccharomyces cerevisiae Pex5p sequence that interconnect the receptor-cargo complex with the docking complex. Site-directed mutagenesis of the conserved tryptophan residue within a reverse WXXXF motif abolished two-hybrid binding with the N-terminal half of Pex14p. In combination with an additional mutation introduced into the Pex13p-binding site, we generated a Pex5p mutant defective in a stable association not only with the docking complex but also with the RING finger peroxins at the membrane. Surprisingly, PTS1 proteins are still imported into peroxisomes in these mutant cells. Because these mutations had no significant effect on the membrane binding properties of Pex5p, we examined yeast and human Pex5p for intrinsic lipid binding activity. In vitro analyses demonstrated that both proteins have the potential to insert spontaneously into phospholipid membranes. Altogether, these data strongly suggest that a translocation-competent state of the PTS1 receptor enters the membrane via protein-lipid interactions before it tightly associates with other peroxins.

Peroxisome membrane biogenesis: The stage is set

Pex3p and Pex19p are key players in the post-translational import of peroxisomal membrane proteins. New data suggest that these peroxins act in tandem, Pex19p as a cytosolic chaperone and import receptor for peroxisomal membrane proteins, and Pex3p as docking factor at the peroxisomal membrane.