Multicopy suppressor analysis of thermosensitive YIP1 alleles implicates GOT1 in transport from the ER

Yip1p belongs to a conserved family of membrane-spanning proteins that are involved in intracellular trafficking. Studies have shown that Yip1p forms a heteromeric integral membrane complex, is required for biogenesis of ER-derived COPII vesicles, and can interact with Rab GTPases. However, the role of the Yip1 complex in vesicle budding is not well understood. To gain further insight, we isolated multicopy suppressors of the thermosensitive yip1-2 allele. This screen identified GOT1, FYV8 and TSC3 as novel high-copy suppressors. The strongest suppressor, GOT1, also displayed moderate suppressor activity toward temperature-sensitive mutations in the SEC23 and SEC31 genes, which encode subunits of the COPII coat. Further characterization of Got1p revealed that this protein was efficiently packaged into COPII vesicles and cycled rapidly between the ER and Golgi compartments. Based on the findings we propose that Got1p has an unexpected role in vesicle formation from the ER by influencing membrane properties.

[1]  H. Bussey,et al.  A Saccharomyces cerevisiae genome-wide mutant screen for altered sensitivity to K1 killer toxin. , 2003, Genetics.

[2]  H. Riezman,et al.  A yeast T‐snare involved in endocytosis , 1998, Molecular biology of the cell.

[3]  C. Barlowe,et al.  A Rab requirement is not bypassed in SLY1-20 suppression. , 2005, Molecular biology of the cell.

[4]  J. Boeke,et al.  Designer deletion strains derived from Saccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR‐mediated gene disruption and other applications , 1998, Yeast.

[5]  C. Barlowe.  Coupled ER to Golgi Transport Reconstituted with Purified Cytosolic Proteins , 1997, The Journal of cell biology.

[6]  Gary D Bader,et al.  Systematic Genetic Analysis with Ordered Arrays of Yeast Deletion Mutants , 2001, Science.

[7]  M. Heidtman,et al.  A role for Yip1p in COPII vesicle biogenesis , 2003, The Journal of cell biology.

[8]  H. Schwarz,et al.  Ypt1p is essential for retrograde Golgi-ER transport and for Golgi maintenance in S. cerevisiae , 2008, Journal of Cell Science.

[9]  C. Barlowe,et al.  Erv25p, a Component of COPII-coated Vesicles, Forms a Complex with Emp24p That Is Required for Efficient Endoplasmic Reticulum to Golgi Transport* , 1996, The Journal of Biological Chemistry.

[10]  H. Pelham,et al.  A SNARE-like protein required for traffic through the Golgi complex , 1995, Nature.

[11]  W. J. Belden,et al.  Deletion of Yeast p 24 Genes Activates the Unfolded Protein Response , 2001 .

[12]  R. Schekman,et al.  Bi-directional protein transport between the ER and Golgi. , 2004, Annual review of cell and developmental biology.

[13]  H. Pelham,et al.  The dynamics of golgi protein traffic visualized in living yeast cells. , 1998, Molecular biology of the cell.

[14]  R. Schekman,et al.  A Sec63p-BiP complex from yeast is required for protein translocation in a reconstituted proteoliposome , 1993, The Journal of cell biology.

[15]  E. O’Shea,et al.  Global analysis of protein localization in budding yeast , 2003, Nature.

[16]  Ellson Y. Chen,et al.  Overview of manual and automated DNA sequencing by the dideoxy chain termination method , 1991 .

[17]  Hiroaki Kitano,et al.  The PANTHER database of protein families, subfamilies, functions and pathways , 2004, Nucleic Acids Res..

[18]  R. Schekman,et al.  The Sec13p complex and reconstitution of vesicle budding from the ER with purified cytosolic proteins. , 1993, The EMBO journal.

[19]  K. Gable,et al.  Tsc3p Is an 80-Amino Acid Protein Associated with Serine Palmitoyltransferase and Required for Optimal Enzyme Activity* , 2000, The Journal of Biological Chemistry.

[20]  C. Barlowe,et al.  Deletion of yeast p24 genes activates the unfolded protein response. , 2001, Molecular biology of the cell.

[21]  R. Schekman,et al.  Protein translocation mutants defective in the insertion of integral membrane proteins into the endoplasmic reticulum. , 1992, Molecular biology of the cell.

[22]  J. Bonifacino,et al.  The Mechanisms of Vesicle Budding and Fusion , 2004, Cell.

[23]  R. Sternglanz,et al.  A novel Golgi membrane protein is part of a GTPase‐binding protein complex involved in vesicle targeting , 2000, The EMBO journal.

[24]  W. Wickner,et al.  The Docking Stage of Yeast Vacuole Fusion Requires the Transfer of Proteins from a Cis-Snare Complex to a Rab/Ypt Protein , 2000, The Journal of cell biology.

[25]  Fred Winston,et al.  Construction of a set of convenient saccharomyces cerevisiae strains that are isogenic to S288C , 1995, Yeast.

[26]  P. Novick,et al.  GDI1 encodes a GDP dissociation inhibitor that plays an essential role in the yeast secretory pathway. , 1994, EMBO Journal.

[27]  D. Baker,et al.  Reconstitution of SEC gene product-dependent intercompartmental protein transport , 1988, Cell.

[28]  D. Gallwitz,et al.  Evidence for Overlapping and Distinct Functions in Protein Transport of Coat Protein Sec24p Family Members* , 2000, The Journal of Biological Chemistry.

[29]  R. Pepperkok,et al.  Sterols regulate ER‐export dynamics of secretory cargo protein ts‐O45‐G , 2006, The EMBO journal.

[30]  R. Schekman,et al.  Yeast Sec23p acts in the cytoplasm to promote protein transport from the endoplasmic reticulum to the Golgi complex in vivo and in vitro. , 1989, The EMBO journal.

[31]  Sean R. Collins,et al.  Exploration of the Function and Organization of the Yeast Early Secretory Pathway through an Epistatic Miniarray Profile , 2005, Cell.

[32]  A. Nakano,et al.  Emp47p and its close homolog Emp46p have a tyrosine-containing endoplasmic reticulum exit signal and function in glycoprotein secretion in Saccharomyces cerevisiae. , 2002, Molecular biology of the cell.

[33]  M. Heidtman,et al.  Yos1p is a novel subunit of the Yip1p-Yif1p complex and is required for transport between the endoplasmic reticulum and the Golgi complex. , 2005, Molecular biology of the cell.

[34]  A. Koong,et al.  Identification of Mitogen-Activated Protein Kinase Signaling Pathways That Confer Resistance to Endoplasmic Reticulum Stress in Saccharomyces cerevisiae , 2005, Molecular Cancer Research.

[35]  W. Wickner,et al.  Remodeling of organelle-bound actin is required for yeast vacuole fusion , 2002, The Journal of cell biology.

[36]  R. Elble.  A simple and efficient procedure for transformation of yeasts. , 1992, BioTechniques.

[37]  R. Lester,et al.  Functions and metabolism of sphingolipids in Saccharomyces cerevisiae. , 2006, Progress in lipid research.

[38]  David Botstein,et al.  Two differentially regulated mRNAs with different 5′ ends encode secreted and intracellular forms of yeast invertase , 1982, Cell.

[39]  P. Philippsen,et al.  Additional modules for versatile and economical PCR‐based gene deletion and modification in Saccharomyces cerevisiae , 1998, Yeast.

[40]  C. Barlowe,et al.  Transport of Axl2p Depends on Erv14p, an ER–Vesicle Protein Related to the Drosophila cornichon Gene Product , 1998, The Journal of cell biology.

[41]  R. Pepperkok,et al.  Secretory Cargo Regulates the Turnover of COPII Subunits at Single ER Exit Sites , 2006, Current Biology.

[42]  C. Barlowe,et al.  Erv14p directs a transmembrane secretory protein into COPII-coated transport vesicles. , 2002, Molecular biology of the cell.

[43]  K. Gable,et al.  Mutations in the Lcb2p subunit of serine palmitoyltransferase eliminate the requirement forthe TSC3 gene in Saccharomyces cerevisiae , 2002, Yeast.

[44]  R. Schekman,et al.  New mutants of Saccharomyces cerevisiae affected in the transport of proteins from the endoplasmic reticulum to the Golgi complex. , 1996, Genetics.

[45]  N. Winand,et al.  Identification of the novel proteins Yip4p and Yip5p as Rab GTPase interacting factors , 2002, FEBS letters.

[46]  P. Silver,et al.  Mutants Affecting the Structure of the Cortical Endoplasmic Reticulum in Saccharomyces cerevisiae , 2000, The Journal of cell biology.

[47]  D. Gallwitz,et al.  Specific binding to a novel and essential Golgi membrane protein (Yip1p) functionally links the transport GTPases Ypt1p and Ypt31p , 1998, The EMBO journal.

[48]  F. Sherman.  Getting started with yeast. , 1991, Methods in enzymology.

[49]  Randy Schekman,et al.  Multiple Cargo Binding Sites on the COPII Subunit Sec24p Ensure Capture of Diverse Membrane Proteins into Transport Vesicles , 2003, Cell.

[50]  Benjamin S. Glick,et al.  A Role for Actin, Cdc1p, and Myo2p in the Inheritance of Late Golgi Elements in Saccharomyces cerevisiae , 2001, The Journal of cell biology.

[51]  J. Rothman,et al.  A rab protein is required for the assembly of SNARE complexes in the docking of transport vesicles , 1994, Cell.

[52]  Ronald W. Davis,et al.  Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. , 1999, Science.

[53]  Randy Schekman,et al.  COPII–cargo interactions direct protein sorting into ER-derived transport vesicles , 1998, Nature.

[54]  R. Schekman,et al.  Sec23p and a novel 105-kDa protein function as a multimeric complex to promote vesicle budding and protein transport from the endoplasmic reticulum. , 1992, Molecular biology of the cell.

[55]  Leszek Rychlewski,et al.  FFAS03: a server for profile–profile sequence alignments , 2005, Nucleic Acids Res..

[56]  H. Riezman,et al.  The Golgi-localization of yeast Emp47p depends on its di-lysine motif but is not affected by the ret1-1 mutation in alpha-COP , 1995, The Journal of cell biology.

[57]  C. Barlowe,et al.  Initial docking of ER‐derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins , 1998, The EMBO journal.

[58]  Charles Barlowe,et al.  Analysis of Sec22p in endoplasmic reticulum/Golgi transport reveals cellular redundancy in SNARE protein function. , 2002, Molecular biology of the cell.

[59]  C. Barlowe,et al.  Asymmetric Requirements for a Rab Gtpase and Snare Proteins in Fusion of Copii Vesicles with Acceptor Membranes , 2000, The Journal of cell biology.

[60]  C. Burd,et al.  Dual prenylation is required for Rab protein localization and function. , 2003, Molecular biology of the cell.

[61]  Stefan Wiemann,et al.  High-content screening microscopy identifies novel proteins with a putative role in secretory membrane traffic. , 2004, Genome research.

[62]  J. Thorner,et al.  Pkh1 and Pkh2 differentially phosphorylate and activate Ypk1 and Ykr2 and define protein kinase modules required for maintenance of cell wall integrity. , 2002, Molecular biology of the cell.

[63]  A. Emili,et al.  The ctf13-30/CTF13 Genomic Haploinsufficiency Modifier Screen Identifies the Yeast Chromatin Remodeling Complex RSC, Which Is Required for the Establishment of Sister Chromatid Cohesion , 2004, Molecular and Cellular Biology.

[64]  R. Schekman,et al.  Characteristics of endoplasmic reticulum-derived transport vesicles , 1994, The Journal of cell biology.

[65]  W. Wickner,et al.  The GTPase Ypt7p of Saccharomyces cerevisiae is required on both partner vacuoles for the homotypic fusion step of vacuole inheritance. , 1995, The EMBO journal.

[66]  R. Schekman,et al.  Distinct sets of SEC genes govern transport vesicle formation and fusion early in the secretory pathway , 1990, Cell.

[67]  R. Schekman,et al.  Reconstitution of transport from endoplasmic reticulum to Golgi complex using endoplasmic reticulum-enriched membrane fraction from yeast. , 1992, Methods in enzymology.

[68]  H. Pelham,et al.  Got1p and Sft2p: membrane proteins involved in traffic to the Golgi complex , 1999, The EMBO journal.

[69]  R. Schekman,et al.  Multiple genes are required for proper insertion of secretory proteins into the endoplasmic reticulum in yeast , 1989, The Journal of cell biology.

[70]  Y. Hannun,et al.  Selective Substrate Supply in the Regulation of Yeast de Novo Sphingolipid Synthesis* , 2007, Journal of Biological Chemistry.

[71]  R. Schekman,et al.  Biochemical requirements for the targeting and fusion of ER-derived transport vesicles with purified yeast Golgi membranes , 1996, The Journal of cell biology.

[72]  H. Riezman,et al.  The absence of Emp24p, a component of ER‐derived COPII‐coated vesicles, causes a defect in transport of selected proteins to the Golgi. , 1995, The EMBO journal.

[73]  Charles Barlowe,et al.  Erv26p directs pro-alkaline phosphatase into endoplasmic reticulum-derived coat protein complex II transport vesicles. , 2006, Molecular biology of the cell.

[74]  M. Marzioch,et al.  Erp1p and Erp2p, partners for Emp24p and Erv25p in a yeast p24 complex. , 1999, Molecular biology of the cell.

[75]  S. Emr,et al.  Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum , 1994, Cell.

[76]  K. Gable,et al.  The Saccharomyces cerevisiae TSC10/YBR265w Gene Encoding 3-Ketosphinganine Reductase Is Identified in a Screen for Temperature-sensitive Suppressors of the Ca2+-sensitive csg2Δ Mutant* , 1998, The Journal of Biological Chemistry.

[77]  K. Myung,et al.  Mutator genes for suppression of gross chromosomal rearrangements identified by a genome-wide screening in Saccharomyces cerevisiae , 2004, Proceedings of the National Academy of Sciences of the United States of America.