Charting the Secretory Pathway in a Simple Eukaryote

Department of Molecular and Cell Biology and Howard Hughes Medical Institute, University of California-Berkeley, Berkeley, CA 94720, USA.
Molecular biology of the cell (Impact Factor: 4.47). 11/2010; 21(22):3781-4. DOI: 10.1091/mbc.E10-05-0416
Source: PubMed

ABSTRACT George Palade, a founding father of cell biology and of the American Society for Cell Biology (ASCB), established the ultrastructural framework for an analysis of how proteins are secreted and membranes are assembled in eukaryotic cells. His vision inspired a generation of investigators to probe the molecular mechanisms of protein transport. My laboratory has dissected these pathways with complementary genetic and biochemical approaches. Peter Novick, one of my first graduate students, isolated secretion mutants of Saccharomyces cerevisiae, and through cytological analysis of single and double mutants and molecular cloning of the corresponding SEC genes, we established that yeast cells use a secretory pathway fundamentally conserved in all eukaryotes. A biochemical reaction that recapitulates the first half of the secretory pathway was used to characterize Sec proteins that comprise the polypeptide translocation channel in the endoplasmic reticulum (ER) membrane (Sec61) and the cytoplasmic coat protein complex (COPII) that captures cargo proteins into transport vesicles that bud from the ER.

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    • "Eukaryotic molecules are exported through different cellular pathways, including conventional and unconventional secretory routes67. Conventionally secreted proteins follow the classical endoplasmic reticulum (ER)/Golgi-dependent secretory pathway7. Protein secretion through conventional mechanisms is dependent of an N-terminus-linked signal peptide, which is responsible for translocation of polypeptides into the lumen of the ER7. "
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    ABSTRACT: Fungal pathogenesis requires a number of extracellularly released virulence factors. Recent studies demonstrating that most fungal extracellular molecules lack secretory tags suggest that unconventional secretion mechanisms and fungal virulence are strictly connected. Proteins of the endosomal sorting complex required for transport (ESCRT) have been recently associated with polysaccharide export in the yeast-like human pathogen Cryptococcus neoformans. Snf7 is a key ESCRT operator required for unconventional secretion in Eukaryotes. In this study we generated snf7Δ mutant strains of C. neoformans and its sibling species C. gattii. Lack of Snf7 resulted in important alterations in polysaccharide secretion, capsular formation and pigmentation. This phenotype culminated with loss of virulence in an intranasal model of murine infection in both species. Our data support the notion that Snf7 expression regulates virulence in C. neoformans and C. gattii by ablating polysaccharide and melanin traffic. These results are in agreement with the observation that unconventional secretion is essential for cryptococcal pathogenesis and strongly suggest the occurrence of still obscure mechanisms of exportation of non-protein molecules in Eukaryotes.
    Scientific Reports 09/2014; 4:6198. DOI:10.1038/srep06198 · 5.58 Impact Factor
    • "The availability of temperature-sensitive (ts) secretion mutants in S. cerevisiae has formed a strong basis for the understanding and identification of secretion pathway genes, including the Sec components of the exocyst complex (Novick et al., 1980; Schekman, 2010). However, tools and strategies for selecting secretion mutants in filamentous fungi are lacking so far, which is one reason why little is known about the regulation of the secretory pathway in filamentous fungi. "
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    ABSTRACT: The filamentous fungus Aspergillus niger is an industrially exploited protein expression platform, well known for its capacity to secrete high levels of proteins. To study the process of protein secretion in A. niger, we established a GFP-v-SNARE reporter strain in which the trafficking and dynamics of secretory vesicles can be followed in vivo. The biological role of putative A. niger orthologs of seven secretion-specific genes, known to function in key aspects of the protein secretion machinery in S. cerevisiae, was analyzed by constructing respective gene deletion mutants in the GFP-v-SNARE reporter strain. Comparison of the deletion phenotype of conserved proteins functioning in the secretory pathway revealed common features but also interesting differences between S. cerevisiae and A. niger. Deletion of the S. cerevisiae Sec2p ortholog in A. niger (SecB), encoding a guanine exchange factor for the GTPase Sec4p (SrgA in A. niger), did not have an obvious phenotype, while SEC2 deletion in baker's yeast is lethal. Similarly, deletion of the A. niger ortholog of the S. cerevisiae exocyst subunit Sec3p (SecC) did not result in a lethal phenotype as in S. cerevisiae, although severe growth reduction of A. niger was observed. Deletion of secA, secH and ssoA (the A. niger orthologs of S. cerevisiae Sec1p, Sec8p and Sso1/2p, respectively) showed that these genes are essential for A. niger, similarly to the situation in S. cerevisiae. These data demonstrate that the orchestration of exocyst-mediated vesicle transport is only partially conserved in S. cerevisiae and A. niger.
    Microbiology 12/2013; 160(Pt_2). DOI:10.1099/mic.0.074252-0 · 2.56 Impact Factor
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    ABSTRACT: In eukaryotic cells, the trans-Golgi network serves as a sorting station for post-Golgi traffic. In addition to coat- and adaptor-mediated mechanisms, studies in mammalian epithelial cells and yeast have provided evidence for lipid-dependent protein sorting as a major delivery mechanism for cargo sorting to the cell surface. The mechanism for lipid-mediated sorting is the generation of raft platforms of sphingolipids, sterols and specific sets of cargo proteins by phase segregation in the TGN. Here, we review the evidence for such lipid-raft-based sorting at the TGN, as well as their involvement in the formation of TGN-to-PM transport carriers. This article is part of a Special Issue entitled Lipids and Vesicular Transport.
    Biochimica et Biophysica Acta 12/2011; 1821(8):1059-67. DOI:10.1016/j.bbalip.2011.12.008 · 4.66 Impact Factor
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