Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway

Cell (Impact Factor: 33.12). 09/1980; 21(1):205-15. DOI: 10.1016/0092-8674(80)90128-2
Source: PubMed

ABSTRACT 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.

Download full-text


Available from: Peter Novick, Mar 24, 2014
  • Source
    [Show abstract] [Hide abstract]
    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.
    Biochimica et Biophysica Acta 03/2015; 1848(7). DOI:10.1016/j.bbamem.2015.03.026 · 4.66 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Over the past decades, studies using zebrafish have significantly advanced our understanding of the cellular basis for development and human diseases. Zebrafish have rapidly developing transparent embryos that allow comprehensive imaging of embryogenesis combined with powerful genetic approaches. However, forward genetic screens in zebrafish have generated unanticipated findings that are mirrored by human genetic studies: disruption of genes implicated in basic cellular processes, such as protein secretion or cytoskeletal dynamics, causes discrete developmental or disease phenotypes. This is surprising because many processes that were assumed to be fundamental to the function and survival of all cell types appear instead to be regulated by cell-specific mechanisms. Such discoveries are facilitated by experiments in whole animals, where zebrafish provides an ideal model for visualization and manipulation of organelles and cellular processes in a live vertebrate. Here, we review well-characterized mutants and newly developed tools that underscore this notion. We focus on the secretory pathway and microtubule-based trafficking as illustrative examples of how studying cell biology in vivo using zebrafish has broadened our understanding of the role fundamental cellular processes play in embryogenesis and disease.
    Journal of Cell Science 02/2014; 127(Pt 3):485-495. DOI:10.1242/jcs.140194 · 5.33 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Delivery and final fusion of the secretory vesicles with the relevant target membrane are hierarchically organized and reciprocally interconnected multi-step processes involving not only specific protein-protein interactions, but also specific protein-phospholipid interactions. The exocyst was discovered as a tethering complex mediating initial encounter of arriving exocytic vesicles with the plasma membrane. The exocyst complex is regulated by Rab and Rho small GTPases, resulting in docking of exocytic vesicles to the plasma membrane (PM) and finally their fusion mediated by specific SNARE complexes. In model Opisthokont cells, the exocyst was shown to directly interact with both microtubule and microfilament cytoskeleton and related motor proteins as well as with the PM via phosphatidylinositol 4, 5-bisphosphate specific binding, which directly affects cortical cytoskeleton and PM dynamics. Here we summarize the current knowledge on exocyst-cytoskeleton-PM interactions in order to open a perspective for future research in this area in plant cells.
    Frontiers in Plant Science 01/2014; 4:543. DOI:10.3389/fpls.2013.00543 · 3.64 Impact Factor