Transport to the plasma membrane is regulated differently early and late in the cell cycle in Saccharomyces cerevisiae

Using the monoclonal antibody MA-01, which recognizes a 210-kDa protein in cell-free extracts, spindle and cytoplasmic microtubules were visualized in budding yeast, Saccharomyces cerevisiae. In additional, a spot-like staining was found beneath the plasma membrane, revealing in part correlation with F-actin distribution. This pattern was common for cells of all cell-cycle stages. The interaction of the protein recognized by MA-01 with microtubules was confirmed in the double labeling with a polyclonal antitubulin antibody and by the sensitivity of intranuclear structures stained by MA-01 to the microtubule disrupting drug nocodazole. Key words: immunoblotting, immunofluorescence, microtubule-interacting protein, Saccharomyces cerevisiae.

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Use of Bimolecular Fluorescence Complementation To Study In Vivo Interactions between Cdc42p and Rdi1p of Saccharomyces cerevisiae

Eukaryotic Cell ◽

10.1128/ec.00368-06 ◽

2007 ◽

Vol 6 (3) ◽

pp. 378-387 ◽

Cited By ~ 32

Author(s):

Karen C. Cole ◽

Heather W. McLaughlin ◽

Douglas I. Johnson

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Plasma Membrane ◽

Cell Cycle Progression ◽

Neck Region ◽

Bimolecular Fluorescence Complementation ◽

Phase Cell ◽

Fluorescence Complementation ◽

Bimolecular Fluorescence

ABSTRACT Saccharomyces cerevisiae Cdc42p functions as a GTPase molecular switch, activating multiple signaling pathways required to regulate cell cycle progression and the actin cytoskeleton. Regulatory proteins control its GTP binding and hydrolysis and its subcellular localization, ensuring that Cdc42p is appropriately activated and localized at sites of polarized growth during the cell cycle. One of these, the Rdi1p guanine nucleotide dissociation inhibitor, negatively regulates Cdc42p by extracting it from cellular membranes. In this study, the technique of bimolecular fluorescence complementation (BiFC) was used to study the dynamic in vivo interactions between Cdc42p and Rdi1p. The BiFC data indicated that Cdc42p and Rdi1p interacted in the cytoplasm and around the periphery of the cell at the plasma membrane and that this interaction was enhanced at sites of polarized cell growth during the cell cycle, i.e., incipient bud sites, tips and sides of small- and medium-sized buds, and the mother-bud neck region. In addition, a ring-like structure containing the Cdc42p-Rdi1p complex transiently appeared following release from G1-phase cell cycle arrest. A homology model of the Cdc42p-Rdi1p complex was used to introduce mutations that were predicted to affect complex formation. These mutations resulted in altered BiFC interactions, restricting the complex exclusively to either the plasma membrane or the cytoplasm. Data from these studies have facilitated the temporal and spatial modeling of Rdi1p-dependent extraction of Cdc42p from the plasma membrane during the cell cycle.

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Ability of Sit4p To Promote K+ Efflux via Nha1p Is Modulated by Sap155p and Sap185p

Eukaryotic Cell ◽

10.1128/ec.4.6.1041-1049.2005 ◽

2005 ◽

Vol 4 (6) ◽

pp. 1041-1049 ◽

Cited By ~ 12

Author(s):

Cara Marie A. Manlandro ◽

Devon H. Haydon ◽

Anne G. Rosenwald

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Plasma Membrane ◽

Wild Type ◽

Positive Modulator ◽

Negative Modulator

ABSTRACT We demonstrate here that SAP155 encodes a negative modulator of K+ efflux in the yeast Saccharomyces cerevisiae. Overexpression of SAP155 decreases efflux, whereas deletion increases efflux. In contrast, a homolog of SAP155, called SAP185, encodes a positive modulator of K+ efflux: overexpression of SAP185 increases efflux, whereas deletion decreases efflux. Two other homologs, SAP4 and SAP190, are without effect on K+ homeostasis. Both SAP155 and SAP185 require the presence of SIT4 for function, which encodes a PP2A-like phosphatase important for the G1-S transition through the cell cycle. Overexpression of either the outwardly rectifying K+ channel, Tok1p, or the putative plasma membrane K+/H+ antiporter, Kha1p, increases efflux in both wild-type and sit4Δ strains. However, overexpression of the Na+-K+/H+ antiporter, Nha1p, is without effect in a sit4Δ strain, suggesting that Sit4p signals to Nha1p. In summary, the combined activities of Sap155p and Sap185p appear to control the function of Nha1p in K+ homeostasis via Sit4p.

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The inner plasma membrane protein Plac8 regulates cell cycle progression in pancreatic ductal adenocarcinoma (PDAC)

Zeitschrift für Gastroenterologie ◽

10.1055/s-0035-1559251 ◽

2015 ◽

Vol 53 (08) ◽

Author(s):

M Buchholz ◽

B Kaistha ◽

H Lorenz ◽

H Schmidt ◽

N Weber-Lassalle ◽

...

Keyword(s):

Cell Cycle ◽

Plasma Membrane ◽

Membrane Protein ◽

Pancreatic Ductal Adenocarcinoma ◽

Cell Cycle Progression ◽

Ductal Adenocarcinoma ◽

Plasma Membrane Protein ◽

Cycle Progression

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Faculty Opinions recommendation of In vitro fusion between Saccharomyces cerevisiae secretory vesicles and cytoplasmic-side-out plasma membrane vesicles.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1010821.167908 ◽

2002 ◽

Author(s):

Miguel Penalva

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Membrane Vesicles ◽

Secretory Vesicles ◽

Plasma Membrane Vesicles ◽

Cytoplasmic Side

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Faculty Opinions recommendation of Plasma membrane localization of Ras requires class C Vps proteins and functional mitochondria in Saccharomyces cerevisiae.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1018473.371838 ◽

2006 ◽

Author(s):

Robert Parton

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Membrane Localization ◽

Plasma Membrane Localization ◽

Class C

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Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

Biochemical Journal ◽

10.1042/bj3390299 ◽

1999 ◽

Vol 339 (2) ◽

pp. 299-307 ◽

Cited By ~ 33

Author(s):

Arthur L. KRUCKEBERG ◽

Ling YE ◽

Jan A. BERDEN ◽

Karel van DAM

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Green Fluorescent Protein ◽

Glucose Transport ◽

Cellular Localization ◽

Fluorescent Protein ◽

Hexose Transporter ◽

High Concentrations ◽

Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

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High yield expression of the Neurospora crassa plasma membrane H(+)-ATPase in Saccharomyces cerevisiae

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)32498-5 ◽

1994 ◽

Vol 269 (26) ◽

pp. 17705-17712

Author(s):

S.K. Mahanty ◽

U.S. Rao ◽

R.A. Nicholas ◽

G.A. Scarborough

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Neurospora Crassa ◽

High Yield

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Yeast dom34 Mutants Are Defective in Multiple Developmental Pathways and Exhibit Decreased Levels of Polyribosomes

Genetics ◽

10.1093/genetics/149.1.45 ◽

1998 ◽

Vol 149 (1) ◽

pp. 45-56

Author(s):

Luther Davis ◽

JoAnne Engebrecht

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Heterologous Expression ◽

Protein Translation ◽

Developmental Pathways ◽

G1 Phase ◽

Growth Defect ◽

Wild Type ◽

Drosophila Homolog ◽

Mutant Cells

Abstract The DOM34 gene of Saccharomyces cerevisiae is similar togenes found in diverse eukaryotes and archaebacteria. Analysis of dom34 strains shows that progression through the G1 phase of the cell cycle is delayed, mutant cells enter meiosis aberrantly, and their ability to form pseudohyphae is significantly diminished. RPS30A, which encodes ribosomal protein S30, was identified in a screen for high-copy suppressors of the dom34Δ growth defect. dom34Δ mutants display an altered polyribosome profile that is rescued by expression of RPS30A. Taken together, these data indicate that Dom34p functions in protein translation to promote G1 progression and differentiation. A Drosophila homolog of Dom34p, pelota, is required for the proper coordination of meiosis and spermatogenesis. Heterologous expression of pelota in dom34Δ mutants restores wild-type growth and differentiation, suggesting conservation of function between the eukaryotic members of the gene family.

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Recombination of Ty elements in yeast can be induced by a double-strand break.

Genetics ◽

10.1093/genetics/140.1.67 ◽

1995 ◽

Vol 140 (1) ◽

pp. 67-77 ◽

Cited By ~ 1

Author(s):

A Parket ◽

O Inbar ◽

M Kupiec

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Strand Break ◽

Double Strand Break ◽

The Other ◽

Direct Repeats ◽

Yeast Saccharomyces Cerevisiae ◽

Low Frequencies ◽

Ty Elements ◽

Main Family

Abstract The Ty retrotransposons are the main family of dispersed repeated sequences in the yeast Saccharomyces cerevisiae. These elements are flanked by a pair of long terminal direct repeats (LTRs). Previous experiments have shown that Ty elements recombine at low frequencies, despite the fact that they are present in 30 copies per genome. This frequency is not highly increased by treatments that cause DNA damage, such as UV irradiation. In this study, we show that it is possible to increase the recombination level of a genetically marked Ty by creating a double-strand break in it. This break is repaired by two competing mechanisms: one of them leaves a single LTR in place of the Ty, and the other is a gene conversion event in which the marked Ty is replaced by an ectopically located one. In a strain in which the marked Ty has only one LTR, the double-strand break is repaired by conversion. We have also measured the efficiency of repair and monitored the progression of the cells through the cell-cycle. We found that in the presence of a double-strand break in the marked Ty, a proportion of the cells is unable to resume growth.

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