Location of sequences within rotavirus SA11 glycoprotein VP7 which direct it to the endoplasmic reticulum

The Simian 11 rotavirus glycoprotein VP7 is directed to the endoplasmic reticulum (ER) of the cell and retained as an integral membrane protein. The gene coding for VP7 predicts two potential initiation codons, each of which precedes a hydrophobic region of amino acids (H1 and H2) with the characteristics of a signal peptide. Using the techniques of gene mutagenesis and expression, we have determined that either hydrophobic domain alone can direct VP7 to the ER. A protein lacking both hydrophobic regions was not transported to the ER. Some polypeptides were directed across the ER membrane and then into the secretory pathway of the cell. For a variant retaining only the H1 domain, secretion was cleavage dependent, since an amino acid change which prevented cleavage also stopped secretion. However, secretion of two other deletion mutants lacking H1 and expressing truncated H2 domains was unaffected by this mutation, suggesting that these proteins were secreted without cleavage of their NH2-terminal hydrophobic regions or secreted after cleavage at a site(s) not predicted by current knowledge.

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The yeast SLY gene products, suppressors of defects in the essential GTP-binding Ypt1 protein, may act in endoplasmic reticulum-to-Golgi transport

Molecular and Cellular Biology ◽

10.1128/mcb.11.6.2980-2993.1991 ◽

1991 ◽

Vol 11 (6) ◽

pp. 2980-2993

Author(s):

R Ossig ◽

C Dascher ◽

H H Trepte ◽

H D Schmitt ◽

D Gallwitz

Keyword(s):

Endoplasmic Reticulum ◽

Secretory Pathway ◽

Protein Transport ◽

Single Copy ◽

Integral Membrane Protein ◽

Carboxypeptidase Y ◽

Null Mutant ◽

Yeast Saccharomyces Cerevisiae ◽

Golgi Transport ◽

Gtp Binding

It has been shown previously that defects in the essential GTP-binding protein, Ypt1p, lead to a block in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus in the yeast Saccharomyces cerevisiae. Here we report that four newly discovered suppressors of YPT1 deletion (SLY1-20, SLY2, SLY12, and SLY41) to a varying degree restore ER-to-Golgi transport defects in cells lacking Ypt1p. These suppressors also partially complement the sec21-1 and sec22-3 mutants which lead to a defect early in the secretory pathway. Sly1p-depleted cells, as well as a conditional lethal sly2 null mutant at nonpermissive temperatures, accumulate ER membranes and core-glycosylated invertase and carboxypeptidase Y. The sly2 null mutant under restrictive conditions (37 degrees C) can be rescued by the multicopy suppressor SLY12 and the single-copy suppressor SLY1-20, indicating that these three SLY genes functionally interact. Sly2p is shown to be an integral membrane protein.

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Deletions into an NH2-terminal hydrophobic domain result in secretion of rotavirus VP7, a resident endoplasmic reticulum membrane glycoprotein.

The Journal of Cell Biology ◽

10.1083/jcb.101.6.2199 ◽

1985 ◽

Vol 101 (6) ◽

pp. 2199-2209 ◽

Cited By ~ 58

Author(s):

M S Poruchynsky ◽

C Tyndall ◽

G W Both ◽

F Sato ◽

A R Bellamy ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Integral Membrane Protein ◽

Endoplasmic Reticulum Membrane ◽

Wild Type ◽

Positive Signal ◽

Complex Type ◽

Immunofluorescent Localization ◽

Hydrophobic Domain ◽

Rotavirus A ◽

Altered Protein

Rotavirus, a non-enveloped reovirus, buds into the rough endoplasmic reticulum and transiently acquires a membrane. The structural glycoprotein, VP7, a 38-kD integral membrane protein of the endoplasmic reticulum (ER), presumably transfers to virus in this process. The gene for VP7 potentially encodes a protein of 326 amino acids which has two tandem hydrophobic domains at the NH2-terminal, each preceded by an in-frame ATG codon. A series of deletion mutants constructed from a full-length cDNA clone of the Simian 11 rotavirus VP7 gene were expressed in COS 7 cells. Products from wild-type, and mutants which did not affect the second hydrophobic domain of VP7, were localized by immunofluorescence to elements of the ER only. However, deletions affecting the second hydrophobic domain (mutants 42-61, 43-61, 47-61) showed immunofluorescent localization of VP7 which coincided with that of wheat germ agglutinin, indicating transport to the Golgi apparatus. Immunoprecipitable wild-type protein, or an altered protein lacking the first hydrophobic sequence, remained intracellular and endo-beta-N-acetylglucosaminidase H sensitive. In contrast, products of mutants 42-61, 43-61, and 47-61 were transported from the ER, and secreted. Glycosylation of the secreted molecules was inhibited by tunicamycin, resistant to endo-beta-N-acetylglucosaminidase H digestion and therefore of the N-linked complex type. An unglycosylated version of VP7 was also secreted. We suggest that the second hydrophobic domain contributes to a positive signal for ER location and a membrane anchor function. Secretion of the mutant glycoprotein implies that transport can be constitutive with the destination being dictated by an overriding compartmentalization signal.

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Erv14p Directs a Transmembrane Secretory Protein into COPII-coated Transport Vesicles

Molecular Biology of the Cell ◽

10.1091/mbc.01-10-0499 ◽

2002 ◽

Vol 13 (3) ◽

pp. 880-891 ◽

Cited By ~ 81

Author(s):

Jacqueline Powers ◽

Charles Barlowe

Keyword(s):

Endoplasmic Reticulum ◽

Secretory Pathway ◽

Integral Membrane Protein ◽

Secretory Protein ◽

Conserved Residues ◽

Early Secretory Pathway ◽

Transport Vesicles ◽

Copii Vesicle ◽

Copii Vesicles ◽

Selection Of

Erv14p is a conserved integral membrane protein that traffics in COPII-coated vesicles and localizes to the early secretory pathway in yeast. Deletion of ERV14 causes a defect in polarized growth because Axl2p, a transmembrane secretory protein, accumulates in the endoplasmic reticulum and is not delivered to its site of function on the cell surface. Herein, we show that Erv14p is required for selection of Axl2p into COPII vesicles and for efficient formation of these vesicles. Erv14p binds to subunits of the COPII coat and binding depends on conserved residues in a cytoplasmically exposed loop domain of Erv14p. When mutations are introduced into this loop, an Erv14p-Axl2p complex accumulates in the endoplasmic reticulum, suggesting that Erv14p links Axl2p to the COPII coat. Based on these results and further genetic experiments, we propose Erv14p coordinates COPII vesicle formation with incorporation of specific secretory cargo.

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Structure, biosynthesis, and localization of dipeptidyl aminopeptidase B, an integral membrane glycoprotein of the yeast vacuole.

The Journal of Cell Biology ◽

10.1083/jcb.108.4.1363 ◽

1989 ◽

Vol 108 (4) ◽

pp. 1363-1373 ◽

Cited By ~ 100

Author(s):

C J Roberts ◽

G Pohlig ◽

J H Rothman ◽

T H Stevens

Keyword(s):

Membrane Protein ◽

Secretory Pathway ◽

Apparent Molecular Weight ◽

Integral Membrane Protein ◽

Restrictive Temperature ◽

Proteolytic Activation ◽

Hydrophobic Domain ◽

Yeast Vacuole ◽

Dipeptidyl Aminopeptidase

We have characterized the structure, biogenesis, and localization of dipeptidyl aminopeptidase B (DPAP B), a membrane protein of the yeast vacuole. An antibody specific for DPAP B recognizes a 120-kD glycoprotein in yeast that behaves like an integral membrane protein in that it is not removed from membranes by high pH Na2CO3 treatment. Inspection of the deduced amino acid sequence of DPAP B reveals a hydrophobic domain near the NH2 terminus that could potentially span a lipid bilayer. The in vitro enzymatic activity and apparent molecular weight of DPAP B are unaffected by the allelic state of PEP4, a gene essential for the proteolytic activation of a number of soluble vacuolar hydrolases. DPAP B is synthesized as a glycosylated precursor that is converted to the mature 120-kD species by carbohydrate addition. The precursor form of DPAP B accumulates in sec mutants (Novick, P., C. Field, and R. Schekman. 1980. Cell. 21:205-215) that are blocked at the ER (sec18) or Golgi apparatus (sec7), but not at secretory vesicles (sec1). Immunolocalization of DPAP B in wild-type or sec1 mutant cells shows that the protein resides in the vacuolar membrane. However, it is present in non-vacuolar compartments in sec18 and sec7 cells, confirming that the delivery of DPAP B is blocked in these mutants. Interestingly, DPAP B appears to stain the nuclear envelope in a sec18 mutant, which is consistent with the accumulation of DPAP B in the ER membrane at the restrictive temperature. These results suggest that soluble and membrane-bound vacuolar proteins use the same stages of the secretory pathway for their transport.

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The yeast SLY gene products, suppressors of defects in the essential GTP-binding Ypt1 protein, may act in endoplasmic reticulum-to-Golgi transport.

Molecular and Cellular Biology ◽

10.1128/mcb.11.6.2980 ◽

1991 ◽

Vol 11 (6) ◽

pp. 2980-2993 ◽

Cited By ~ 117

Author(s):

R Ossig ◽

C Dascher ◽

H H Trepte ◽

H D Schmitt ◽

D Gallwitz

Keyword(s):

Endoplasmic Reticulum ◽

Secretory Pathway ◽

Protein Transport ◽

Single Copy ◽

Integral Membrane Protein ◽

Carboxypeptidase Y ◽

Null Mutant ◽

Yeast Saccharomyces Cerevisiae ◽

Golgi Transport ◽

Gtp Binding

It has been shown previously that defects in the essential GTP-binding protein, Ypt1p, lead to a block in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus in the yeast Saccharomyces cerevisiae. Here we report that four newly discovered suppressors of YPT1 deletion (SLY1-20, SLY2, SLY12, and SLY41) to a varying degree restore ER-to-Golgi transport defects in cells lacking Ypt1p. These suppressors also partially complement the sec21-1 and sec22-3 mutants which lead to a defect early in the secretory pathway. Sly1p-depleted cells, as well as a conditional lethal sly2 null mutant at nonpermissive temperatures, accumulate ER membranes and core-glycosylated invertase and carboxypeptidase Y. The sly2 null mutant under restrictive conditions (37 degrees C) can be rescued by the multicopy suppressor SLY12 and the single-copy suppressor SLY1-20, indicating that these three SLY genes functionally interact. Sly2p is shown to be an integral membrane protein.

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Yos1p Is a Novel Subunit of the Yip1p–Yif1p Complex and Is Required for Transport between the Endoplasmic Reticulum and the Golgi Complex

Molecular Biology of the Cell ◽

10.1091/mbc.e04-10-0873 ◽

2005 ◽

Vol 16 (4) ◽

pp. 1673-1683 ◽

Cited By ~ 31

Author(s):

Matthew Heidtman ◽

Catherine Z. Chen ◽

Ruth N. Collins ◽

Charles Barlowe

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Complex ◽

Secretory Pathway ◽

Integral Membrane Protein ◽

Rab Gtpases ◽

Reading Frame ◽

Multicopy Suppressor ◽

Transport Vesicles ◽

Identification And Characterization

Yeast Yip1p is a member of a conserved family of transmembrane proteins that interact with Rab GTPases. Previous studies also have indicated a role for Yip1p in the biogenesis of endoplasmic reticulum (ER)-derived COPII transport vesicles. In this report, we describe the identification and characterization of the uncharacterized open reading frame YER074W-A as a novel multicopy suppressor of the thermosensitive yip1-4 strain. We have termed this gene Yip One Suppressor 1 (YOS1). Yos1p is essential for growth and for function of the secretory pathway; depletion or inactivation of Yos1p blocks transport between the ER and the Golgi complex. YOS1 encodes an integral membrane protein of 87 amino acids that is conserved in eukaryotes. Yos1p localizes to ER and Golgi membranes and is efficiently packaged into ER-derived COPII transport vesicles. Yos1p associates with Yip1p and Yif1p, indicating Yos1p is a novel subunit of the Yip1p–Yif1p complex.

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Calnexin: a molecular chaperone with a taste for carbohydrate

Biochemistry and Cell Biology ◽

10.1139/o95-015 ◽

1995 ◽

Vol 73 (3-4) ◽

pp. 123-132 ◽

Cited By ~ 51

Author(s):

David B. Williams

Keyword(s):

Quality Control ◽

Endoplasmic Reticulum ◽

Molecular Chaperone ◽

Molecular Chaperones ◽

Secretory Pathway ◽

Quaternary Structure ◽

Integral Membrane Protein ◽

Secretory Proteins ◽

Chaperone Function ◽

Folded Proteins

Calnexin is an integral membrane protein of the endoplasmic reticulum (ER) that binds transiently to a wide array of newly synthesized membrane and secretory proteins. It also exhibits prolonged binding to misfolded or incompletely folded proteins. Recent studies have demonstrated that calnexin functions as a molecular chaperone to facilitate the folding and assembly of proteins in the ER. It is also a component of the quality control system that prevents proteins from progressing along the secretory pathway until they have acquired proper tertiary or quaternary structure. Most proteins that are translocated into the ER are glycosylated at Asn residues, and calnexin's interactions are almost exclusively restricted to proteins that possess this posttranslational modification. The preference for glycoproteins resides in calnexin's ability to function as a lectin with specificity for the GlC1Man9GlcNAc2 oligosaccharide, an early intermediate in the processing of Asn-linked oligosaccharides. Calnexin also has the capacity to bind to polypeptide segments of unfolded glycoproteins. Available evidence suggests that calnexin utilizes its lectin property during initial capture of a newly synthesized glycoprotein and that subsequent association (and chaperone function) is mediated through polypeptide interactions. Unlike other molecular chaperones that are soluble proteins, calnexin is an intrinsic component of the ER membrane. Its unique ability to capture unfolded glycoproteins through their large oligosaccharide moieties may have evolved as a means to overcome accessibility problems imposed by being constrained within a lipid bilayer.Key words: protein folding, molecular chaperones, calnexin, quality control, endoplasmic reticulum.

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Erf4p and Erf2p Form an Endoplasmic Reticulum-associated Complex Involved in the Plasma Membrane Localization of Yeast Ras Proteins

Journal of Biological Chemistry ◽

10.1074/jbc.m209760200 ◽

2002 ◽

Vol 277 (51) ◽

pp. 49352-49359 ◽

Cited By ~ 62

Author(s):

Lihong Zhao ◽

Sandra Lobo ◽

Xiangwen Dong ◽

Addison D. Ault ◽

Robert J. Deschenes

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Membrane Protein ◽

Secretory Pathway ◽

Integral Membrane Protein ◽

Genetic Screen ◽

Membrane Localization ◽

Ras Proteins ◽

Transport Pathway ◽

Plasma Membrane Localization

Ras oncogene proteins are plasma membrane-associated signal transducers that are found in all eukaryotes. Posttranslational addition of lipid to a carboxyl-terminal CaaXbox (where “C” represents a cysteine, “a” is generally an aliphatic residue, andXcan be any amino acid) is required to target Ras proteins to the cytosolic surface of the plasma membrane. The pathway by which Ras translocates from the endoplasmic reticulum to the plasma membrane is currently not clear. We have performed a genetic screen to identify components of the Ras plasma membrane localization pathway. Mutations in two genes,ERF2andERF4/SHR5, have been shown to affect the palmitoylation and subcellular localization of Ras proteins. In this report, we show that Erf4p is localized on the endoplasmic reticulum as a peripheral membrane protein in a complex with Erf2p, an integral membrane protein that was identified from the same genetic screen. Erf2p has been shown to be required for the plasma membrane localization of GFP-Ras2p via a pathway distinct from the classical secretory pathway (X. Dong and R. J. Deschenes, manuscript in preparation). We show here that Erf4p, like Erf2p, is involved in the plasma membrane localization of Ras2p. Erf2p and Erf4p represent components of a previously uncharacterized subcellular transport pathway involved in the plasma membrane targeting of Ras proteins.

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Assembly of the Yeast Vacuolar H+-ATPase Occurs in the Endoplasmic Reticulum and Requires a Vma12p/Vma22p Assembly Complex

The Journal of Cell Biology ◽

10.1083/jcb.142.1.39 ◽

1998 ◽

Vol 142 (1) ◽

pp. 39-49 ◽

Cited By ~ 70

Author(s):

Laurie A. Graham ◽

Kathryn J. Hill ◽

Tom H. Stevens

Keyword(s):

Endoplasmic Reticulum ◽

Protein Interactions ◽

Secretory Pathway ◽

Integral Membrane Protein ◽

Subcellular Fractionation ◽

Cross Linking ◽

Physical Interaction ◽

Protein Protein Interactions ◽

Atpase Subunit ◽

Mutant Cells

Three previously identified genes from Saccharomyces cerevisiae, VMA12, VMA21, and VMA22, encode proteins localized to the endoplasmic reticulum (ER). These three proteins are required for the biogenesis of a functional vacuolar ATPase (V-ATPase), but are not part of the final enzyme complex. Subcellular fractionation and chemical cross-linking studies have revealed that Vma12p and Vma22p form a stable membrane associated complex. Cross-linking analysis also revealed a direct physical interaction between the Vma12p/Vma22p assembly complex and Vph1p, the 100-kD integral membrane subunit of the V-ATPase. The interaction of the Vma12p/Vma22p complex with Vph1p was transient (half-life of ∼5 min), reflecting trafficking of this V-ATPase subunit through the ER en route to the vacuolar membrane. Analysis of these protein–protein interactions in ER-blocked sec12 mutant cells indicated that the Vph1p-Vma12p/Vma22p interactions are quite stable when transport of the V-ATPase out of the ER is blocked. Fractionation of solubilized membrane proteins on a density gradient revealed comigration of Vma22p and Vma12p, indicating that they form a complex even in the absence of cross-linker. Vma12p and Vma22p migrated to fractions separate from Vma21p. Loss of Vph1p caused the Vma12p/Vma22p complex to sediment to less dense fractions, consistent with association of Vma12p/ Vma22p with nascent Vph1p in ER membranes. This is the first evidence for a dedicated assembly complex in the ER required for the assembly of an integral membrane protein complex (V-ATPase) as it is transported through the secretory pathway.

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