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

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.

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

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.

scholarly journals Two Endoplasmic Reticulum (ER) Membrane Proteins That Facilitate ER-to-Golgi Transport of Glycosylphosphatidylinositol-anchored Proteins

1999 ◽  
Vol 10 (4) ◽  
pp. 1043-1059 ◽  
Author(s):  
Wolfgang P. Barz ◽  
Peter Walter
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Protein Translocation ◽  
Sequence Similarity ◽  
Surface Proteins ◽  
Golgi Transport ◽  
Er Membrane

Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI). GPI anchors are covalently attached in the endoplasmic reticulum (ER). The modified proteins are then transported through the secretory pathway to the cell surface. We have identified two genes inSaccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for “delayed GPI-anchored protein transport”), encoding structurally related proteins with multiple membrane-spanning domains. Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy. Deletion of either gene caused no detectable phenotype, whereas lag1Δ dgt1Δ cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting thatLAG1 and DGT1 encode functionally redundant or overlapping proteins. The rate of GPI anchor attachment was not affected, nor was the transport rate of several non–GPI-anchored proteins. Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Δ dgt1Δ cells deposit abnormal, multilayered cell walls. Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane. In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Δ dgt1Δcells, suggesting that neither yeast gene plays a role in this process. Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins.

scholarly journals Reconstitution of GTP-binding Sar1 protein function in ER to Golgi transport.

1991 ◽  
Vol 114 (4) ◽  
pp. 671-679 ◽  
Author(s):  
T Oka ◽  
S Nishikawa ◽  
A Nakano
Keyword(s):  
Temperature Sensitivity ◽  
Protein Function ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Temperature Sensitive ◽  
Transport Reaction ◽  
Golgi Transport ◽  
Gtp Binding

In the yeast secretory pathway, two genes SEC12 and SAR1, which encode a 70-kD integral membrane protein and a 21-kD GTP-binding protein, respectively, cooperate in protein transport from the ER to the Golgi apparatus. In vivo, the elevation of the SAR1 dosage suppresses temperature sensitivity of the sec12 mutant. In this paper, we show cell-free reconstitution of the ER-to-Golgi transport that depends on both of these gene products. First, the membranes from the sec12 mutant cells reproduce temperature sensitivity in the in vitro ER-to-Golgi transport reaction. Furthermore, the addition of the Sar1 protein completely suppresses this temperature-sensitive defect of the sec12 membranes. The analysis of Sar1p partially purified by E. coli expression suggests that GTP hydrolysis is essential for Sar1p to execute its function.

scholarly journals Erg30, a Vap-33–Related Protein, Functions in Protein Transport Mediated by Copi Vesicles

1999 ◽  
Vol 146 (2) ◽  
pp. 301-312 ◽  
Author(s):  
Lior Soussan ◽  
Darya Burakov ◽  
Mathew P. Daniels ◽  
Mira Toister-Achituv ◽  
Amir Porat ◽  
...  
Keyword(s):  
Intracellular Transport ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Structural Characteristics ◽  
Coiled Coil ◽  
Integral Membrane Protein ◽  
Type Ii ◽  
Golgi Transport ◽  
Protein Functions

Intracellular transport of newly synthesized and mature proteins via vesicles is controlled by a large group of proteins. Here we describe a ubiquitous rat protein—endoplasmic reticulum (ER) and Golgi 30-kD protein (ERG30)—which shares structural characteristics with VAP-33, a 33-kD protein from Aplysia californica which was shown to interact with the synaptic protein VAMP. The transmembrane topology of the 30-kD ERG30 corresponds to a type II integral membrane protein, whose cytoplasmic NH2 terminus contains a predicted coiled-coil motif. We localized ERG30 to the ER and to pre-Golgi intermediates by biochemical and immunocytochemical methods. Consistent with a role in vesicular transport, anti-ERG30 antibodies specifically inhibit intra-Golgi transport in vitro, leading to significant accumulation of COPI-coated vesicles. It appears that ERG30 functions early in the secretory pathway, probably within the Golgi and between the Golgi and the ER.

scholarly journals Inhibition of endoplasmic reticulum (ER)-to-Golgi transport induces relocalization of binding protein (BiP) within the ER to form the BiP bodies.

1994 ◽  
Vol 5 (10) ◽  
pp. 1129-1143 ◽  
Author(s):  
S Nishikawa ◽  
A Hirata ◽  
A Nakano
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Binding Protein ◽  
Yeast Cells ◽  
Restrictive Temperature ◽  
Golgi Transport ◽  
Coa Reductase ◽  
Vacuolar Protein ◽  
Er Membrane

Immunofluorescence staining of yeast cells with anti-binding protein (BiP) antibodies shows uniform staining of the endoplasmic reticulum (ER). We have found that overproduction of Sec12p, an ER membrane protein, causes a change of BiP distribution within the cell. Upon induction of Sec12p by the GAL1 promoter, the staining pattern of BiP turns into bright dots scattering in the cell, whereas the staining of Sec12p remains to be the typical ER figure. Overproduction of other ER membrane proteins, HMG-CoA reductase or Sed4 protein, does not induce such relocalization of BiP. Pulse-chase experiments and electron microscopy have revealed that the overproduction of Sec12p inhibits protein transport from the ER to the Golgi apparatus. When the transport is arrested by one of the sec mutations that block the ER-to-Golgi step at the restrictive temperature, the BiP staining also changes into the punctate pattern. In contrast, the sec mutants that block later or earlier steps of the secretory pathway do not induce such change of BiP localization. These observations indicate that relocalization of BiP is caused by the inhibition of ER-to-Golgi transport. Using immunoelectron microscopy, we have found that the punctate staining is because of the accumulation of BiP in the restricted region of the ER, which we propose to call the "BiP body." This implicates existence of ER subdomains in yeast. A vacuolar protein, proteinase A, appears to colocalize in the BiP body when the ER-to-Golgi transport is blocked, suggesting that the BiP body may have a role as the site of accumulation of cargo molecules before exit from the ER.

scholarly journals Suppression of Coatomer Mutants by a New Protein Family with COPI and COPII Binding Motifs in Saccharomyces cerevisiae

2003 ◽  
Vol 14 (8) ◽  
pp. 3097-3113 ◽  
Author(s):  
Thomas Sandmann ◽  
Johannes M. Herrmann ◽  
Jörn Dengjel ◽  
Heinz Schwarz ◽  
Anne Spang
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Trafficking ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Integral Membrane Protein ◽  
Eukaryotic Cell ◽  
Vesicle Formation ◽  
Binding Motifs ◽  
Early Secretory Pathway ◽  
Direct Binding

Protein trafficking is achieved by a bidirectional vesicle flow between the various compartments of the eukaryotic cell. COPII coated vesicles mediate anterograde protein transport from the endoplasmic reticulum to the Golgi apparatus, whereas retrograde Golgi-to-endoplasmic reticulum vesicles use the COPI coat. Inactivation of COPI vesicle formation in conditional sec21 (γ-COP) mutants rapidly blocks transport of certain proteins along the early secretory pathway. We have identified the integral membrane protein Mst27p as a strong suppressor of sec21-3 and ret1-1 mutants. A C-terminal KKXX motif of Mst27p that allows direct binding to the COPI complex is crucial for its suppression ability. Mst27p and its homolog Yar033w (Mst28p) are part of the same complex. Both proteins contain cytoplasmic exposed C termini that have the ability to interact directly with COPI and COPII coat complexes. Site-specific mutations of the COPI binding domain abolished suppression of the sec21 mutants. Our results indicate that overexpression of MST27 provides an increased number of coat binding sites on membranes of the early secretory pathway and thereby promotes vesicle formation. As a consequence, the amount of cargo that can bind COPI might be important for the regulation of the vesicle flow in the early secretory pathway.

scholarly journals The unfolded protein response coordinates the production of endoplasmic reticulum protein and endoplasmic reticulum membrane.

1997 ◽  
Vol 8 (9) ◽  
pp. 1805-1814 ◽  
Author(s):  
J S Cox ◽  
R E Chapman ◽  
P Walter
Keyword(s):  
Endoplasmic Reticulum ◽  
Unfolded Protein Response ◽  
Secretory Pathway ◽  
Lipid Synthesis ◽  
Secretory Proteins ◽  
Endoplasmic Reticulum Membrane ◽  
Yeast Saccharomyces Cerevisiae ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

The endoplasmic reticulum (ER) is a multifunctional organelle responsible for production of both lumenal and membrane components of secretory pathway compartments. Secretory proteins are folded, processed, and sorted in the ER lumen and lipid synthesis occurs on the ER membrane itself. In the yeast Saccharomyces cerevisiae, synthesis of ER components is highly regulated: the ER-resident proteins by the unfolded protein response and membrane lipid synthesis by the inositol response. We demonstrate that these two responses are intimately linked, forming different branches of the same pathway. Furthermore, we present evidence indicating that this coordinate regulation plays a role in ER biogenesis.

scholarly journals A High Copy Suppressor Screen Reveals Genetic Interactions Between BET3 and a New Gene: Evidence for a Novel Complex in ER-to-Golgi Transport

Genetics ◽  
1998 ◽  
Vol 149 (2) ◽  
pp. 833-841
Author(s):  
Yu Jiang ◽  
Al Scarpa ◽  
Li Zhang ◽  
Shelly Stone ◽  
Ed Feliciano ◽  
...  
Keyword(s):  
Growth Defect ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Specific Suppression ◽  
Golgi Transport ◽  
New Gene ◽  
Insight Into ◽  
Suppressor Screen

Abstract The BET3 gene in the yeast Saccharomyces cerevisiae encodes a 22-kD hydrophilic protein that is required for vesicular transport between the ER and Golgi complex. To gain insight into the role of Bet3p, we screened for genes that suppress the growth defect of the temperature-sensitive bet3 mutant at 34°. This high copy suppressor screen resulted in the isolation of a new gene, called BET5. BET5 encodes an essential 18-kD hydrophilic protein that in high copy allows growth of the bet3-1 mutant, but not other ER accumulating mutants. This strong and specific suppression is consistent with the fact that Bet3p and Bet5p are members of the same complex. Using PCR mutagenesis, we generated a temperature-sensitive mutation in BET5 (bet5-1) that blocks the transport of carboxypeptidase Y to the vacuole and prevents secretion of the yeast pheromone α-factor at 37°. The precursor forms of these proteins that accumulate in this mutant are indicative of a block in membrane traffic between the ER and Golgi apparatus. High copy suppressors of the bet5-1 mutant include several genes whose products are required for ER-to-Golgi transport (BET1, SEC22, USO1 and DSS4) and the maintenance of the Golgi (ANP1). These findings support the hypothesis that Bet5p acts in conjunction with Bet3p to mediate a late stage in ER-to-Golgi transport. The identification of mammalian homologues of Bet3p and Bet5p implies that the Bet3p/Bet5p complex is highly conserved in evolution.

scholarly journals Rab1b regulates vesicular transport between the endoplasmic reticulum and successive Golgi compartments.

1991 ◽  
Vol 115 (1) ◽  
pp. 31-43 ◽  
Author(s):  
H Plutner ◽  
A D Cox ◽  
S Pind ◽  
R Khosravi-Far ◽  
J R Bourne ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Essential Role ◽  
Secretory Pathway ◽  
Binding Protein ◽  
Vesicular Transport ◽  
Initial Step ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Golgi Compartment

We report an essential role for the ras-related small GTP-binding protein rab1b in vesicular transport in mammalian cells. mAbs detect rab1b in both the ER and Golgi compartments. Using an assay which reconstitutes transport between the ER and the cis-Golgi compartment, we find that rab1b is required during an initial step in export of protein from the ER. In addition, it is also required for transport of protein between successive cis- and medial-Golgi compartments. We suggest that rab1b may provide a common link between upstream and downstream components of the vesicular fission and fusion machinery functioning in early compartments of the secretory pathway.

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