SED5 encodes a 39-kD integral membrane protein required for vesicular transport between the ER and the Golgi complex

The ERD2 gene, which encodes the yeast HDEL (His-Asp-Glu-Leu) receptor, is essential for growth (Semenza, J. C., K. G. Hardwick, N. Dean, and H. R. B. Pelham. 1990. Cell. 61:1349-1357; Lewis, M. J., D. J. Sweet, and H. R. B. Pelham. 1990. Cell. 61:1359-1363). SED5, when present in multiple copies, enables cells to grow in the absence of Erd2p. Sequence analysis of SED5 reveals no significant homology with ERD2 or other known genes. We have raised antibodies to Sed5p which specifically recognize a 39-kD integral membrane protein. A stretch of hydrophobic residues at the COOH terminus is predicted to hold Sed5p on the cytoplasmic face of intracellular membranes. Cells that are depleted of Sed5p are unable to transport carboxypeptidase Y to the Golgi complex, and stop growing after a dramatic accumulation of ER membranes and vesicles. We conclude that the SED5 gene is essential for growth and that Sed5p is required for ER to Golgi transport. When Sed5p is overexpressed the efficiency of ER to Golgi transport is reduced, vesicles accumulate, and cellular morphology is perturbed. Immunofluorescence studies reveal that the bulk of Sed5p is not found on ER membranes but on punctate structures throughout the cytoplasm, the number of which increases upon SED5 overexpression. We suggest that Sed5p has an essential role in vesicular transport between ER and Golgi compartments and that it may itself cycle between these organelles.

[1]  George Palade,et al.  Intracellular Aspects of the Process of Protein Synthesis , 1975, Science.

[2]  S. Ferro-Novick,et al.  Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection , 1987, The Journal of cell biology.

[3]  D. Botstein,et al.  ADP-ribosylation factor is functionally and physically associated with the Golgi complex. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[4]  R. Higuchi.  Using PCR to Engineer DNA , 1989 .

[5]  G. Fink,et al.  Methods in yeast genetics , 1979 .

[6]  R. Schekman,et al.  Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway , 1980, Cell.

[7]  S. Munro,et al.  A C-terminal signal prevents secretion of luminal ER proteins , 1987, Cell.

[8]  R. Schekman,et al.  Sec12p-dependent membrane binding of the small GTP-binding protein Sar1p promotes formation of transport vesicles from the ER , 1991, The Journal of cell biology.

[9]  G K Lewis,et al.  Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product , 1985, Molecular and cellular biology.

[10]  S. Ferro-Novick,et al.  The BOS1 gene encodes an essential 27-kD putative membrane protein that is required for vesicular transport from the ER to the Golgi complex in yeast , 1991, The Journal of cell biology.

[11]  R. Schekman,et al.  A membrane glycoprotein, Sec12p, required for protein transport from the endoplasmic reticulum to the Golgi apparatus in yeast , 1988, The Journal of cell biology.

[12]  H. Pelham,et al.  The Saccharomyces cerevisiae SEC20 gene encodes a membrane glycoprotein which is sorted by the HDEL retrieval system. , 1992, The EMBO journal.

[13]  H. Pelham,et al.  Recycling of proteins from the Golgi compartment to the ER in yeast , 1990, The Journal of cell biology.

[14]  H. Pelham.  Evidence that luminal ER proteins are sorted from secreted proteins in a post‐ER compartment. , 1988, The EMBO journal.

[15]  R. Schekman,et al.  Order of events in the yeast secretory pathway , 1981, Cell.

[16]  M. Muramatsu,et al.  A novel GTP-binding protein, Sar1p, is involved in transport from the endoplasmic reticulum to the Golgi apparatus , 1989, The Journal of cell biology.

[17]  Henry A. Erlich,et al.  PCR Technology: Principles and Applications for DNA Amplification , 1989 .

[18]  D. Botstein,et al.  The yeast GTP-binding YPT1 protein and a mammalian counterpart are associated with the secretion machinery , 1988, Cell.

[19]  R. Schekman,et al.  Distinct biochemical requirements for the budding, targeting, and fusion of ER-derived transport vesicles , 1991, The Journal of cell biology.

[20]  H. Pelham,et al.  Sorting of soluble ER proteins in yeast. , 1988, The EMBO journal.

[21]  R. Schekman,et al.  Localization of components involved in protein transport and processing through the yeast Golgi apparatus , 1991, The Journal of cell biology.

[22]  H. Pelham.  Control of protein exit from the endoplasmic reticulum. , 1989, Annual review of cell biology.

[23]  K. Murata,et al.  Transformation of intact yeast cells treated with alkali cations. , 1984, Journal of bacteriology.

[24]  H. Pelham,et al.  The ERD2 gene determines the specificity of the luminal ER protein retention system , 1990, Cell.

[25]  C. Yanisch-Perron,et al.  Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. , 1985, Gene.

[26]  R. Doolittle,et al.  A simple method for displaying the hydropathic character of a protein. , 1982, Journal of molecular biology.

[27]  J. Rothman,et al.  Molecular dissection of the secretory pathway , 1992, Nature.

[28]  P. Robbins,et al.  Glycosylation in Saccharomyces cerevisiae: cloning and characterization of an alpha-1,2-mannosyltransferase structural gene. , 1992, Glycobiology.

[29]  J. Rothman,et al.  A multisubunit particle implicated in membrane fusion , 1992, The Journal of cell biology.

[30]  K. Becherer,et al.  Isolation and characterization of PEP3, a gene required for vacuolar biogenesis in Saccharomyces cerevisiae , 1991, Molecular and cellular biology.

[31]  R. Schekman,et al.  Functional compartments of the yeast Golgi apparatus are defined by the sec7 mutation. , 1989, The EMBO journal.

[32]  Jan C. Semenza,et al.  ERD2, a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway , 1990, Cell.

[33]  R. Scheller,et al.  Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. , 1992, Science.

[34]  H. Pelham,et al.  Genes that allow yeast cells to grow in the absence of the HDEL receptor. , 1992, The EMBO journal.