Structure of the yeast endoplasmic reticulum: Localization of ER proteins using immunofluorescence and immunoelectron microscopy

The endoplasmic reticulum (ER) and other secretory compartments of Saccharomyces cerevisiae have biochemical functions that closely parallel those described in higher eukaryotic cells, yet the morphology of the yeast organelles is quite distinct. In order to associate ER functions with the corresponding cellular structures, we localized several proteins, each of which is expected to be associated with the ER on the basis of enzymatic activity, biological function, or oligosaccharide content. These marker proteins were visualized by immunofluorescence or immunoelectron microscopy, allowing definition of the S. cerevisiae ER structure, both in intact cells and at the ultrastructural level. Each marker protein was most abundant within the membranes that envelop the nucleus and several were also found in extensions of the ER that frequently juxtapose the plasma membrane. Double‐labeling experiments were entirely consistent with the idea that the marker proteins reside within the same compartment. This analysis has permitted, for the first time, a detailed characterization of the ER morphology as yeast cells proceed through their growth and division cycles.

[1]  W. Tanner,et al.  Localization of dolichyl phosphate- and pyrophosphate-dependent glycosyl transfer reactions in Saccharomyces cerevisiae , 1979, Journal of bacteriology.

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

[3]  B. Byers.  Cytology of the Yeast Life Cycle , 1981 .

[4]  M. Osumi,et al.  Transmission and scanning electron microscopic examination of intracellular organelles in freeze-substituted Kloeckera and Saccharomyces cerevisiae Yeast Cells , 1987 .

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

[6]  J. Sambrook,et al.  The Saccharomyces cerevisiae DPM1 gene encoding dolichol-phosphate-mannose synthase is able to complement a glycosylation-defective mammalian cell line , 1990, Molecular and cellular biology.

[7]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[8]  W. Tanner,et al.  The role of dolicholmonophosphate in glycoprotein biosynthesis in Saccharomyces cerevisiae. , 1974, European journal of biochemistry.

[9]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[10]  H. Lodish,et al.  Transport of secretory and membrane glycoproteins from the rough endoplasmic reticulum to the Golgi. A rate-limiting step in protein maturation and secretion. , 1988, The Journal of biological chemistry.

[11]  K Verner,et al.  Protein translocation across membranes. , 1988, Science.

[12]  M. Basson,et al.  Increased amounts of HMG-CoA reductase induce "karmellae": a proliferation of stacked membrane pairs surrounding the yeast nucleus , 1988, The Journal of cell biology.

[13]  G. Palade,et al.  THE ENDOPLASMIC RETICULUM , 1956, The Journal of biophysical and biochemical cytology.

[14]  G. Blobel,et al.  Secretory protein translocation in a yeast cell-free system can occur posttranslationally and requires ATP hydrolysis , 1986, The Journal of cell biology.

[15]  M. Rose,et al.  KAR2, a karyogamy gene, is the yeast homolog of the mammalian BiP/GRP78 gene , 1989, Cell.

[16]  C. Vorgias,et al.  The ras‐related mouse ypt1 protein can functionally replace the YPT1 gene product in yeast. , 1989, The EMBO journal.

[17]  G. Fink,et al.  The NUP1 gene encodes an essential component of the yeast nuclear pore complex , 1990, Cell.

[18]  J. Rine,et al.  Transmission electron microscopy and immunocytochemical studies of yeast: analysis of HMG-CoA reductase overproduction by electron microscopy. , 1989, Methods in cell biology.

[19]  R. Schekman,et al.  Localized secretion of acid phosphatase reflects the pattern of cell surface growth in saccharomyces cerevisiae , 1980, The Journal of cell biology.

[20]  J. Lampen,et al.  Tunicamycin--an inhibitor of yeast glycoprotein synthesis. , 1974, Biochemical and biophysical research communications.

[21]  P. Orlean,et al.  Cloning and sequencing of the yeast gene for dolichol phosphate mannose synthase, an essential protein. , 1988, The Journal of biological chemistry.

[22]  A. Claude,et al.  A STUDY OF TISSUE CULTURE CELLS BY ELECTRON MICROSCOPY , 1945, The Journal of experimental medicine.

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

[24]  J. Lampen,et al.  [76] β-d-Fructofuranoside fructohydrolase from yeast , 1975 .

[25]  R. Schekman.  Protein localization and membrane traffic in yeast. , 1985, Annual review of cell biology.

[26]  M. Wabl,et al.  Immunoglobulin heavy chain binding protein , 1983, Nature.

[27]  J. Sambrook,et al.  Correction of a defect in mammalian GPI anchor biosynthesis by a transfected yeast gene. , 1990, Science.

[28]  E. Hurt,et al.  A novel nucleoskeletal‐like protein located at the nuclear periphery is required for the life cycle of Saccharomyces cerevisiae. , 1988, The EMBO journal.

[29]  D. Meyer,et al.  Secretion in yeast: Reconstitution of the translocation and glycosylation of α-factor and invertase in a homologous cell-free system , 1986, Cell.

[30]  F. Képès,et al.  Genetic dissection of the early stages of protein secretion in yeast. , 1989, Trends in genetics : TIG.

[31]  S. Kornfeld,et al.  Assembly of asparagine-linked oligosaccharides. , 1985, Annual review of biochemistry.

[32]  W. Linnemans,et al.  Localization of acid phosphatase in protoplasts from Saccharomyces cerevisiae , 1975, Journal of bacteriology.

[33]  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.

[34]  P. Walter,et al.  In vitro protein translocation across the yeast endoplasmic reticulum: ATP-dependent post-translational translocation of the prepro-α-factor , 1986, Cell.

[35]  J. Sambrook,et al.  Expression of wild-type and mutant forms of influenza hemagglutinin: The role of folding in intracellular transport , 1986, Cell.

[36]  D. Botstein,et al.  Mutants of yeast defective in sucrose utilization. , 1981, Genetics.

[37]  R. Carrell,et al.  α1-Antitrypsin and the serpins: variation and countervariation , 1985, Trends in Biochemical Sciences.

[38]  M. Rose,et al.  Loss of BiP/GRP78 function blocks translocation of secretory proteins in yeast , 1990, The Journal of cell biology.

[39]  J. Rothman.  Polypeptide chain binding proteins: Catalysts of protein folding and related processes in cells , 1989, Cell.

[40]  K. Redding,et al.  Immunolocalization of Kex2 protease identifies a putative late Golgi compartment in the yeast Saccharomyces cerevisiae , 1991, The Journal of cell biology.

[41]  N. Ruderman,et al.  Electron microscopic and biochemical study of lipoprotein synthesis in the isolated perfused rat liver. , 1967, Journal of lipid research.

[42]  R. Doms,et al.  Regulation of protein export from the endoplasmic reticulum. , 1988, Annual review of cell biology.

[43]  L. Chen,et al.  Dynamic behavior of endoplasmic reticulum in living cells , 1988, Cell.

[44]  J. Broach,et al.  The Molecular biology of the yeast Saccharomyces : metabolism and gene expression , 1982 .

[45]  R. Schekman,et al.  A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum , 1987, The Journal of cell biology.

[46]  P. Silver,et al.  A yeast gene important for protein assembly into the endoplasmic reticulum and the nucleus has homology to DnaJ, an Escherichia coli heat shock protein , 1989, The Journal of cell biology.

[47]  L. Lehle,et al.  Protein glycosylation in yeast. , 1987, Antonie van Leeuwenhoek.

[48]  R. Schekman,et al.  Compartmentalized assembly of oligosaccharides on exported glycoproteins in yeast , 1981, Cell.

[49]  J. Sambrook,et al.  S. cerevisiae encodes an essential protein homologous in sequence and function to mammalian BiP , 1989, Cell.

[50]  R. Schekman,et al.  Invertase signal and mature sequence substitutions that delay intercompartmental transport of active enzyme , 1985, The Journal of cell biology.

[51]  R. Schekman,et al.  Invertase beta-galactosidase hybrid proteins fail to be transported from the endoplasmic reticulum in Saccharomyces cerevisiae , 1984, Molecular and cellular biology.

[52]  E. Chen,et al.  A fusion protein required for vesicle-mediated transport in both mammalian cells and yeast , 1989, Nature.

[53]  W. Lennarz,et al.  Localization of the enzyme system for glycosylation of proteins via the lipid-linked pathway in rough endoplasmic reticulum. , 1977, The Journal of biological chemistry.

[54]  G. Fink,et al.  Laboratory course manual for methods in yeast genetics , 1986 .

[55]  Catherine A. Lee,et al.  Genetic studies on the inability of beta-galactosidase to be translocated across the Escherichia coli cytoplasmic membrane , 1989, Journal of bacteriology.

[56]  J. Rothman,et al.  SNAPs, a family of NSF attachment proteins involved in intracellular membrane fusion in animals and yeast , 1990, Cell.

[57]  N. Walworth,et al.  A GTP-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast , 1988, Cell.