scholarly journals Targeting of the yeast plasma membrane [H+]ATPase: a novel gene AST1 prevents mislocalization of mutant ATPase to the vacuole.

1995 ◽  
Vol 128 (1) ◽  
pp. 39-49 ◽  
Author(s):  
A Chang ◽  
G R Fink
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Growth Defect ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Plasma Membrane Atpase ◽  
Novel Gene ◽  
Multicopy Suppressors ◽  
Proteinase A ◽  
Synthetic Growth

We have characterized a class of mutations in PMA1, (encoding plasma membrane ATPase) that is ideal for the analysis of membrane targeting in Saccharomyces cerevisiae. This class of pma1 mutants undergoes growth arrest at the restrictive temperature because newly synthesized ATPase fails to be targeted to the cell surface. Instead, mutant ATPase is delivered to the vacuole, where it is degraded. Delivery to the vacuole occurs without previous arrival at the plasma membrane because degradation of mutant ATPase is not prevented when internalization from the cell surface is blocked. Disruption of PEP4, encoding vacuolar proteinase A, blocks ATPase degradation, but fails to restore growth because the ATPase is still improperly targeted. One of these pma1 mutants was used to select multicopy suppressors that would permit growth at the nonpermissive temperature. A novel gene, AST1, identified by this selection, suppresses several pma1 alleles defective for targeting. The basis for suppression is that multicopy AST1 causes rerouting of mutant ATPase from the vacuole to the cell surface. pma1 mutants deleted for AST1 have a synthetic growth defect at the permissive temperature, providing genetic evidence for interaction between AST1 and PMA1. Ast1 is a cytoplasmic protein that associates with membranes, and is localized to multiple compartments, including the plasma membrane. The identification of AST1 homologues suggests that Ast1 belongs to a novel family of proteins that participates in membrane traffic.

The fission yeast bromodomain protein Bdf2 is required for growth of cells with circular chromosomes

2021 ◽  
Author(s):  
Misaki Yasuda ◽  
Ahmed G K Habib ◽  
Kanako Sugiura ◽  
Hossain Mohammad Shamim ◽  
Masaru Ueno
Keyword(s):  
Fission Yeast ◽  
High Temperatures ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
M Phase ◽  
Ts Mutant ◽  
Synthetically Lethal ◽  
Circular Chromosomes ◽  
Synthetic Growth

Abstract Circular chromosomes have frequently been observed in tumors of mesenchymal origin. In the fission yeast Schizosaccharomyces pombe, deletion of pot1+ results in rapid telomere loss, and the resulting survivors have circular chromosomes. Fission yeast has two bromodomains and extra-terminal (BET) proteins, Bdf1 and Bdf2; both are required for maintaining acetylated histones. Here, we found that bdf2, but not bdf1, was synthetically lethal with pot1. We also obtained a temperature-sensitive bdf2-ts mutant, which can grow at high temperatures but becomes camptothecin sensitive. This suggests that Bdf2 is defective at high temperatures. The cell cycle of the pot1 bdf2-ts mutant was delayed in the G2 and/or M phase at a semi-permissive temperature. Furthermore, a temperature-sensitive mutant of mst1, which encodes histone acetyltransferase, showed a synthetic growth defect with a pot1 disruptant at a semi-permissive temperature. Our results suggest that Bdf2 and Mst1 are required for the growth of cells with circular chromosomes.

scholarly journals An Endosome-to-Plasma Membrane Pathway Involved in Trafficking of a Mutant Plasma Membrane ATPase in Yeast

2000 ◽  
Vol 11 (2) ◽  
pp. 579-592 ◽  
Author(s):  
Wen-jie Luo ◽  
Amy Chang
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Secretory Pathway ◽  
Protein Sorting ◽  
Temperature Sensitive ◽  
Plasma Membrane Atpase ◽  
Vacuolar Protein Sorting ◽  
Membrane Atpase ◽  
Vacuolar Protein ◽  
Endosomal System

The plasma membrane ATPase, encoded by PMA1, is delivered to the cell surface via the secretory pathway. Previously, we characterized a temperature-sensitive pma1 mutant in which newly synthesized Pma1-7 is not delivered to the plasma membrane but is mislocalized instead to the vacuole at 37°C. Severalvps mutants, which are defective in vacuolar protein sorting, suppress targeting-defective pma1 by allowing mutant Pma1 to move once again to the plasma membrane. In this study, we have analyzed trafficking in the endosomal system by monitoring the movement of Pma1-7 in vps36, vps1, andvps8 mutants. Upon induction of expression, mutant Pma1 accumulates in the prevacuolar compartment in vps36cells. After chase, a fraction of newly synthesized Pma1-7 is delivered to the plasma membrane. In both vps1 andvps8 cells, newly synthesized mutant Pma1 appears in small punctate structures before arrival at the cell surface. Nevertheless, biosynthetic membrane traffic appears to follow different routes in vps8 and vps1: the vacuolar protein-sorting receptor Vps10p is stable in vps8 but not in vps1. Furthermore, a defect in endocytic delivery to the vacuole was revealed in vps8 (andvps36) but not vps1 by endocytosis of the bulk membrane marker FM 4-64. Moreover, in vps8 cells, there is defective down-regulation from the cell surface of the mating receptor Ste3, consistent with persistent receptor recycling from an endosomal compartment to the plasma membrane. These data support a model in which mutant Pma1 is diverted from the Golgi to the surface invps1 cells. We hypothesize that in vps8and vps36, in contrast to vps1, mutant Pma1 moves to the surface via endosomal intermediates, implicating an endosome-to-surface traffic pathway.

scholarly journals A Large PEST-like Sequence Directs the Ubiquitination, Endocytosis, and Vacuolar Degradation of the Yeast a-Factor Receptor

1998 ◽  
Vol 142 (4) ◽  
pp. 949-961 ◽  
Author(s):  
Amy F. Roth ◽  
Daniel M. Sullivan ◽  
Nicholas G. Davis
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Broad Class ◽  
Surface Protein ◽  
Sequence Motif ◽  
Plasma Membrane Atpase ◽  
Alanine Scanning Mutagenesis ◽  
Entire Interval ◽  
Sequence Elements ◽  
In The Wild

The yeast a-factor receptor (encoded by STE3) is subject to two modes of endocytosis, a ligand-dependent endocytosis as well as a constitutive, ligand-independent mode. Both modes are associated with receptor ubiquitination (Roth, A.F., and N.G. Davis. 1996. J. Cell Biol. 134:661–674) and both depend on sequence elements within the receptor's regulatory, cytoplasmically disposed, COOH-terminal domain (CTD). Here, we concentrate on the Ste3p sequences required for constitutive endocytosis. Constitutive endocytosis is rapid. Receptor is synthesized, delivered to the cell surface, endocytosed, and then delivered to the vacuole where it is degraded, all with a t1/2 of 15 min. Deletion analysis has defined a 36-residue-long sequence mapping near the COOH-terminal end of the Ste3p CTD that is the minimal sequence required for this rapid turnover. Deletions intruding into this interval block or severely slow the rate of endocytic turnover. Moreover, the same 36-residue sequence directs receptor ubiquitination. Mutants deleted for this sequence show undetectable levels of ubiquitination, and mutants having intermediate endocytosis defects show a correlated reduced level of ubiquitination. Not only necessary for ubiquitination and endocytosis, this sequence also is sufficient. When transplanted to a stable cell surface protein, the plasma membrane ATPase Pma1p, the 36-residue STE3 signal directs both ubiquitination of the PMA1-STE3 fusion protein as well as its endocytosis and consequent vacuolar degradation. Alanine scanning mutagenesis across the 36-residue-long interval highlights its overall complexity—no singular sequence motif or signal is found, instead required sequence elements distribute throughout the entire interval. The high proportion of acidic and hydroxylated amino acid residues in this interval suggests a similarity to PEST sequences—a broad class of sequences which have been shown to direct the ubiquitination and subsequent proteosomal degradation of short-lived nuclear and cytoplasmic proteins. A likely possibility, therefore, is that this sequence, responsible for both endocytosis and ubiquitination, may be first and foremost a ubiquitination signal. Finally, we present evidence suggesting that the true signal in the wild-type receptor extends beyond the 36-residue-long sequence defined as a minimal signal to include contiguous PEST-like sequences which extend another 21 residues to the COOH terminus of Ste3p. Together with sequences identified in two other yeast plasma membrane proteins, the STE3 sequence defines a new class of ubiquitination/endocytosis signal.

scholarly journals Ubiquitin-mediated Targeting of a Mutant Plasma Membrane ATPase, Pma1-7, to the Endosomal/Vacuolar System in Yeast

2004 ◽  
Vol 15 (5) ◽  
pp. 2401-2409 ◽  
Author(s):  
Maddalena Pizzirusso ◽  
Amy Chang
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Ubiquitin Ligase ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Plasma Membrane Atpase ◽  
Membrane Atpase ◽  
Endosomal Pathway ◽  
Quality Control Mechanism ◽  
Vacuolar System

Pma1-7 is a mutant plasma membrane ATPase that is impaired in targeting to the cell surface at 37°C and is delivered instead to the endosomal/vacuolar pathway for degradation. We have proposed that Pma1-7 is a substrate for a Golgibased quality control mechanism. By contrast with wild-type Pma1, Pma1-7 is ubiquitinated. Ubiquitination and endosomal targeting of Pma1-7 is dependent on the Rsp5-Bul1-Bul2 ubiquitin ligase protein complex but not the transmembrane ubiquitin ligase Tul1. Analysis of Pma1-7 ubiquitination in mutants blocked in protein transport at various steps of the secretory pathway suggests that ubiquitination occurs after ER exit but before endosomal entry. In the absence of ubiquitination in rsp5-1 cells, Pma1-7 is delivered to the cell surface and remains stable. Nevertheless, Pma1-7 remains impaired in association with detergent-insoluble glycolipid-enriched complexes in rsp5-1 cells, suggesting that ubiquitination is not the cause of Pma1-7 exclusion from rafts. In vps1 cells in which protein transport into the endosomal pathway is blocked, Pma1-7 is routed to the cell surface. On arrival at the plasma membrane in vps1 cells, Pma1-7 remains stable and its ubiquitination disappears, suggesting deubiquitination activity at the cell surface. We suggest that Pma1-7 sorting and fate are regulated by ubiquitination.

scholarly journals The yeast actin-related protein Arp2p is required for the internalization step of endocytosis.

1997 ◽  
Vol 8 (7) ◽  
pp. 1361-1375 ◽  
Author(s):  
V Moreau ◽  
J M Galan ◽  
G Devilliers ◽  
R Haguenauer-Tsapis ◽  
B Winsor
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Synthetic Lethality ◽  
Endocytic Pathway ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Related Protein ◽  
Pathway Inhibition

The Saccharomyces cerevisiae actin-related protein Arp2p is an essential component of the actin cytoskeleton. We have tested its potential role in the endocytic and exocytic pathways by using a temperature-sensitive allele, arp2-1. The fate of the plasma membrane transporter uracil permease was followed to determine whether Arp2p plays a role in the endocytic pathway. Inhibition of normal endocytosis as revealed by maintenance of active uracil permease at the plasma membrane and strong protection against subsequent vacuolar degradation of the protein were observed in the mutant at the restrictive temperature. Furthermore, arp2-1 cells accumulated ubiquitin-permease conjugates, formed prior to internalization. These effects were also visible at permissive temperature, whereas the actin cytoskeleton appeared to be normally polarized. The soluble hydrolase carboxypeptidase Y and the lipophilic dye FM 4-64 were targeted normally to the vacuole in arp2-1 cells. Thus, Arp2p is required for internalization but does not play a major role in later steps of endocytosis. Synthetic lethality was demonstrated between arp2-1 and the endocytic mutant end3-1, suggesting participation of Arp2p and End3p in the same process. Finally, no evidence for a major defect in secretion was apparent; invertase secretion and delivery of uracil permease to the plasma membrane were unaffected in arp2-1 cells.

scholarly journals Monensin and FCCP inhibit the intracellular transport of alphavirus membrane glycoproteins

1980 ◽  
Vol 87 (3) ◽  
pp. 783-791 ◽  
Author(s):  
L Kaariainen ◽  
K Hashimoto ◽  
J Saraste ◽  
I Virtanen ◽  
K Penttinen
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Intracellular Transport ◽  
Semliki Forest Virus ◽  
Marginal Effect ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Membrane Glycoproteins ◽  
Infected Cells

Temperature-sensitive mutants of semliki forest virus (SFV) and sindbis virus (SIN) were used to study the intracellular transport of virus membrane glycoproteins in infected chicken embryo fibroblasts. When antisera against purified glycoproteins and (125)I- labeled protein A from staphylococcus aureus were used only small amounts of virus glycoproteins were detected at the surface of SFV ts-1 and SIN Ts-10 infected cells incubated at the restrictive temperature (39 degrees C). When the mutant-infected cells were shifted to the permissive temperature (28 degrees C), in the presence of cycloheximide, increasing amounts of virus glycoproteins appeared at the cell surface from 20 to 80 min after the shift. Both monensin (10muM) and carbonylcyanide-p- trifluoromethoxyphenylhydrazone (FCCP; 10-20 muM) inhibited the appearance of virus membrane glycoproteins at the cell surface. Vinblastine sulfate (10 μg/ml) inhibited the transport by approximately 50 percent, whereas cytochalasin B (1 μg/ml) had only a marginal effect. Intracellular distribution of virus glycoproteins in the mutant-infected cells was visualized in double-fluorescence studies using lectins as markers for endoplasmic reticulum and Golgi apparatus. At 39 degrees C, the virus membrane glycoproteins were located at the endoplasmic reticulum, whereas after shift to 28 degrees C, a bright juxtanuclear reticular fluorescence was seen in the location of the Golgi apparatus. In the presence of monensin, the virus glycoproteins could migrate to the Golgi apparatus, although transport to the cell surface did not take place. When the shift was carried out in the presence of FCCP, negligible fluorescence was seen in the Golgi apparatus and the glycoproteins apparently remained in the rough endoplasmic reticulum. A rapid inhibition in the accumulation of virus glycoproteins at the cell surface was obtained when FCCP was added during the active transport period, whereas with monensin there was a delay of approximately 10 min. These results suggest a similar intracellular pathway in the maturation of both plasma membrane and secretory glycoproteins.

scholarly journals GPI7Involved in Glycosylphosphatidylinositol Biosynthesis Is Essential for Yeast Cell Separation

2004 ◽  
Vol 279 (50) ◽  
pp. 51869-51879 ◽  
Author(s):  
Morihisa Fujita ◽  
Takehiko Yoko-o ◽  
Michiyo Okamoto ◽  
Yoshifumi Jigami
Keyword(s):  
Cell Separation ◽  
Specific Growth ◽  
Growth Defect ◽  
Cell Cortex ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Loss Of Function ◽  
Septal Region ◽  
Multicopy Suppressors ◽  
Specific Proteins

GPI7is involved in adding ethanolaminephosphate to the second mannose in the biosynthesis of glycosylphosphatidylinositol (GPI) inSaccharomyces cerevisiae. We isolatedgpi7mutants, which have defects in cell separation and a daughter cell-specific growth defect at the non-permissive temperature.WSC1,RHO2,ROM2,GFA1, andCDC5genes were isolated as multicopy suppressors ofgpi7-2mutant. Multicopy suppressors could suppress the growth defect ofgpi7mutants but not the cell separation defect. Loss of function mutations of genes involved in the Cbk1p-Ace2p pathway, which activates the expression of daughter-specific genes for cell separation after cytokinesis, bypassed the temperature-sensitive growth defect ofgpi7mutants. Furthermore, deletion ofEGT2, one of the genes controlled by Ace2p and encoding a GPI-anchored protein required for cell separation, ameliorated the temperature sensitivity of thegpi7mutant. In this mutant, Egt2p was displaced from the septal region to the cell cortex, indicating thatGPI7plays an important role in cell separation via the GPI-based modification of daughter-specific proteins inS. cerevisiae.

scholarly journals Synthesis of Sphingolipids with Very Long Chain Fatty Acids but Not Ergosterol Is Required for Routing of Newly Synthesized Plasma Membrane ATPase to the Cell Surface of Yeast

2005 ◽  
Vol 280 (23) ◽  
pp. 22515-22522 ◽  
Author(s):  
Barbara Gaigg ◽  
Birgit Timischl ◽  
Linda Corbino ◽  
Roger Schneiter
Keyword(s):  
Plasma Membrane ◽  
Fatty Acid ◽  
Cell Surface ◽  
Secretory Pathway ◽  
Chain Fatty Acid ◽  
Proton Pumping ◽  
Sterol Synthesis ◽  
Plasma Membrane Atpase ◽  
Long Chain Fatty Acid ◽  
Long Chain

The proton pumping H+-ATPase, Pma1p, is an abundant and very long-lived polytopic protein of the Saccharomyces cerevisiae plasma membrane. Pma1p constitutes a major cargo of the secretory pathway and thus serves as an excellent model to study plasma membrane biogenesis. We have previously shown that newly synthesized Pma1p is mistargeted to the vacuole in an elo3Δ mutant that affects the synthesis of the ceramide-bound C26 very long chain fatty acid (Eisenkolb, M., Zenzmaier, C., Leitner, E., and Schneiter, R. (2002) Mol. Biol. Cell 13, 4414–4428) and now describe a more detailed analysis of the role of lipids in Pma1p biogenesis. Remarkably, a block at various steps of sterol biosynthesis, a complete block in sterol synthesis, or the substitution of internally synthesized ergosterol by externally supplied ergosterol or even by cholesterol does not affect Pma1p biogenesis or its association with detergent-resistant membrane domains (lipid “rafts”). However, a block in sphingolipid synthesis or any perturbation in the synthesis of the ceramide-bound C26 very long chain fatty acid results in mistargeting of newly synthesized Pma1p to the vacuole. Mistargeting correlates with a lack of newly synthesized Pma1p to acquire detergent resistance, suggesting that sphingolipids with very long acyl chains affect sorting of Pma1p to the cell surface.

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