The peroxisome represents a ubiquitous single membrane-bound key organelle that executes various metabolic pathways such as fatty acid degradation by ␣-and ␤-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 ϳ50 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.

The current view of peroxisome inheritance provides for the formation of new peroxisomes by both budding from the endoplasmic reticulum and autonomous division. Here we investigate peroxisome-cytoskeleton interactions and show by proteomics, biochemical and immunofluorescence analyses that actin, non-muscle myosin IIA (NMM IIA), RhoA, Rho kinase II (ROCKII) and Rab8 associate with peroxisomes. Our data provide evidence that (i) RhoA in its inactive state, maintained for example by C. botulinum toxin exoenzyme C3, dissociates from peroxisomes enabling microtubule-based peroxisomal movements and (ii) dominant-active RhoA targets to peroxisomes, uncouples the organelles from microtubules and favors Rho kinase recruitment to peroxisomes. We suggest that ROCKII activates NMM IIA mediating local peroxisomal constrictions. Although our understanding of peroxisome-cytoskeleton interactions is still incomplete, a picture is emerging demonstrating alternate RhoA-dependent association of peroxisomes to the microtubular and actin cytoskeleton. Whereas association of peroxisomes to microtubules clearly serves bidirectional, long-range saltatory movements, peroxisome-acto-myosin interactions may support biogenetic functions balancing peroxisome size, shape, number, and clustering.

a b s t r a c tA proteomics screen was initiated to identify Rab proteins regulating transport to and away from peroxisomes. Mass spectrometry-based protein correlation profiling of rat liver organelles and immunofluorescence analysis of the peroxisome candidate Rab proteins revealed Rab6, Rab10, Rab14 and Rab18 to associate with the peroxisomal membrane. While Rab14 localized to peroxisomes predominantly in its dominant-active form, other Rab proteins associated with peroxisomes in both their GTP-and GDP-bound state. In summary, our data suggest that Rab6, Rab10, Rab14 and Rab18 associate with the peroxisomal compartment and similar as previously shown for Rab8, Rab18 in its GDP-bound state favors peroxisome proliferation. Structured summary of protein interactions:bifunctional enzyme, PEX11alpha, Rab-18, Rab-14, Rab-6A, Rab-10 and Rab-2A colocalize by cosedimentation through density gradient (View interaction) Catalase and Rab18 colocalize by fluorescence microscopy (View interaction) Rab14 and Catalase colocalize by fluorescence microscopy (View interaction) Rab6 and Catalase colocalize by fluorescence microscopy (View interaction) Rab10 and Catalase colocalize by fluorescence microscopy (View interaction)

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