scholarly journals Identification of Novel Vesicles in the Cytosol to Vacuole Protein Degradation Pathway

1997 ◽  
Vol 136 (4) ◽  
pp. 803-810 ◽  
Author(s):  
Pei-Hsin Huang ◽  
Hui-Ling Chiang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Degradation ◽  
High Speed ◽  
Kinetic Studies ◽  
Degradation Pathway ◽  
Proteinase K ◽  
Wild Type ◽  
Fractionation Analysis ◽  
Gluconeogenic Enzyme ◽  
Copii Vesicles

The key gluconeogenic enzyme, fructose1,6-bisphosphatase (FBPase), is induced when Saccharomyces cerevisiae are starved of glucose. FBPase is targeted from the cytosol to the yeast vacuole for degradation when glucose-starved cells are replenished with fresh glucose. Several vid mutants defective in the glucose-induced degradation of FBPase in the vacuole have been isolated. In some vid mutants, FBPase is found in punctate structures in the cytoplasm. When extracts from these cells are fractionated, a substantial amount of FBPase is sedimentable in the high speed pellet, suggesting that FBPase is associated with intracellular structures in these vid mutants. In this paper we investigated whether FBPase association with intracellular structures also existed in wild-type cells. We report the purification of novel FBPase-associated vesicles from wild-type cells to near homogeneity. Kinetic studies indicate that FBPase association with these vesicles is stimulated by glucose and occurs only transiently, suggesting that these vesicles are intermediate in the FBPase degradation pathway. Fractionation analysis demonstrates that these vesicles are distinct from known organelles such as the vacuole, ER, Golgi, mitochondria, peroxisomes, endosomes, COPI, or COPII vesicles. Under EM, these vesicles are 30–40 nm in diam. Proteinase K experiments indicate that the majority of FBPase is sequestered inside the vesicles. We propose that FBPase is imported into these vesicles before entering the vacuole.

scholarly journals Domain structure and functional analysis of the carboxyl-terminal polyacidic sequence of the RAD6 protein of Saccharomyces cerevisiae.

1988 ◽  
Vol 8 (3) ◽  
pp. 1179-1185 ◽  
Author(s):  
A Morrison ◽  
E J Miller ◽  
L Prakash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Proteinase K ◽  
Intrinsic Activity ◽  
Structural Domain ◽  
Globular Domain ◽  
Wild Type ◽  
Calculated Mass ◽  
Stokes Radius ◽  
Carboxyl Terminal

The RAD6 gene of Saccharomyces cerevisiae, which is required for normal tolerance of DNA damage and for sporulation, encodes a 172-residue protein whose 23 carboxyl-terminal residues are almost all acidic. We show that this polyacidic sequence appends to RAD6 protein as a polyanionic tail and that its function in vivo does not require stoichiometry of length. RAD6 protein was purified to near homogeneity from a yeast strain carrying a RAD6 overproducing plasmid. Approximately the first 150 residues of RAD6 protein composed a structural domain that was resistant to proteinase K and had a Stokes radius typical of a globular protein of its calculated mass. The carboxyl-terminal polyacidic sequence was sensitive to proteinase K, and it endowed RAD6 protein with an aberrantly large Stokes radius that indicates an asymmetric shape. We deduce that RAD6 protein is monomeric and comprises a globular domain with a freely extending polyacidic tail. We tested the phenotypic effects of partial or complete deletion of the polyacidic sequence, demonstrating the presence of the shortened proteins in the cell by using antibody to RAD6 protein. Removal of the entire polyacidic sequence severely reduced sporulation but only slightly affected survival after UV irradiation or UV-induced mutagenesis. Strains with deletions of all but the first 4 or 15 residues of the polyacidic sequence were phenotypically almost wild type or wild type, respectively. We conclude that the intrinsic activity of RAD6 protein resides in the globular domain, that the polyacidic sequence has a stimulatory or modifying role evident primarily in sporulation, and that only a short section apparently functions as effectively as the entire polyacidic sequence.

scholarly journals Vid24p, a Novel Protein Localized to the Fructose-1,6-bisphosphatase–containing Vesicles, Regulates Targeting of Fructose-1,6-bisphosphatase from the Vesicles to the Vacuole for Degradation

1998 ◽  
Vol 140 (6) ◽  
pp. 1347-1356 ◽  
Author(s):  
Meng-Chieh Chiang ◽  
Hui-Ling Chiang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Protein ◽  
Critical Role ◽  
Carbon Sources ◽  
Degradation Pathway ◽  
Yeast Cells ◽  
Fructose 1,6 Bisphosphatase ◽  
Peripheral Membrane Protein ◽  
Gluconeogenic Enzyme ◽  
Novel Protein

Glucose regulates the degradation of the key gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase), in Saccharomyces cerevisiae. FBPase is targeted from the cytosol to a novel type of vesicle, and then to the vacuole for degradation when yeast cells are transferred from medium containing poor carbon sources to fresh glucose. To identify proteins involved in the FBPase degradation pathway, we cloned our first VID (vacuolar import and degradation) gene. The VID24 gene was identified by complementation of the FBPase degradation defect of the vid24-1 mutant. Vid24p is a novel protein of 41 kD and is synthesized in response to glucose. Vid24p is localized to the FBPase-containing vesicles as a peripheral membrane protein. In the absence of functional Vid24p, FBPase accumulates in the vesicles and fails to move to the vacuole, suggesting that Vid24p regulates FBPase targeting from the vesicles to the vacuole. FBPase sequestration into the vesicles is not affected in the vid24-1 mutant, indicating that Vid24p acts after FBPase sequestration into the vesicles has occurred. Vid24p is the first protein identified that marks the FBPase-containing vesicles and plays a critical role in delivering FBPase from the vesicles to the vacuole for degradation.

scholarly journals A Saccharomyces cerevisiae Mutant Lacking a K+/H+ Exchanger

1998 ◽  
Vol 180 (22) ◽  
pp. 5860-5865 ◽  
Author(s):  
Jorge Ramírez ◽  
Oscar Ramírez ◽  
Carlos Saldaña ◽  
Roberto Coria ◽  
Antonio Peña
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Core Protein ◽  
Kinetic Studies ◽  
Flow Cytometry Analysis ◽  
Wild Type ◽  
Reading Frame ◽  
Duplication Rate ◽  
Significant Difference ◽  
Internal Ph ◽  
Mutant Cells

ABSTRACT The KHA1 gene corresponding to the open reading frame YJL094c (2.62 kb) encoding a putative K+/H+antiporter (873 amino acids) in Saccharomyces cerevisiaewas disrupted by homologous recombination. The core protein is similar to the putative Na+/H+ antiporters fromEnterococcus hirae (NAPA gene) andLactococcus lactis (LLUPP gene) and the putative K+/H+ exchanger from Escherichia coli (KEFC gene). Disruption of the KHA1gene resulted in an increased K+ accumulation and net influx without a significant difference in efflux, as well as an increased growth rate, smaller cells, and twice the cell yield per glucose used. Flow cytometry analysis showed an increase of the DNA duplication rate in the mutant. Kinetic studies of86Rb+ uptake showed the same saturable system for wild-type and disruptant strains. Mutant cells also produced a greater acidification of the medium coincident with an internal pH alkalinization and showed a higher oxygen consumption velocity. We speculate that higher K+ accumulation and increased osmotic pressure accelerate the cell cycle and metabolic activity.

scholarly journals The multiubiquitin-chain-binding protein Mcb1 is a component of the 26S proteasome in Saccharomyces cerevisiae and plays a nonessential, substrate-specific role in protein turnover.

1996 ◽  
Vol 16 (11) ◽  
pp. 6020-6028 ◽  
Author(s):  
S van Nocker ◽  
S Sadis ◽  
D M Rubin ◽  
M Glickman ◽  
H Fu ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Protein ◽  
Degradation Pathway ◽  
26S Proteasome ◽  
Wild Type ◽  
Cell Extracts ◽  
Steady State Levels ◽  
Type Rate ◽  
Beta Galactosidase

The 26S proteasome is an essential proteolytic complex that is responsible for degrading proteins conjugated with ubiquitin. It has been proposed that the recognition of substrates by the 26S proteasome is mediated by a multiubiquitin-chain-binding protein that has previously been characterized in both plants and animals. In this study, we identified a Saccharomyces cerevisiae homolog of this protein, designated Mcb1. Mcb1 copurified with the 26S proteasome in both conventional and nickel chelate chromatography. In addition, a significant fraction of Mcb1 in cell extracts was present in a low-molecular-mass form free of the 26S complex. Recombinant Mcb1 protein bound multiubiquitin chains in vitro and, like its plant and animal counterparts, exhibited a binding preference for longer chains. Surprisingly, (delta)mcb1 deletion mutants were viable, grew at near-wild-type rates, degraded the bulk of short-lived proteins normally, and were not sensitive to UV radiation or heat stress. These data indicate that Mcb1 is not an essential component of the ubiquitin-proteasome pathway in S.cerevisiae. However, the (delta)mcb1 mutant exhibited a modest sensitivity to amino acid analogs and had increased steady-state levels of ubiquitin-protein conjugates. Whereas the N-end rule substrate, Arg-beta-galactosidase, was degraded at the wild-type rate in the (delta)mcb1 strain, the ubiquitin fusion degradation pathway substrate, ubiquitin-Pro-beta-galactosidase, was markedly stabilized. Collectively, these data suggest that Mcb1 is not the sole factor involved in ubiquitin recognition by the 26S proteasome and that Mcb1 may interact with only a subset of ubiquitinated substrates.

Domain structure and functional analysis of the carboxyl-terminal polyacidic sequence of the RAD6 protein of Saccharomyces cerevisiae

1988 ◽  
Vol 8 (3) ◽  
pp. 1179-1185 ◽  
Author(s):  
A Morrison ◽  
E J Miller ◽  
L Prakash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Proteinase K ◽  
Intrinsic Activity ◽  
Structural Domain ◽  
Globular Domain ◽  
Wild Type ◽  
Calculated Mass ◽  
Stokes Radius ◽  
Carboxyl Terminal

The RAD6 gene of Saccharomyces cerevisiae, which is required for normal tolerance of DNA damage and for sporulation, encodes a 172-residue protein whose 23 carboxyl-terminal residues are almost all acidic. We show that this polyacidic sequence appends to RAD6 protein as a polyanionic tail and that its function in vivo does not require stoichiometry of length. RAD6 protein was purified to near homogeneity from a yeast strain carrying a RAD6 overproducing plasmid. Approximately the first 150 residues of RAD6 protein composed a structural domain that was resistant to proteinase K and had a Stokes radius typical of a globular protein of its calculated mass. The carboxyl-terminal polyacidic sequence was sensitive to proteinase K, and it endowed RAD6 protein with an aberrantly large Stokes radius that indicates an asymmetric shape. We deduce that RAD6 protein is monomeric and comprises a globular domain with a freely extending polyacidic tail. We tested the phenotypic effects of partial or complete deletion of the polyacidic sequence, demonstrating the presence of the shortened proteins in the cell by using antibody to RAD6 protein. Removal of the entire polyacidic sequence severely reduced sporulation but only slightly affected survival after UV irradiation or UV-induced mutagenesis. Strains with deletions of all but the first 4 or 15 residues of the polyacidic sequence were phenotypically almost wild type or wild type, respectively. We conclude that the intrinsic activity of RAD6 protein resides in the globular domain, that the polyacidic sequence has a stimulatory or modifying role evident primarily in sporulation, and that only a short section apparently functions as effectively as the entire polyacidic sequence.

scholarly journals Yeast Mutants Deficient in ER-Associated Degradation of the Z Variant of Alpha-1-Protease Inhibitor

Genetics ◽  
1996 ◽  
Vol 144 (4) ◽  
pp. 1355-1362 ◽  
Author(s):  
Ardythe A McCracken ◽  
Igor V Karpichev ◽  
James E Ernaga ◽  
Eric D Werner ◽  
Andrew G Dillin ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Degradation ◽  
Proteinase Inhibitor ◽  
Parent Strain ◽  
Mendelian Inheritance ◽  
Wild Type ◽  
Mutant Strains ◽  
Complementation Groups ◽  
Yeast Mutants ◽  
Wild Type Yeast

Saccharomyces cerevisiae mutants deficient in degradation of alpha-1-proteinase inhibitor Z (A1PiZ) have been isolated and genetically characterized. Wild-type yeast expressing A1PiZ synthesize an ER form of this protein that is rapidly degraded by an intracellular proteolytic process known as ER-associated protein degradation (ERAD). The mutant strains were identified after treatment with EMS using a colony blot immunoassay to detect colonies that accumulated high levels of A1PiZ. A total of 120,000 colonies were screened and 30 putative mutants were identified. The level of A1PiZ accumulation in these mutants, measured by ELISA, ranged from two to 11 times that of A1PiZ in the parent strain. Further studies demonstrated that the increased levels of A1PiZ in most of the mutant strains was not the result of defective secretion or elevated A1PiZ mRNA. Pulse chase experiments indicated that A1PiZ was stabilized in several strains, evidence that these mutants are defective in ER-associated protein degradation. Genetic analyses revealed that most of the mutations were recessive, ∼30% of the mutants characterized conformed to simple Mendelian inheritance, and at least seven complementation groups were identified.

scholarly journals PROTEIN DEGRADATION, MEIOSIS AND SPORULATION IN PROTEINASE-DEFICIENT MUTANTS OF SACCHAROMYCES CEREVISIAE

Genetics ◽  
1981 ◽  
Vol 97 (1) ◽  
pp. 45-64 ◽  
Author(s):  
George S Zubenko ◽  
Elizabeth W Jones
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drug Resistance ◽  
Protein Degradation ◽  
Mating Type ◽  
Carboxypeptidase Y ◽  
Wild Type ◽  
Resistance Marker ◽  
Drug Resistance Marker

ABSTRACT During the process of sporulation, a/α diploids degrade about 50% of their vegetative proteins. This degradation is not sporulation specific, for asporogenous diploids of a/a mating type degrade their vegetative proteins in a fashion similar to that of their a/α counterparts. Diploids lacking carboxypeptidase Y activity, prc1/prc1, show about 80% of wild-type levels of protein degradation, but are unimpaired in the production of normal asci. Diploids lacking proteinase B activity, prb1/prb1, show about 50% of wild-type levels of protein degradation. The effect on degradation of the proteinase B deficiency is epistatic to the degradation deficit attributable to the carboxypeptidase Y deficiency. The prb1 homozygotes undergo meiosis and produce spores, but the asci and, possibly, the spores are abnormal. Diploids homozygous for the pleiotropic pep4-3 mutation show only 30% of the wild-type levels of degradation when exposed to a sporulation regimen, and do not undergo meiosis or sporulation. Neither proteinase B nor carboxypeptidase Y is necessary for germination of spores. ——Approximately half of the colonies arising from a/a or α/α diploids exposed to the sporulation regiment that express an initially heterozygous drug-resistance marker (can1) appear to arise from mating-type switches followed by meiosis and sporulation.

scholarly journals Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 383-391 ◽  
Author(s):  
Yasumasa Tsukamoto ◽  
Jun-ichi Kato ◽  
Hideo Ikeda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quantitative Analysis ◽  
Structural Analysis ◽  
Recombination Rate ◽  
Type Strain ◽  
Negative Selection ◽  
Wild Type Strain ◽  
Illegitimate Recombination ◽  
Wild Type ◽  
In The Wild

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

scholarly journals DDRE-09. DEVELOPING IDO-PROTACS TO IMPROVE IMMUNOTHERAPEUTIC EFFICACY IN PATIENTS WITH GLIOBLASTOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii63-ii63
Author(s):  
Lakshmi Bollu ◽  
Derek Wainwright ◽  
Lijie Zhai ◽  
Erik Ladomersky ◽  
Kristen Lauing ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Time Course ◽  
Immune Cell ◽  
Enzyme Inhibitors ◽  
Tumor Development ◽  
Ubiquitin Proteasome System ◽  
Degradation Pathway ◽  
Specific Protein ◽  
Cell Functions

Abstract INTRODUCTION Indoleamine 2,3-dioxygenase 1 (IDO; IDO1) is a rate-limiting enzyme that metabolizes the essential amino acid tryptophan into kynurenine. Recent work by our group has revealed that IDO promotes tumor development and suppresses immune cell functions independent of its enzyme activity. Moreover, pharmacologic IDO enzyme inhibitors that currently serve as the only class of drugs available for targeting immunosuppressive IDO activity, fail to improve the survival of patients with GBM. Here, we developed IDO-Proteolysis Targeting Chimeras (IDO-PROTACs). PROTACs bind to a specific protein and recruit an E3 ubiquitin ligase that enhance proteasome-mediated degradation of the target protein. METHODS A library of ≥100 IDO-PROTACs were developed by joining BMS986205 (IDO binder) with a linker group to various E3-ligase ligands. Western blot analysis of PROTAC-induced IDO degradation was tested in vitro among multiple human and mouse GBM cell lines including U87, GBM6, GBM43 and GL261 along a time course ranging between 1–96 hours of treatment and at varying concentrations. The mechanism of IDO protein degradation was investigated using pharmacologic ligands that inhibit or compete with the proteasome-mediated protein degradation pathway. RESULTS Primary screening identified several IDO-PROTACs with IDO protein degradation potential. Secondary screening showed that our lead compound has a DC50 value of ~0.5µM with an ability to degrade IDO in all GBM cells analyzed, and an initial activity within 12 hours of treatment that extended for up to 96 hours. Mutating the CRBN-binding ligand, pretreatment with the ubiquitin proteasome system inhibitors MG132 or MLN4924 or using unmodified parental compound all inhibited IDO protein degradation. CONCLUSIONS This study developed an initial IDO-PROTAC technology that upon further optimization, can neutralize both IDO enzyme and non-enzyme immunosuppressive effects. When combined with other forms of immunotherapy, IDO-PROTACs have the potential to substantially enhance immunotherapeutic efficacy in patients with GBM.

scholarly journals Nonrecombinant meiosis I nondisjunction in Saccharomyces cerevisiae induced by tRNA ochre suppressors.

Genetics ◽  
1989 ◽  
Vol 123 (1) ◽  
pp. 81-95 ◽  
Author(s):  
E J Louis ◽  
J E Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Chromosome ◽  
Stop Codon ◽  
Fold Increase ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Nonsense Mutations ◽  
Chromosome Iii ◽  
Meiosis I ◽  
Chromosome Nondisjunction

Abstract The presence of the tRNA ochre suppressors SUP11 and SUP5 is found to induce meiosis I nondisjunction in the yeast Saccharomyces cerevisiae. The induction increases with increasing dosage of the suppressor and decreases in the presence of an antisuppressor. The effect is independent of the chromosomal location of SUP11. Each of five different chromosomes monitored exhibited nondisjunction at frequencies of 0.1%-1.1% of random spores, which is a 16-160-fold increase over wild-type levels. Increased nondisjunction is reflected by a marked increase in tetrads with two and zero viable spores. In the case of chromosome III, for which a 50-cM map interval was monitored, the resulting disomes are all in the parental nonrecombinant configuration. Recombination along chromosome III appears normal both in meioses that have no nondisjunction and in meioses for which there was nondisjunction of another chromosome. We propose that a proportion of one or more proteins involved in chromosome pairing, recombination or segregation are aberrant due to translational read-through of the normal ochre stop codon. Hygromycin B, an antibiotic that can suppress nonsense mutations via translational read-through, also induces nonrecombinant meiosis I nondisjunction. Increases in mistranslation, therefore, increase the production of aneuploids during meiosis. There was no observable effect of SUP11 on mitotic chromosome nondisjunction; however some disomes caused SUP11 ade2-ochre strains to appear white or red, instead of pink.

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