Proteomics Characterization of Mouse Kidney Peroxisomes by Tandem Mass Spectrometry and Protein Correlation Profiling

Abstract

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.

A role for Mitochondrial Rho GTPase 1 (MIRO1) in motility and membrane dynamics of peroxisomes

“…A dual mitochondrial and peroxisomal localisation of MIRO1 was confirmed by immunofluorescence after expression of Myc‐MIRO1 in COS‐7 cells (Figure 1A). Furthermore, we previously reported endogenous MIRO1 in highly purified peroxisomal and mitochondrial fractions,34 in agreement with proteomics data 35, 36…”

Getting a camel through the eye of a needle: the import of folded proteins by peroxisomes

“…50 proteins (Yi et al, 2002;Wiese et al, 2007), whereas plant peroxisomes appear to have approximately double this number (Eubel et al, 2008). However, new functions for peroxisomes are still being discovered by proteomic (Reumann et al, 2007;Wiese et al, 2007;Arai et al, 2008;Eubel et al, 2008;Reumann et al, 2009) and genetic (Lorenz and Fink, 2001;Lipka et al, 2005;Boisson-Dernier et al, 2008) studies.…”

PEX14 is required for microtubule-based peroxisome motility in human cells

“…Processing of gels, in-gel tryptic digestion and online reversed-phase capillary HPLC/ESI-MS/MS analyses of peptide samples were performed as described previously (Wiese et al, 2007). In brief, mass spectrometric analyses were performed on a Bruker Daltonics HCT plus ion trap instrument (Bremen, Germany).…”

“…Thus, because of the functional redundancy of peroxins and the sharing of peroxins by different organelles, other strategies have to be employed to identify such peroxins with overlapping function. To fill this gap, proteomic approaches have been applied; however, so far the complete analyses of whole peroxisomal proteomes did not result in the identification of new biogenesis factors (Saleem et al, 2006;Wiese et al, 2007). This might be explained by the low abundance of such proteins or the fact that they might be mistaken as contaminants.…”