scholarly journals Wss1 Is a SUMO-Dependent Isopeptidase That Interacts Genetically with the Slx5-Slx8 SUMO-Targeted Ubiquitin Ligase

2010 ◽  
Vol 30 (15) ◽  
pp. 3737-3748 ◽  
Author(s):  
Janet R. Mullen ◽  
Chi-Fu Chen ◽  
Steven J. Brill
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Protein ◽  
Ubiquitin Ligase ◽  
Copy Number ◽  
Dna Helicase ◽  
Genome Integrity ◽  
Growth Defect ◽  
Sumo Fusion ◽  
Isopeptidase Activity

ABSTRACT Protein sumoylation plays an important but poorly understood role in controlling genome integrity. In Saccharomyces cerevisiae, the Slx5-Slx8 SUMO-targeted Ub ligase appears to be needed to ubiquitinate sumoylated proteins that arise in the absence of the Sgs1 DNA helicase. WSS1, a high-copy-number suppressor of a mutant SUMO, was implicated in this pathway because it shares phenotypes with SLX5-SLX8 mutants, including a wss1Δ sgs1Δ synthetic-fitness defect. Here we show that Wss1, a putative metalloprotease, physically binds SUMO and displays in vitro isopeptidase activity on poly-SUMO chains. Like that of SLX5, overexpression of WSS1 suppresses sgs1Δ slx5Δ lethality and the ulp1ts growth defect. Interestingly, although Wss1 is relatively inactive on ubiquitinated substrates and poly-Ub chains, it efficiently deubiquitinates a Ub-SUMO isopeptide conjugate and a Ub-SUMO fusion protein. Wss1 was further implicated in Ub metabolism on the basis of its physical association with proteasomal subunits. The results suggest that Wss1 is a SUMO-dependent isopeptidase that acts on sumoylated substrates as they undergo proteasomal degradation.

scholarly journals Overlapping function of Hrd1 and Ste24 in translocon quality control provides robust channel surveillance

2020 ◽  
Vol 295 (47) ◽  
pp. 16113-16120
Author(s):  
Avery M. Runnebohm ◽  
Kyle A. Richards ◽  
Courtney Broshar Irelan ◽  
Samantha M. Turk ◽  
Halie E. Vitali ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Ubiquitin Ligase ◽  
Biological Membranes ◽  
The Other ◽  
Growth Defect ◽  
Zinc Metalloprotease

Translocation of proteins across biological membranes is essential for life. Proteins that clog the endoplasmic reticulum (ER) translocon prevent the movement of other proteins into the ER. Eukaryotes have multiple translocon quality control (TQC) mechanisms to detect and destroy proteins that persistently engage the translocon. TQC mechanisms have been defined using a limited panel of substrates that aberrantly occupy the channel. The extent of substrate overlap among TQC pathways is unknown. In this study, we found that two TQC enzymes, the ER-associated degradation ubiquitin ligase Hrd1 and zinc metalloprotease Ste24, promote degradation of characterized translocon-associated substrates of the other enzyme in Saccharomyces cerevisiae. Although both enzymes contribute to substrate turnover, our results suggest a prominent role for Hrd1 in TQC. Yeast lacking both Hrd1 and Ste24 exhibit a profound growth defect, consistent with overlapping function. Remarkably, two mutations that mildly perturb post-translational translocation and reduce the extent of aberrant translocon engagement by a model substrate diminish cellular dependence on TQC enzymes. Our data reveal previously unappreciated mechanistic complexity in TQC substrate detection and suggest that a robust translocon surveillance infrastructure maintains functional and efficient translocation machinery.

scholarly journals Cns1 Is an Essential Protein Associated with the Hsp90 Chaperone Complex in Saccharomyces cerevisiae That Can Restore Cyclophilin 40-Dependent Functions in cpr7ΔCells

1998 ◽  
Vol 18 (12) ◽  
pp. 7353-7359 ◽  
Author(s):  
James A. Marsh ◽  
Helen M. Kalton ◽  
Richard F. Gaber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Chaperone ◽  
Single Gene ◽  
Normal Growth ◽  
Maximal Activity ◽  
Negative Regulation ◽  
Growth Defect ◽  
Chaperone Complex ◽  
Cyclophilin 40

ABSTRACT Saccharomyces cerevisiae harbors two cyclophilin 40-type enzymes, Cpr6 and Cpr7, which are components of the Hsp90 molecular chaperone machinery. Cpr7 is required for normal growth and is required for maximal activity of heterologous Hsp90-dependent substrates, including glucocorticoid receptor (GR) and the oncogenic tyrosine kinase pp60v-src . In addition, it has recently been shown that Cpr7 plays a major role in negative regulation of the S. cerevisiae heat shock transcription factor (HSF). To better understand functions associated with Cpr7, a search was undertaken for multicopy suppressors of the cpr7Δ slow-growth phenotype. The screen identified a single gene, designatedCNS1 (for cyclophilin seven suppressor), capable of suppressing the cpr7Δ growth defect. Overexpression ofCNS1 in cpr7Δ cells also largely restored GR activity and negative regulation of HSF. In vitro protein retention experiments in which Hsp90 heterocomplexes were precipitated resulted in coprecipitation of Cns1. Interaction between Cns1 and the carboxy terminus of Hsp90 was also shown by two-hybrid analysis. The functional consequences of CNS1 overexpression and its physical association with the Hsp90 machinery indicate that Cns1 is a previously unidentified component of molecular chaperone complexes. Thus far, Cns1 is the only tetratricopeptide repeat-containing component of Hsp90 heterocomplexes found to be essential for cell viability under all conditions tested.

scholarly journals Transcriptional repression in Saccharomyces cerevisiae by a SIN3-LexA fusion protein

1993 ◽  
Vol 13 (3) ◽  
pp. 1805-1814
Author(s):  
H Wang ◽  
D J Stillman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Fusion Protein ◽  
Protein Interactions ◽  
Transcriptional Repression ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Predicted Amino Acid Sequence

The yeast SIN3 gene (also known as SDI1, UME4, RPD1, and GAM2) has been identified as a transcriptional regulator. Previous work has led to the suggestion that SIN3 regulates transcription via interactions with DNA-binding proteins. Although the SIN3 protein is located in the nucleus, it does not bind directly to DNA in vitro. We have expressed a LexA-SIN3 fusion protein in Saccharomyces cerevisiae and show that this fusion protein represses transcription from heterologous promoters that contain lexA operators. The predicted amino acid sequence of the SIN3 protein contains four copies of a paired amphipathic helix (PAH) motif, similar to motifs found in HLH (helix-loop-helix) and TPR (tetratricopeptide repeat) proteins, and these motifs are proposed to be involved in protein-protein interactions. We have conducted a deletion analysis of the SIN3 gene and show that the PAH motifs are required for SIN3 activity. Additionally, the C-terminal region of the SIN3 protein is sufficient for repression activity in a LexA-SIN3 fusion, and deletion of a PAH motif in this region inactivates this repression activity. A model is presented in which SIN3 recognizes specific DNA-binding proteins in vivo in order to repress transcription.

The Saccharomyces cerevisiae ADP/ATP carrier-iso 1 cytochrome c fusion protein: One-step purification and functional analysis in vitro

Biochimie ◽  
2007 ◽  
Vol 89 (9) ◽  
pp. 1070-1079 ◽  
Author(s):  
M REY ◽  
X BRAZZOLOTTO ◽  
B CLEMENCON ◽  
A AFCHAIN ◽  
G BRANDOLIN ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Functional Analysis ◽  
Cytochrome C ◽  
Fusion Protein ◽  
One Step

scholarly journals The CDC25 protein of Saccharomyces cerevisiae promotes exchange of guanine nucleotides bound to ras.

1991 ◽  
Vol 11 (5) ◽  
pp. 2641-2646 ◽  
Author(s):  
S Jones ◽  
M L Vignais ◽  
J R Broach
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cyclic Amp ◽  
Genetic Analysis ◽  
Fusion Protein ◽  
Guanine Nucleotides ◽  
Biochemical Activity ◽  
Cdc25 Protein ◽  
Beta Galactosidase

The product of the CDC25 gene of Saccharomyces cerevisiae, in its capacity as an activator of the RAS/cyclic AMP pathway, is required for initiation of the cell cycle. In this report, we provide an identification of Cdc25p, the product of the CDC25 gene, and evidence that it promotes exchange of guanine nucleotides bound to Ras in vitro. Extracts of strains containing high levels of Cdc25p catalyze both removal of GDP from and the concurrent binding of GTP to Ras. This same activity is also obtained with an immunopurified Cdc25p-beta-galactosidase fusion protein, suggesting that Cdc25p participates directly in the exchange reaction. This biochemical activity is consistent with previous genetic analysis of CDC25 function.

scholarly journals Bni5p, a Septin-Interacting Protein, Is Required for Normal Septin Function and Cytokinesis in Saccharomyces cerevisiae

2002 ◽  
Vol 22 (19) ◽  
pp. 6906-6920 ◽  
Author(s):  
Philip R. Lee ◽  
Sukgil Song ◽  
Hyeon-Su Ro ◽  
Chong J. Park ◽  
John Lippincott ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Localization ◽  
Growth Defect ◽  
Dependent Manner ◽  
Interacting Protein ◽  
Growth Defects ◽  
Bud Emergence ◽  
Bud Neck ◽  
And Function

ABSTRACT In the budding yeast Saccharomyces cerevisiae, the Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Sep7p/Shs1p septins assemble early in the cell cycle in a ring that marks the future cytokinetic site. The septins appear to be major structural components of a set of filaments at the mother-bud neck and function as a scaffold for recruiting proteins involved in cytokinesis and other processes. We isolated a novel gene, BNI5, as a dosage suppressor of the cdc12-6 growth defect. Overexpression of BNI5 also suppressed the growth defects of cdc10-1, cdc11-6, and sep7Δ strains. Loss of BNI5 resulted in a cytokinesis defect, as evidenced by the formation of connected cells with shared cytoplasms, and deletion of BNI5 in a cdc3-6, cdc10-1, cdc11-6, cdc12-6, or sep7Δ mutant strain resulted in enhanced defects in septin localization and cytokinesis. Bni5p localizes to the mother-bud neck in a septin-dependent manner shortly after bud emergence and disappears from the neck approximately 2 to 3 min before spindle disassembly. Two-hybrid, in vitro binding, and protein-localization studies suggest that Bni5p interacts with the N-terminal domain of Cdc11p, which also appears to be sufficient for the localization of Cdc11p, its interaction with other septins, and other critical aspects of its function. Our data suggest that the Bni5p-septin interaction is important for septin ring stability and function, which is in turn critical for normal cytokinesis.

scholarly journals Saccharomyces cerevisiae RAI1 (YGL246c) Is Homologous to Human DOM3Z and Encodes a Protein That Binds the Nuclear Exoribonuclease Rat1p

2000 ◽  
Vol 20 (11) ◽  
pp. 4006-4015 ◽  
Author(s):  
Yang Xue ◽  
Xinxue Bai ◽  
Insuk Lee ◽  
George Kallstrom ◽  
Jennifer Ho ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Interaction ◽  
Rna Degradation ◽  
Growth Defect ◽  
Nuclear Targeting ◽  
Interacting Protein ◽  
Rrna Species ◽  
Synthetically Lethal

ABSTRACT The RAT1 gene of Saccharomyces cerevisiaeencodes a 5′→3′ exoribonuclease which plays an essential role in yeast RNA degradation and/or processing in the nucleus. We have cloned a previously uncharacterized gene (YGL246c) that we refer to asRAI1 (Rat1p interacting protein 1). RAI1 is homologous to Caenorhabditis elegans DOM-3 and humanDOM3Z. Deletion of RAI1 confers a growth defect which can be complemented by an additional copy of RAT1 on a centromeric vector or by directing Xrn1p, the cytoplasmic homolog of Rat1p, to the nucleus through the addition of a nuclear targeting sequence. Deletion of RAI1 is synthetically lethal with therat1-1ts mutation and shows genetic interaction with a deletion of SKI2 but not XRN1. Polysome analysis of an rai1 deletion mutant indicated a defect in 60S biogenesis which was nearly fully reversed by high-copyRAT1. Northern blot analysis of rRNAs revealed thatrai1 is required for normal 5.8S processing. In the absence of RAI1, 5.8SL was the predominant form of 5.8S and there was an accumulation of 3′-extended forms but not 5′-extended species of 5.8S. In addition, a 27S pre-rRNA species accumulated in therai1 mutant. Thus, deletion of RAI1 affects both 5′ and 3′ processing reactions of 5.8S rRNA. Consistent with the in vivo data suggesting that RAI1 enhances RAT1function, purified Rai1p stabilized the in vitro exoribonuclease activity of Rat1p.

scholarly journals LEM domain proteins control the efficiency of adaptation through copy number variation

2018 ◽  
Author(s):  
Paolo Colombi ◽  
Diane E. King ◽  
Jessica F. Williams ◽  
C. Patrick Lusk ◽  
Megan C. King
Keyword(s):  
Copy Number ◽  
Genome Instability ◽  
Genetic Interaction ◽  
Transcriptional Response ◽  
Genome Integrity ◽  
Changing Environment ◽  
Number Variation ◽  
Transcriptional Induction

AbstractWhile loss of genome integrity is at the basis of numerous pathologies, including cancer, genome plasticity is necessary to adapt to a changing environment and thus is essential for long-term organismal success. Here we present data supporting a targeted mechanism that promotes adaptation to environmental stress by driving site-specific genome instability tied to transcriptional induction and the formation of RNA-DNA hybrids. Using an in vitro evolution assay we observe that the inner nuclear membrane LEM domain proteins Heh1 and Heh2 play antagonistic roles in inhibiting or promoting adaptation through copy number expansion, respectively, which is also reflected in their genetic interaction networks with genes responsible for transcription-dependent genome instability. Taken together, our data suggest the existence of a LEM domain protein-mediated mechanism by which an immediate transcriptional response to a changing environment drives targeted genome instability to promote increased variation on which selection can act to support long-term adaptation.

scholarly journals Molecular Cloning of XYL3 (d-Xylulokinase) from Pichia stipitis and Characterization of Its Physiological Function

2002 ◽  
Vol 68 (3) ◽  
pp. 1232-1239 ◽  
Author(s):  
Yong-Su Jin ◽  
Sharon Jones ◽  
Nian-Qing Shi ◽  
Thomas W. Jeffries
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ethanol Production ◽  
Parental Strain ◽  
Copy Number ◽  
Genomic Dna ◽  
Physiological Function ◽  
Pichia Stipitis ◽  
Single Copy

ABSTRACT XYL3, which encodes a d-xylulokinase (EC 2.7.1.17), was isolated from Pichia stipitis CBS 6054 genomic DNA by using primers designed against conserved motifs. Disruption of XYL3 eliminated d-xylulokinase activity, but d-ribulokinase activity was still present. Southern analysis of P. stipitis genomic DNA with XYL3 as a probe confirmed the disruption and did not reveal additional related genes. Disruption of XYL3 stopped ethanol production from xylose, but the resulting mutant still assimilated xylose slowly and formed xylitol and arabinitol. These results indicate that XYL3 is critical for ethanol production from xylose but that P. stipitis has another pathway for xylose assimilation. Expression of XYL3 using its P. stipitis promoter increased Saccharomyces cerevisiae d-xylulose consumption threefold and enabled the transformants to produce ethanol from a mixture of xylose and xylulose, whereas the parental strain only accumulated xylitol. In vitro, d-xylulokinase activity in recombinant S. cerevisiae was sixfold higher with a multicopy than with a single-copy XYL3 plasmid, but ethanol production decreased with increased copy number. These results confirmed the function of XYL3 in S. cerevisiae.

scholarly journals Saccharomyces cerevisiae Srs2 DNA Helicase Selectively Blocks Expansions of Trinucleotide Repeats

2004 ◽  
Vol 24 (17) ◽  
pp. 7324-7330 ◽  
Author(s):  
Saumitri Bhattacharyya ◽  
Robert S. Lahue
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Recombination ◽  
Dna Helicase ◽  
Trinucleotide Repeats ◽  
Model Organisms ◽  
Helicase Activity ◽  
Dna Polymerase Delta ◽  
Dinucleotide Repeats ◽  
Cgg Repeats

ABSTRACT Trinucleotide repeats (TNRs) undergo frequent mutations in families afflicted with certain neurodegenerative disorders and in model organisms. TNR instability is modulated both by the repeat tract itself and by cellular proteins. Here we identified the Saccharomyces cerevisiae DNA helicase Srs2 as a potent and selective inhibitor of expansions. srs2 mutants had up to 40-fold increased expansion rates of CTG, CAG, and CGG repeats. The expansion phenotype was specific, as mutation rates at dinucleotide repeats, at unique sequences, or for TNR contractions in srs2 mutants were not altered. Srs2 is known to suppress inappropriate genetic recombination; however, the TNR expansion phenotype of srs2 mutants was largely independent of RAD51 and RAD52. Instead, Srs2 mainly functioned with DNA polymerase delta to block expansions. The helicase activity of Srs2 was important, because a point mutant lacking ATPase function was defective in blocking expansions. Purified Srs2 was substantially better than bacterial UvrD helicase at in vitro unwinding of a DNA substrate that mimicked a TNR hairpin. Disruption of the related helicase gene SGS1 did not lead to excess expansions, nor did wild-type SGS1 suppress the expansion phenotype of an srs2 strain. We conclude that Srs2 selectively blocks triplet repeat expansions through its helicase activity and primarily in conjunction with polymerase delta.

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