A vesicle carrier that mediates peroxisome protein traffic from the endoplasmic reticulum

Department of Molecular and Cell Biology and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 11/2010; 107(50):21523-8. DOI: 10.1073/pnas.1013397107
Source: PubMed


Pex19p, a soluble cytoplasmic transport protein, is required for the traffic of the peroxisomal membrane proteins Pex3p and Pex15p from the endoplasmic reticulum (ER) to the peroxisome. We documented Pex15p traffic from the ER using a chimeric protein containing a C-terminal glycosylation acceptor peptide. Pex15Gp expressed in wild-type yeast cells is N-glycosylated and functions properly in the peroxisome. In contrast, pex19Δ-mutant cells accumulate the glycoprotein Pex15Gp in the ER. We developed a cell-free preperoxisomal vesicle-budding reaction in which Pex15Gp and Pex3p are packaged into small vesicles in the presence of cytosol, Pex19p, and ATP. Secretory vesicle budding (COPII) detected by the packaging of a SNARE protein (soluble N-ethylmaleimide-sensitive attachment protein receptor) occurs in the same incubation but does not depend on Pex19p. Conversely a dominant GTPase mutant Sar1p which inhibits COPII has no effect on Pex3p packaging. Pex15Gp and Pex3p budded vesicles sediment as low-buoyant-density membranes on a Nycodenz gradient and copurify by affinity isolation using native but not Triton X-100-treated budded vesicles. ER-peroxisome transport vesicles appear to rely on a novel budding mechanism requiring Pex19p and additional unknown factors.

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    • "Both the coat protein complex (LDBPK_ 322150) and ARF (LDBPK_312350 and LDBPK_312890) proteins have been detected in the present leishmanial glycosomal preparation. A complete set of proteins essential for heterotypic fusion reactions requires N-ethylmaleimidesensitive factor (NSF; LDBPK_200820), and soluble NSF attachment protein receptors (SNAREs; LDBPK_352760 and LDBPK_070570) (Lam et al., 2010) were also found in glycosomal fraction. Several Rab proteins, ras-like small GTPase, and GTP binding proteins that are essential regulators of vesicle trafficking (Gronemeyer et al., 2013) were also found in the proteomic analysis of glycosomes. "
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    ABSTRACT: Leishmania donovani is a kinetoplastid protozoan that causes a severe and fatal disease kala-azar, or visceral leishmaniasis. L. donovani infects human host after the phlebotomine sandfly takes a blood meal and resides within the phagolysosome of infected macrophages. Previous studies on host-parasite interactions have not focused on Leishmania organelles and the role that they play in the survival of this parasite within macrophages. Leishmania possess glycosomes that are unique and specialized subcellular microbody organelles. Glycosomes are known to harbor most peroxisomal enzymes and, in addition, they also possess nine glycolytic enzymes. In the present study, we have carried out proteomic profiling using high resolution mass spectrometry of a sucrose density gradient-enriched glycosomal fraction isolated from L. donovani promastigotes. This study resulted in the identification of 4022 unique peptides, leading to the identification of 1355 unique proteins from a preparation enriched in L. donovani glycosomes. Based on protein annotation, 566 (41.8%) were identified as hypothetical proteins with no known function. A majority of the identified proteins are involved in metabolic processes such as carbohydrate, lipid, and nucleic acid metabolism. Our present proteomic analysis is the most comprehensive study to date to map the proteome of L. donovani glycosomes.
    Omics A Journal of Integrative Biology 03/2015; 19(3):157-70. DOI:10.1089/omi.2014.0163 · 2.36 Impact Factor
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    • "First, fluorescently tagged, membrane anchored Pex proteins, notably Pex3 and Pex16, have been observed emerging from the ER in conditions where peroxisomes are either induced by growth conditions or in pulse-chase type of rescue experiments (Titorenko and Rachubinski, 1998; Hoepfner et al., 2005; Kragt et al., 2005; Tam et al., 2005; Kim et al., 2006; Motley and Hettema, 2007). Second, cell free budding assays from isolated ER have established some of the machinery required to bud Pex-containing vesicles in yeast Saccharomyces cerevisiae (Lam et al., 2010). In this case, the authors showed both Pex3p and Pex15p emerging within vesicles in a manner that depended on ATP and Pex19p, but not Sar1, a GTPase essential for anterograde COPII budding events. "
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    ABSTRACT: In the last century peroxisomes were thought to have an endosymbiotic origin. Along with mitochondria and chloroplasts, peroxisomes primarily regulate their numbers through the growth and division of pre-existing organelles, and they house specific machinery for protein import. These features were considered unique to endosymbiotic organelles, prompting the idea that peroxisomes were key cellular elements that helped facilitate the evolution of multicellular organisms. The functional similarities to mitochondria within mammalian systems expanded these ideas, as both organelles scavenge peroxide and reactive oxygen species, both organelles oxidize fatty acids, and at least in higher eukaryotes, the biogenesis of both organelles is controlled by common nuclear transcription factors of the PPAR family. Over the last decade it has been demonstrated that the fission machinery of both organelles is also shared, and that both organelles act as critical signaling platforms for innate immunity and other pathways. Taken together it is clear that the mitochondria and peroxisomes are functionally coupled, regulating cellular metabolism and signaling through a number of common mechanisms. However, recent work has focused primarily on the role of the ER in the biogenesis of peroxisomes, potentially overshadowing the critical importance of the mitochondria as a functional partner. In this review, we explore the mechanisms of functional coupling of the peroxisomes to the mitochondria/ER networks, providing some new perspectives on the potential contribution of the mitochondria to peroxisomal biogenesis.
    Frontiers in Physiology 09/2013; 4:268. DOI:10.3389/fphys.2013.00268 · 3.53 Impact Factor
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    • "The N-terminal segment of Pex3 sorts an ER membrane protein to the peroxisomal ER subdomain Pex3 is sorted via a subdomain of the ER to peroxisomes. In pex19D cells, Pex3-GFP accumulates in this ER subdomain, as exit from the ER requires Pex19 (Agrawal et al., 2011; Hoepfner et al., 2005; Lam et al., 2011; Tam et al., 2005). In contrast, 66-3- 3-GFP and 0-3-3-GFP are initially distributed throughout the ER, as indicated by the striking perinuclear ER staining, and chase to the vacuole in both wild type (Fig. 2D) and pex19D cells (Fig. 3A). "
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    ABSTRACT: Pex3 is an evolutionarily conserved type III peroxisomal membrane protein required for peroxisome formation. It is inserted into the ER membrane and sorted via an ER subdomain (the peroxisomal ER, or pER) to peroxisomes. By constructing chimeras between Pex3 and the type III ER membrane protein Sec66, we have been able to separate the signals that mediate insertion of Pex3 into the ER from those that mediate sorting within the ER to the pER subdomain. The N-terminal 17-amino acid segment of Pex3 contains two signals that are each sufficient for sorting to the pER: a chimeric protein containing the N-terminal domain of Pex3 fused to the transmembrane and cytoplasmic segments of Sec66 sorts to the pER in wild type cells, and does not colocalise with peroxisomes. Subsequent transport to existing peroxisomes requires the Pex3 transmembrane segment. When expressed in Drosophila S2R+ cells, ScPex3 targeting to peroxisomes is dependent on the intra-ER sorting signals in the N-terminal segment. The N-terminal segments of both human and Drosophila Pex3 contain intra-ER sorting information and can replace that of ScPex3. Our analysis has uncovered the signals within Pex3 required for the various steps of its transport to peroxisomes. Our generation of versions of Pex3 that are blocked at each stage along its transport pathway provides a tool to dissect the mechanism, as well as the molecular machinery required at each step of the pathway.
    Biology Open 08/2013; 2(8):829-37. DOI:10.1242/bio.20134788 · 2.42 Impact Factor
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