Novick, P., Field, C. & Schekman, R. Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell 21, 205−215

Cell (Impact Factor: 32.24). 09/1980; 21(1):205-15. DOI: 10.1016/0092-8674(80)90128-2
Source: PubMed


Cells of a Saccharomyces cerevisiae mutant that is temperature-sensitive for secretion and cell surface growth become dense during incubation at the non-permissive temperature (37 degrees C). This property allows the selection of additional secretory mutants by sedimentation of mutagenized cells on a Ludox density gradient. Colonies derived from dense cells are screened for conditional growth and secretion of invertase and acid phosphatase. The sec mutant strains that accumulate an abnormally large intracellular pool of invertase at 37 degrees C (188 mutant clones) fall into 23 complementation groups, and the distribution of mutant alleles suggests that more complementation groups could be found. Bud emergence and incorporation of a plasma membrane sulfate permease activity stop quickly after a shift to 37 degrees C. Many of the mutants are thermoreversible; upon return to the permissive temperature (25 degrees C) the accumulated invertase is secreted. Electron microscopy of sec mutant cells reveals, with one exception, the temperature-dependent accumulation of membrane-enclosed secretory organelles. We suggest that these structures represent intermediates in a pathway in which secretion and plasma membrane assembly are colinear.

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    • "The tethering step, described as the initial contact of the secretory vesicle with the target PM prior to the SNARE-catalyzed membrane fusion [5], is in many cases mediated by the evolutionary conserved multiprotein complex, the exocyst. All subunits of the exocyst were discovered in the budding yeast by genetic screens for mutants in secretion [6] and the budding yeast has been an invaluable model in identifying basic principles of the exocyst functions. In addition to its mechanistic role in tethering, the exocyst also determines the region of PM, where exocytosis takes place. "
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    ABSTRACT: The exocytosis is a process of fusion of secretory vesicles with plasma membrane, which plays a prominent role in many crucial cellular processes, e.g. secretion of neurotransmitters, cytokinesis or yeast budding. Prior to the SNARE-mediated fusion, the initial contact of secretory vesicle with the target membrane is mediated by an evolutionary conserved vesicle tethering protein complex, the exocyst. In all eukaryotic cells, the exocyst is composed of eight subunits - Sec5, Sec6, Sec8, Sec10, Sec15, Exo84 and two membrane-targeting landmark subunits Sec3 and Exo70, which have been described to directly interact with phosphatidylinositol (4,5)-bisphosphate (PIP2) of the plasma membrane. In this work, we utilized coarse-grained molecular dynamics simulations to elucidate structural details of the interaction of yeast Sec3p and Exo70p with lipid bilayers containing PIP2. We found that PIP2 is coordinated by the positively charged pocket of N-terminal part of Sec3p, which folds into unique Pleckstrin homology domain. Conversely, Exo70p interacts with the lipid bilayer by several binding sites distributed along the structure of this exocyst subunit. Moreover, we observed that the interaction of Exo70p with the membrane causes clustering of PIP2 in the adjacent leaflet. We further revealed that PIP2 is required for the correct positioning of small GTPase Rho1p, a direct Sec3p interactor, prior to the formation of the functional Rho1p-exocyst-membrane assembly. Our results show the critical importance of the plasma membrane pool of PIP2 for the exocyst function and suggest that specific interaction with acidic phospholipids represents an ancestral mechanism for the exocyst regulation. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Mar 2015 · Biochimica et Biophysica Acta
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    • "Sec12 is a type II transmembrane protein localizing at the ER and exclusively acts as the GEF for Sar1. Sec12 was originally identified from yeast mutants defective in ER-Golgi transport (Novick et al., 1980). Its catalytic domain facing the cytosol is composed of a seven-bladed β-barrel structure, from which a loop with K+ bound (termed the K loop) extends (McMahon et al., 2012). "
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    ABSTRACT: Small GTPase proteins play essential roles in the regulation of vesicular trafficking systems in eukaryotic cells. Two types of small GTPases, secretion-associated Ras-related protein (Sar) and ADP-ribosylation factor (Arf), act in the biogenesis of transport vesicles. Sar/Arf GTPases function as molecular switches by cycling between active, GTP-bound and inactive, GDP-bound forms, catalyzed by guanine nucleotide exchange factors and GTPase-activating proteins, respectively. Activated Sar/Arf GTPases undergo a conformational change, exposing the N-terminal amphipathic α-helix for insertion into membranes. The process triggers the recruitment and assembly of coat proteins to the membranes, followed by coated vesicle formation and scission. In higher plants, Sar/Arf GTPases also play pivotal roles in maintaining the dynamic identity of organelles in the secretory pathway. Sar1 protein strictly controls anterograde transport from the endoplasmic reticulum (ER) through the recruitment of plant COPII coat components onto membranes. COPII vesicle transport is responsible for the organization of highly conserved polygonal ER networks. In contrast, Arf proteins contribute to the regulation of multiple trafficking routes, including transport through the Golgi complex and endocytic transport. These transport systems have diversified in the plant kingdom independently and exhibit several plant-specific features with respect to Golgi organization, endocytic cycling, cell polarity and cytokinesis. The functional diversification of vesicular trafficking systems ensures the multicellular development of higher plants. This review focuses on the current knowledge of Sar/Arf GTPases, highlighting the molecular details of GTPase regulation in vesicle formation in yeast and advances in knowledge of the characteristics of vesicle trafficking in plants.
    Full-text · Article · Aug 2014 · Frontiers in Plant Science
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    • "The exocyst complex (a.k.a. the exocyst) is a ~750 kDa octameric protein complex initially identified in S. cerevisiae and highly conserved from yeast to mammals (Novick et al. 1980; Hsu et al. 1996). The mammalian exocyst comprises Exoc1/Sec3, Exoc2/Sec5, Exoc3/Sec6, Exoc4/ Sec8, Exoc5/Sec10, Exoc6/Sec15, Exoc7/Exo70, and Exoc8/ Exo84 (Novick et al. 1980; Hsu et al. 1996) and is best known for its role in targeting and docking vesicles carrying membrane proteins from the trans-Golgi network (TGN) (Lipschutz and Mostov 2002). Importantly, we previously showed, in renal tubule cells, that exocyst components are localized to primary cilia (Rogers et al. 2004), that the exocyst is required for ciliogenesis (Zuo et al. 2009), and that the exocyst genetically interacts with poly- cystin-2 in zebrafish (Fogelgren et al. 2011; Choi et al. 2013). "
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    ABSTRACT: Cilia, organelles that function as cellular antennae, are central to the pathogenesis of “ciliopathies”, including various forms of polycystic kidney disease (PKD). To date, however, the molecular mechanisms controlling ciliogenesis and ciliary function remain incompletely understood. A recently proposed model of cell–cell communication, called “urocrine signaling”, hypothesizes that a subset of membrane bound vesicles that are secreted into the urinary stream (termed exosome-like vesicles, or ELVs), carry cilia-specific proteins as cargo, interact with primary cilia, and affect downstream cellular functions. This study was undertaken to determine the role of the exocyst, a highly conserved eight-protein trafficking complex, in the secretion and/or retrieval of ELVs. We used Madin–Darby canine kidney (MDCK) cells expressing either Sec10-myc (a central component of the exocyst complex) or Smoothened-YFP (a ciliary protein found in ELVs) in experiments utilizing electron gold microscopy and live fluorescent microscopy, respectively. Additionally, human urinary exosomes were isolated via ultracentrifugation and subjected to mass-spectrometry-based proteomics analysis to determine the composition of ELVs. We found, as determined by EM, that the exocyst localizes to primary cilia, and is present in vesicles attached to the cilium. Furthermore, the entire exocyst complex, as well as most of its known regulatory GTPases, are present in human urinary ELVs. Finally, in living MDCK cells, ELVs appear to interact with primary cilia using spinning disc confocal microscopy. These data suggest that the exocyst complex, in addition to its role in ciliogenesis, is centrally involved in the secretion and/or retrieval of urinary ELVs.
    Full-text · Article · Aug 2014
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