248 research outputs found
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Pex36, a Novel Peroxin Implicated in de novo Peroxisome Biogenesis
No full textABSTRACT OF THE THESISPex36, a Novel Peroxin Implicated in de novo Peroxisome BiogenesisByKrypton CarolinoMasters of Science in Biology University of California, San Diego, 2017 Professor Suresh Subramani, ChairIt was previously believed that peroxisomes could only form through the growth and division of pre-existing peroxisomes. In the past decade, there has been increasing evidence suggesting that peroxisomes can also form de novo. In Pichia pastoris, peroxisomal membrane proteins (PMPs) are sorted within the endoplasmic reticulum (ER) to two distinct pre-peroxisomal ER (pER) sites, from which bud two types of pre- peroxisomal vesicles (ppVs). These ppVs then fuse heterotypically to produce import-viicompetent peroxisomes. Currently, only Pex3 and Pex19 are implicated in ppV budding in P. pastoris, but their exact roles are not defined. In this study, we characterized a novel P. pastoris Pex36 protein, whose loss causes cells to display a dramatic growth delay in methanol medium due to slow peroxisome biogenesis. This growth defect in methanol is enhanced with the simultaneous deletion of another peroxin, Pex25, previously implicated in peroxisome division. Using an in vitro budding assay and fluorescence microscopy of P. pastoris Δpex36 Δpex25 cells, we found that PMPs are able to sort to the pER, but are unable to bud out, suggesting that Pex36 has a role that is redundant with Pex25, in de novo peroxisomal biogenesis, specifically in ppV budding.vii
Components Involved in Peroxisome Import, Biogenesis, Proliferation, Turnover, and Movement
No full textSubramani, Suresh. Components Involved in Peroxisome Import, Biogenesis, Proliferation, Turnover, and Movement. Physiol. Rev. 78: 171–188, 1998. — In the decade that has elapsed since the discovery of the first peroxisomal targeting signal (PTS), considerable information has been obtained regarding the mechanism of protein import into peroxisomes. The PTSs responsible for the import of matrix and membrane proteins to peroxisomes, the receptors for several of these PTSs, and docking proteins for the PTS1 and PTS2 receptors are known. Many peroxins involved in peroxisomal protein import and biogenesis have been characterized genetically and biochemically. These studies have revealed important new insights regarding the mechanism of protein translocation across the peroxisomal membrane, the conservation of PEX genes through evolution, the role of peroxins in fatal human peroxisomal disorders, and the biogenesis of the organelle. It is clear that peroxisomal protein import and biogenesis have many features unique to this organelle alone. More recent studies on peroxisome degradation, division, and movement highlight newer aspects of the biology of this organelle that promise to be just as exciting and interesting as import and biogenesis.</jats:p
Turnover of organelles by autophagy in yeast
No full textEfficient detection and removal of superfluous or damaged organelles are crucial to maintain cellular homeostasis and to assure cell survival. Growing evidence shows that organelles or parts of them can be removed by selective subtypes of otherwise unselective macroautophagy and microautophagy. This requires both the adaptation of the core autophagic machinery and sophisticated mechanisms to recognize organelles destined for turnover. We review the current knowledge on autophagic removal of peroxisomes, mitochondria, ER and parts of the nucleus with an emphasis on yeasts as a model eukaryote.'Deutsche Forschungsgemeinschaft'; NIH [GM069373
The importomer—A peroxisomal membrane complex involved in protein translocation into the peroxisome matrix
No full textAbstractThe import of proteins into the peroxisome matrix is an essential step in peroxisome biogenesis, which is critical for normal functioning of most eukaryotic cells. The translocation of proteins across the peroxisome membrane and the dynamic behavior of the import receptors during the import cycle is facilitated by several peroxisome–membrane-associated protein complexes, one of which is called the importomer complex [B. Agne, N.M. Meindl, K. Niederhoff, H. Einwachter, P. Rehling, A. Sickmann, H.E. Meyer, W. Girzalsky, W.H. Kunau, Pex8p: an intraperoxisomal organizer of the peroxisomal import machinery, Mol. Cell 11 (2003) 635–646; P.P. Hazra, I. Suriapranata, W.B. Snyder, S. Subramani, Peroxisome remnants in pex3Δ cells and the requirement of Pex3p for interactions between the peroxisomal docking and translocation subcomplexes, Traffic 3 (2002) 560–574. [1,2]]. We provide below a brief historical perspective regarding the importomer and its role in peroxisome biogenesis. We also identify areas in which further work is needed to uncover the physiological role of the importomer
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Functional characterization of proteins essential for de novo peroxisome biogenesis
No full textRecent discoveries suggest a role for the endoplasmic reticulum (ER) in peroxisome formation. Following the intra-ER sorting of peroxisomal membrane proteins (PMPs) to a site called the pre-peroxisomal ER (pER), the PMPs bud into pre-peroxisomal vesicles (ppVs). The de novo model of peroxisome biogenesis involves the budding of ppVs from the pER and their subsequent fusion to generate import-competent peroxisomes. Although some of the important PMP cargos in the de novo pathway have been identified, their role and the mechanistic details of their actions remain unclear. In this study we investigate the trafficking and subcellular localization of docking and RING subcomplex PMPs, Pex17 and Pex12, respectively, in Pichia pastoris. By performing subcellular fractionation procedures and fluorescence microscopy imaging of endogenously expressed Pex17-GFP, we demonstrate an exclusive ER origin of Pex17-GFP during the de novo pathway. Additionally, we show the co-packaging of Pex12 with the docking subcomplex vesicles, in a Pex3- and Pex19-dependent manner. To further investigate the role of Pex3 in de novo peroxisome biogenesis, we have created and characterized several Pex3 mutations. Site-directed mutagenesis is revealing the essential role of Pex3 domains in peroxisome biogenesis, although further analysis is required to uncover the mechanistic details of this complex process
The dynamic Atg13-free conformation of the Atg1 EAT domain is required for phagophore expansion
No full text Yeast macroautophagy begins with the de novo formation of a double-membrane phagophore at the preautophagosomal structure/phagophore assembly site (PAS), followed by its expansion into the autophagosome responsible for cargo engulfment. The kinase Atg1 is recruited to the PAS by Atg13 through interactions between the EAT domain of the former and the tMIM motif of the latter. Mass-spectrometry data have shown that, in the absence of Atg13, the EAT domain structure is strikingly dynamic, but the function of this Atg13-free dynamic state has been unclear. We used structure-based mutational analysis and quantitative and superresolution microscopy to show that Atg1 is present on autophagic puncta at, on average, twice the stoichiometry of Atg13. Moreover, Atg1 colocalizes with the expanding autophagosome in a manner dependent on Atg8 but not Atg13. We used isothermal titration calorimetry and crystal structure information to design an EAT domain mutant allele ATG1DD that selectively perturbs the function of the Atg13-free state. Atg1DD shows reduced PAS formation and does not support phagophore expansion, showing that the EAT domain has an essential function that is separate from its Atg13-dependent role in autophagy initiation. </jats:p
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Localization of NADH Shuttling Proteins Implicated in Peroxisome Biogenesis in Pichia pastoris
No full textPeroxisomes proliferate in media whose utilization requires peroxisomal metabolic pathways. In methylotrophic yeast, such as Pichia pastoris, oleate and methanol are the most common carbon sources used for peroxisome proliferation studies. When grown in these conditions, peroxisome metabolism is essential for carbon assimilation and energy production. In Saccharomyces cerevisiae, during growth in oleate, NADH shuttling from the peroxisome to mitochondria, via the cytosol, maintains the cellular redox balance during fatty acid β-oxidation and contributes to energy production. In P. pastoris, during growth in methanol, NADH produced by methanol oxidation shuttles to the mitochondria becoming the only source of energy; however, the NADH shuttling mechanism, which typically requires enzymes in peroxisomes, cytosol and mitochondria, has not been studied yet in this yeast. We used fluorescence microscopy to determine the subcellular localization of the homologous P. pastoris NADH shuttling proteins (malate dehydrogenases and glycerol 3-phosphate dehydrogenases). Surprisingly, none of the NADH shuttling proteins fused to GFP showed peroxisomal localization, although this was expected. To improve the detection, we developed a divergent bimolecular fluorescence complementation (BiFC) assay to detect low levels of peroxisomally localized proteins which could be masked by a strong cytosolic localization. Using this assay, we confirmed that one of the malate dehydrogenases has a dual localization, cytosolic and peroxisomal, but only when grown in oleate, but it was exclusively cytosolic when grown in methanol. These localizations can be rationalized in terms of the NADH produced by oleate metabolism in the peroxisome matrix and in the cytosol during methanol metabolism. Finally, we elucidated the pathway responsible for targeting the malate dehydrogenase to the peroxisome; however, no obvious peroxisomal targeting signal was found in the enzyme suggesting an alternative translocation mechanism, such as piggyback import with a peroxisomal protein containing a peroxisomal targeting signal
A mammalian pexophagy target
No full textProtein ubiquitylation in mammals is known to trigger selective autophagy of peroxisomes through a process termed pexophagy. The physiological peroxisomal target for pexophagy-related ubiquitylation has been controversial, but two studies have now identified the protein PEX5 as the real candidate
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