Recycling of the yeast v-SNARE Sec22p involves COPI-proteins and the ER transmembrane proteins Ufe1p and Sec20p

1998 ◽  
Vol 111 (11) ◽  
pp. 1507-1520 ◽  
Author(s):  
W. Ballensiefen ◽  
D. Ossipov ◽  
H.D. Schmitt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Immunofluorescence Microscopy ◽  
Transmembrane Proteins ◽  
Retrograde Transport ◽  
Subcellular Fractionation ◽  
Hybrid Protein ◽  
Wild Type ◽  
Alpha Factor ◽  
Membrane Compartments

Vesicle-specific SNAP receptors (v-SNAREs) are believed to cycle between consecutive membrane compartments. The v-SNARE Sec22(Sly2)p mediates the targeting of vesicles between endoplasmic reticulum (ER) and early Golgi of Saccharomyces cerevisiae. To analyze factors involved in targeting of Sec22(Sly2)p, an alpha-factor-tagged Sec22 protein (Sec22-alpha) was employed. Only on reaching the late Golgi, can alpha-factor be cleaved from this hybrid protein by Kex2p, a protease localized in this compartment. In wild-type cells Kex2p-cleavage is observed only when Sec22-alpha is greatly overproduced. Immunofluorescence microscopy and subcellular fractionation studies showed that Sec22-alpha is returned to the ER from the late Golgi (Kex2p) compartment. When Sec22-alpha is expressed in wild-type cells at levels comparable to the quantities of endogenous Sec22p, very little of this protein is cleaved by Kex2p. Efficient cleavage, however, occurs in mutants defective in the retrograde transport of different ER-resident proteins indicating that Sec22-alpha rapidly reaches the late Golgi of these cells. These mutants (sec20-1, sec21-1, sec27-1 and ufe1-1) reveal Golgi structures when stained for Sec22-alpha and do not show the ER-immunofluorescence observed in wild-type cells. These results show consistently that Sec22p recycles from the Golgi back to the ER and that this recycling involves retrograde COPI vesicles.

scholarly journals Yeast ER-Golgi v-SNAREs Bos1p and Bet1p differ in steady-state localization and targeting

1999 ◽  
Vol 112 (22) ◽  
pp. 4135-4142
Author(s):  
D. Ossipov ◽  
S. Schroder-Kohne ◽  
H.D. Schmitt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Cellular Localization ◽  
Immunofluorescence Microscopy ◽  
Cell Fractionation ◽  
Dependent Manner ◽  
Hybrid Proteins ◽  
Golgi Compartment ◽  
Alpha Factor ◽  
Golgi Protein

Vesicle specific SNAP receptors (v-SNAREs) Bos1p and Bet1p are involved in targeting of anterograde vesicles between the endoplasmic reticulum (ER) and early Golgi of Saccharomyces cerevisiae. To analyze factors that influence the targeting of these proteins, alpha-factor tagged versions of Bos1p and Bet1p were employed. The alpha-factor can be cleaved off by the Kex2p protease as soon as the hybrid proteins reach the late Golgi compartment. The data obtained by monitoring of Kex2p cleavage, by immunofluorescence microscopy and cell fractionation showed that Bos1-alpha and Bet1-alpha have different cellular localization and dynamics. Bos1-alpha is an ER protein, which recycles between the Golgi and the ER in COPI-dependent manner. Bet1-alpha is an early Golgi protein and it does not change its localization under conditions when other recycling Golgi proteins can be trapped in the ER.

Efficiency and diversity of protein localization by random signal sequences

1990 ◽  
Vol 10 (6) ◽  
pp. 3163-3173
Author(s):  
C A Kaiser ◽  
D Botstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Signal Peptide ◽  
Signal Sequence ◽  
Protein Localization ◽  
Random Signal ◽  
Perinuclear Region ◽  
Hybrid Protein ◽  
Random Sequences ◽  
Beta Galactosidase

Three randomly derived sequences that can substitute for the signal peptide of Saccharomyces cerevisiae invertase were tested for the efficiency with which they can translocate invertase or beta-galactosidase into the endoplasmic reticulum. The rate of translocation, as measured by glycosylation, was estimated in pulse-chase experiments to be less than 6 min. When fused to beta-galactosidase, these peptides, like the normal invertase signal sequence, direct the hybrid protein to a perinuclear region, consistent with localization to the endoplasmic reticulum. The diversity of function of random peptides was studied further by immunofluorescence localization of proteins fused to 28 random sequences: 4 directed the hybrid to the endoplasmic reticulum, 3 directed it to the mitochondria, and 1 directed it to the nucleus.

A deletion that includes the segment coding for the signal peptidase cleavage site delays release of Saccharomyces cerevisiae acid phosphatase from the endoplasmic reticulum

1986 ◽  
Vol 6 (2) ◽  
pp. 723-729
Author(s):  
R Haguenauer-Tsapis ◽  
M Nagy ◽  
A Ryter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Acid Phosphatase ◽  
Cleavage Site ◽  
Ultrastructural Localization ◽  
Signal Peptidase ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Sugar Chains ◽  
Pho5 Gene

We studied ultrastructural localization of acid phosphatase in derepressed Saccharomyces cerevisiae cells transformed with a multicopy plasmid carrying either the wild-type PHO5 gene or a PHO5 gene deleted in the region overlapping the signal peptidase cleavage site. Wild-type enzyme was located in the cell wall, as was 50% of the modified protein, which carried high-mannose-sugar chains. The remaining 50% of the protein was active and core glycosylated, and it accumulated in the endoplasmic reticulum cisternae. The signal peptide remained uncleaved in both forms. Cells expressing the modified protein exhibited an exaggerated endoplasmic reticulum with dilated lumen.

scholarly journals DAF1, a mutant gene affecting size control, pheromone arrest, and cell cycle kinetics of Saccharomyces cerevisiae.

1988 ◽  
Vol 8 (11) ◽  
pp. 4675-4684 ◽  
Author(s):  
F R Cross
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Critical Size ◽  
Size Control ◽  
Mutant Gene ◽  
G1 Phase ◽  
Wild Type ◽  
Alpha Factor ◽  
Multiple Copies ◽  
Kinetics Of

The mating pheromone alpha-factor arrests Saccharomyces cerevisiae MATa cells in the G1 phase of the cell cycle. Size control is also exerted in G1, since cells do not exit G1 until they have attained a critical size. A dominant mutation (DAF1-1) which causes both alpha-factor resistance and small cell size (volume about 0.6-fold that of the wild type) has been isolated and characterized genetically and by molecular cloning. Several alpha-factor-induced mRNAs were induced equivalently in daf1+ and DAF1-1 cells. The DAF1-1 mutation consisted of a termination codon two-thirds of the way through the daf1+ coding sequence. A chromosomal deletion of DAF1 produced by gene transplacement increased cell volume about 1.5-fold; thus, DAF1-1 may be a hyperactive or deregulated allele of a nonessential gene involved in G1 size control. Multiple copies of DAF1-1 also greatly reduced the duration of the G1 phase of the cell cycle.

Expression of a mammalian Na+/H+ antiporter in Saccharomyces cerevisiae

1999 ◽  
Vol 77 (1) ◽  
pp. 25-31 ◽  
Author(s):  
Mónica Montero-Lomelí ◽  
Anna L Okorokova Façanha.
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Subcellular Fractionation ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Coding Region ◽  
Phosphatidylcholine Liposomes ◽  
Yeast Transformants ◽  
Wild Type Yeast ◽  
Membrane Region

The basolateral Na+/H+ antiporter (NHE) from LLC-PK1 cells was expressed in Saccharomyces cerevisiae. Two different strategies were tested for expression. In the first, we used a yeast strain that contains a temperature-sensitive mutation in the SEC-6 gene, whose product is required for the fusion of secretory vesicles with the plasma membrane. This strain was transformed with a vector containing the coding region of the NHE1 isoform under control of a heat shock (HS) promoter (pYNHE1-HS). In the second strategy, we replaced the heat shock promoter from pYNHE1-HS with a galactose (GAL) promoter (pYNHEI-GAL) and transformed wild-type yeast. In both cases, Northern blots demonstrated a transcript that hybridized against a probe containing the membrane region of the exchanger. When an antibody against the last 40 amino acids of the carboxy-terminus of NHE1 was used for immuno-blots, a protein with a Mr of 73 000 was seen in total membranes from both yeast transformants. Subcellular fractionation revealed that NHE1 was expressed in the endoplasmic reticulum. In the case of the pYNHEI-GAL transformant, the 100 000 × g membrane pellet was reconstituted in phosphatidylcholine liposomes, and ethylisopropyl-amiloride-sensitive Na+/H+ exchange was observed. These results have paved the way for expression of the Na+/H+ exchanger in a genetically well-known microorganism.Key words: Na+/H+ exchanger, NHE1, expression, yeast.

scholarly journals Cotranslational Intersection between the SRP and GET Targeting Pathways to the Endoplasmic Reticulum of Saccharomyces cerevisiae

2016 ◽  
Vol 36 (18) ◽  
pp. 2374-2383 ◽  
Author(s):  
Ying Zhang ◽  
Thea Schäffer ◽  
Tina Wölfle ◽  
Edith Fitzke ◽  
Gerhard Thiel ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Transmembrane Domain ◽  
Signal Recognition Particle ◽  
Transmembrane Proteins ◽  
Signal Recognition ◽  
Ribosome Binding ◽  
Channel Proteins ◽  
Membrane Spanning ◽  
Combined Data

Targeting of transmembrane proteins to the endoplasmic reticulum (ER) proceeds via either the signal recognition particle (SRP) or the guided entry of tail-anchored proteins (GET) pathway, consisting of Get1 to -5 and Sgt2. While SRP cotranslationally targets membrane proteins containing one or multiple transmembrane domains, the GET pathway posttranslationally targets proteins containing a single C-terminal transmembrane domain termed the tail anchor. Here, we dissect the roles of the SRP and GET pathways in the sorting of homologous, two-membrane-spanning K+channel proteins termed Kcv, Kesv, and Kesv-VV. We show that Kcv is targeted to the ER cotranslationally via its N-terminal transmembrane domain, while Kesv-VV is targeted posttranslationally via its C-terminal transmembrane domain, which recruits Get4-5/Sgt2 and Get3. Unexpectedly, nascent Kcv recruited not only SRP but also the Get4-5 module of the GET pathway to ribosomes. Ribosome binding of Get4-5 was independent of Sgt2 and was strongly outcompeted by SRP. The combined data indicate a previously unrecognized cotranslational interplay between the SRP and GET pathways.

scholarly journals Emp47p and Its Close Homolog Emp46p Have a Tyrosine-containing Endoplasmic Reticulum Exit Signal and Function in Glycoprotein Secretion in Saccharomyces cerevisiae

2002 ◽  
Vol 13 (7) ◽  
pp. 2518-2532 ◽  
Author(s):  
Ken Sato ◽  
Akihiko Nakano
Keyword(s):  
Endoplasmic Reticulum ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Retrograde Transport ◽  
Subcellular Fractionation ◽  
Terminal Region ◽  
Growth Defect ◽  
Critical Determinant ◽  
Reading Frame ◽  
Glycoprotein Secretion

The yeast open reading frame YLR080w/EMP46 encodes a homolog of the Golgi protein Emp47p. These two proteins are 45% identical and have a single transmembrane domain in their C-terminal regions and a carbohydrate recognition domain signature in the N-terminal region. The C-terminal tail of Emp46p includes a dilysine signal. This protein is localized to Golgi membranes at steady state by subcellular fractionation and green fluorescent protein labeling. On block of forward transport in sec12-4 cells, redistribution of Emp46p from the Golgi to the endoplasmic reticulum is observed. These localization features are similar to those previously reported for Emp47p. In addition, mutagenesis of the C-terminal region identified a tyrosine-containing motif as a critical determinant of the Golgi-localization and interaction with both COPI and COPII components. Similar motifs are also observed in the C-terminal tail of Emp47p and other mammalian homologs. Disruption of Emp47p displays a growth defect at a high temperature or on Ca2+-containing medium, which is rescued by overexpression of Emp46p, suggesting a partially overlapping function between Emp46p and Emp47p. In addition, we found that the disruption of both Emp46p and Emp47p show a marked defect in the secretion of a subset of glycoproteins. Analysis of the C-terminal mutants for Ca2+ sensitivity revealed that the forward transport of Emp46/47p is essential for their function, whereas the retrograde transport is not. We propose that Emp46p and Emp47p are required for the export of specific glycoprotein cargo from the endoplasmic reticulum.

scholarly journals Ascospore Formation in the Yeast Saccharomyces cerevisiae

2005 ◽  
Vol 69 (4) ◽  
pp. 565-584 ◽  
Author(s):  
Aaron M. Neiman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membranes ◽  
Secretory Pathway ◽  
Diploid Cell ◽  
Spore Wall ◽  
Second Phase ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Membrane Compartments ◽  
Two Phases

SUMMARY Sporulation of the baker's yeast Saccharomyces cerevisiae is a response to nutrient depletion that allows a single diploid cell to give rise to four stress-resistant haploid spores. The formation of these spores requires a coordinated reorganization of cellular architecture. The construction of the spores can be broadly divided into two phases. The first is the generation of new membrane compartments within the cell cytoplasm that ultimately give rise to the spore plasma membranes. Proper assembly and growth of these membranes require modification of aspects of the constitutive secretory pathway and cytoskeleton by sporulation-specific functions. In the second phase, each immature spore becomes surrounded by a multilaminar spore wall that provides resistance to environmental stresses. This review focuses on our current understanding of the cellular rearrangements and the genes required in each of these phases to give rise to a wild-type spore.

scholarly journals Retrograde Traffic Out of the Yeast Vacuole to the TGN Occurs via the Prevacuolar/Endosomal Compartment

1998 ◽  
Vol 142 (3) ◽  
pp. 651-663 ◽  
Author(s):  
Nia J. Bryant ◽  
Robert C. Piper ◽  
Lois S. Weisman ◽  
Tom H. Stevens
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Alkaline Phosphatase ◽  
Amino Acid ◽  
Retrograde Transport ◽  
Carboxypeptidase Y ◽  
Hybrid Protein ◽  
Yeast Saccharomyces Cerevisiae ◽  
Endosomal Compartment ◽  
Sorting Motif ◽  
Retrograde Traffic

A large number of trafficking steps occur between the last compartment of the Golgi apparatus (TGN) and the vacuole of the yeast Saccharomyces cerevisiae. To date, two intracellular routes from the TGN to the vacuole have been identified. Carboxypeptidase Y (CPY) travels through a prevacuolar/endosomal compartment (PVC), and subsequently on to the vacuole, while alkaline phosphatase (ALP) bypasses this compartment to reach the same organelle. Proteins resident to the TGN achieve their localization despite a continuous flux of traffic by continually being retrieved from the distal PVC by virtue of an aromatic amino acid–containing sorting motif. In this study we report that a hybrid protein based on ALP and containing this retrieval motif reaches the PVC not by following the CPY sorting pathway, but instead by signal-dependent retrograde transport from the vacuole, an organelle previously thought of as a terminal compartment. In addition, we show that a mutation in VAC7, a gene previously identified as being required for vacuolar inheritance, blocks this trafficking step. Finally we show that Vti1p, a v-SNARE required for the delivery of both CPY and ALP to the vacuole, uses retrograde transport out of the vacuole as part of its normal cellular itinerary.

scholarly journals tRNA thiolation links translation to stress responses in Saccharomyces cerevisiae

2015 ◽  
Vol 26 (2) ◽  
pp. 270-282 ◽  
Author(s):  
Jadyn R. Damon ◽  
David Pincus ◽  
Hidde L. Ploegh
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Elevated Temperature ◽  
Heat Shock ◽  
Stress Responses ◽  
Wild Type ◽  
Trna Modifications ◽  
Shock Response ◽  
Tightly Coupled

Although tRNA modifications have been well catalogued, the precise functions of many modifications and their roles in mediating gene expression are still being elucidated. Whereas tRNA modifications were long assumed to be constitutive, it is now apparent that the modification status of tRNAs changes in response to different environmental conditions. The URM1 pathway is required for thiolation of the cytoplasmic tRNAs tGluUUC, tGlnUUG, and tLysUUU in Saccharomyces cerevisiae. We demonstrate that URM1 pathway mutants have impaired translation, which results in increased basal activation of the Hsf1-mediated heat shock response; we also find that tRNA thiolation levels in wild-type cells decrease when cells are grown at elevated temperature. We show that defects in tRNA thiolation can be conditionally advantageous, conferring resistance to endoplasmic reticulum stress. URM1 pathway proteins are unstable and hence are more sensitive to changes in the translational capacity of cells, which is decreased in cells experiencing stresses. We propose a model in which a stress-induced decrease in translation results in decreased levels of URM1 pathway components, which results in decreased tRNA thiolation levels, which further serves to decrease translation. This mechanism ensures that tRNA thiolation and translation are tightly coupled and coregulated according to need.

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