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Functional characterization of proteins essential for de novo peroxisome biogenesis

  • Author(s): Fassas, Scott
  • Advisor(s): Subramani, Suresh
  • et al.
Abstract

Recent 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.

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