scholarly journals Genetic and biochemical interactions between an essential kinetochore protein, Cbf2p/Ndc10p, and the CDC34 ubiquitin-conjugating enzyme.

1995 ◽  
Vol 15 (9) ◽  
pp. 4835-4842 ◽  
Author(s):  
H J Yoon ◽  
J Carbon
Keyword(s):  
Yeast Cells ◽  
Temperature Sensitive ◽  
Kinetochore Protein ◽  
Ubiquitin Conjugating Enzyme ◽  
Endogenous Substrate ◽  
Growth Phenotype ◽  
Kinetochore Function ◽  
Conjugating Enzyme

CBF2/NDC10/CTF14 encodes the 110-kDa subunit of CBF3, a key component of the yeast centromere/kinetochore. Overexpression of yeast CDC34 specifically suppresses the temperature-sensitive growth phenotype of the ndc10-1 mutation. Mutations in CDC34, which specifies a ubiquitin-conjugating enzyme, arrest yeast cells in the G1 phase of the cell cycle, with no intact spindles formed (M. G. Goebl, J. Yochem, S. Jentsch, J. P. McGrath, A. Varshavsky, and B. Byers, Science 241:1331-1335, 1988). The cdc34-2 mutation drastically alters the pattern of Cbf2p modification. Results of experiments using antibodies against Cbf2p and ubiquitin indicate that Cbf2p is ubiquitinated in vivo. Purified Cdc34p catalyzes the formation of Cbf2p-monoubiquitin conjugate in vitro. These data suggest that Cbf2p is an endogenous substrate of the CDC34 ubiquitin-conjugating enzyme and imply that ubiquitination of a kinetochore protein plays a regulatory role in kinetochore function.

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4084-4092
Author(s):  
P C McCabe ◽  
H Haubruck ◽  
P Polakis ◽  
F McCormick ◽  
M A Innis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Bound States ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Amino Terminal ◽  
Loop Region

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

scholarly journals OTUB1 non-catalytically stabilizes the E2 ubiquitin-conjugating enzyme UBE2E1 by preventing its autoubiquitination

2018 ◽  
Vol 293 (47) ◽  
pp. 18285-18295 ◽  
Author(s):  
Nagesh Pasupala ◽  
Marie E. Morrow ◽  
Lauren T. Que ◽  
Barbara A. Malynn ◽  
Averil Ma ◽  
...  
Keyword(s):  
Polyubiquitin Chains ◽  
Protein Ubiquitination ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzymes ◽  
E2 Enzymes ◽  
Ubiquitin Conjugating Enzymes ◽  
In Cells ◽  
Conjugating Enzyme

OTUB1 is a deubiquitinating enzyme that cleaves Lys-48–linked polyubiquitin chains and also regulates ubiquitin signaling through a unique, noncatalytic mechanism. OTUB1 binds to a subset of E2 ubiquitin-conjugating enzymes and inhibits their activity by trapping the E2∼ubiquitin thioester and preventing ubiquitin transfer. The same set of E2s stimulate the deubiquitinating activity of OTUB1 when the E2 is not charged with ubiquitin. Previous studies have shown that, in cells, OTUB1 binds to E2-conjugating enzymes of the UBE2D (UBCH5) and UBE2E families, as well as to UBE2N (UBC13). Cellular roles have been identified for the interaction of OTUB1 with UBE2N and members of the UBE2D family, but not for interactions with UBE2E E2 enzymes. We report here a novel role for OTUB1–E2 interactions in modulating E2 protein ubiquitination. We observe that Otub1−/− knockout mice exhibit late-stage embryonic lethality. We find that OTUB1 depletion dramatically destabilizes the E2-conjugating enzyme UBE2E1 (UBCH6) in both mouse and human OTUB1 knockout cell lines. Of note, this effect is independent of the catalytic activity of OTUB1, but depends on its ability to bind to UBE2E1. We show that OTUB1 suppresses UBE2E1 autoubiquitination in vitro and in cells, thereby preventing UBE2E1 from being targeted to the proteasome for degradation. Taken together, we provide evidence that OTUB1 rescues UBE2E1 from degradation in vivo.

Dolichol phosphate mannose synthase is required in vivo for glycosyl phosphatidylinositol membrane anchoring, O mannosylation, and N glycosylation of protein in Saccharomyces cerevisiae

1990 ◽  
Vol 10 (11) ◽  
pp. 5796-5805
Author(s):  
P Orlean
Keyword(s):  
Molecular Size ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
In Vitro Mutagenesis ◽  
Nonpermissive Temperature ◽  
Gpi Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Covalent Modifications

Glycosyl phosphatidylinositol (GPI) anchoring, N glycosylation, and O mannosylation of protein occur in the rough endoplasmic reticulum and involve transfer of precursor structures that contain mannose. Direct genetic evidence is presented that dolichol phosphate mannose (Dol-P-Man) synthase, which transfers mannose from GDPMan to the polyisoprenoid dolichol phosphate, is required in vivo for all three biosynthetic pathways leading to these covalent modifications of protein in yeast cells. Temperature-sensitive yeast mutants were isolated after in vitro mutagenesis of the yeast DPM1 gene. At the nonpermissive temperature of 37 degrees C, the dpm1 mutants were blocked in [2-3H]myo-inositol incorporation into protein and accumulated a lipid that could be radiolabeled with both [2-3H]myo-inositol and [2-3H]glucosamine and met existing criteria for an intermediate in GPI anchor biosynthesis. The likeliest explanation for these results is that Dol-P-Man donates the mannose residues needed for completion of the GPI anchor precursor lipid before it can be transferred to protein. Dol-P-Man synthase is also required in vivo for N glycosylation of protein, because (i) dpm1 cells were unable to make the full-length precursor Dol-PP-GlcNAc2Man9Glc3 and instead accumulated the intermediate Dol-PP-GlcNAc2Man5 in their pool of lipid-linked precursor oligosaccharides and (ii) truncated, endoglycosidase H-resistant oligosaccharides were transferred to the N-glycosylated protein invertase after a shift to 37 degrees C. Dol-P-Man synthase is also required in vivo for O mannosylation of protein, because chitinase, normally a 150-kDa O-mannosylated protein, showed a molecular size of 60 kDa, the size predicted for the unglycosylated protein, after shift of the dpm1 mutant to the nonpermissive temperature.

scholarly journals Implication of a novel multiprotein Dam1p complex in outer kinetochore function

2001 ◽  
Vol 155 (7) ◽  
pp. 1137-1146 ◽  
Author(s):  
Iain M. Cheeseman ◽  
Christine Brew ◽  
Michael Wolyniak ◽  
Arshad Desai ◽  
Scott Anderson ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Fluorescent Protein ◽  
Temperature Sensitive ◽  
Temperature Sensitive Mutants ◽  
Phosphorylation Event ◽  
Spindle Microtubules ◽  
Green Fluorescent ◽  
Kinetochore Function

Dam1p, Duo1p, and Dad1p can associate with each other physically and are required for both spindle integrity and kinetochore function in budding yeast. Here, we present our purification from yeast extracts of an ∼245 kD complex containing Dam1p, Duo1p, and Dad1p and Spc19p, Spc34p, and the previously uncharacterized proteins Dad2p and Ask1p. This Dam1p complex appears to be regulated through the phosphorylation of multiple subunits with at least one phosphorylation event changing during the cell cycle. We also find that purified Dam1p complex binds directly to microtubules in vitro with an affinity of ∼0.5 μM. To demonstrate that subunits of the Dam1p complex are functionally important for mitosis in vivo, we localized Spc19–green fluorescent protein (GFP), Spc34-GFP, Dad2-GFP, and Ask1-GFP to the mitotic spindle and to kinetochores and generated temperature-sensitive mutants of DAD2 and ASK1. These and other analyses implicate the four newly identified subunits and the Dam1p complex as a whole in outer kinetochore function where they are well positioned to facilitate the association of chromosomes with spindle microtubules.

scholarly journals Functional Interaction between Human Herpesvirus 6 Immediate-Early 2 Protein and Ubiquitin-Conjugating Enzyme 9 in the Absence of Sumoylation

2006 ◽  
Vol 80 (20) ◽  
pp. 10218-10228 ◽  
Author(s):  
Andru Tomoiu ◽  
Annie Gravel ◽  
Robert M. Tanguay ◽  
Louis Flamand
Keyword(s):  
Human Herpesvirus ◽  
Immediate Early ◽  
Human Herpesvirus 6 ◽  
Interacting Protein ◽  
Ubiquitin Conjugating Enzyme ◽  
Herpesvirus 6 ◽  
Transcriptional Complex ◽  
Conjugating Enzyme

ABSTRACT The immediate-early 2 (IE2) protein of human herpesvirus 6 is a potent transactivator of cellular and viral promoters. To better understand the biology of IE2, we generated a LexA-IE2 fusion protein and screened, using the yeast two-hybrid system, a Jurkat T-cell cDNA library for proteins that could interact with IE2. The most frequently isolated IE2-interacting protein was the human ubiquitin-conjugating enzyme 9 (Ubc9), a protein involved in the small ubiquitin-like modifier (SUMO) conjugation pathway. Using deletion mutants of IE2, we mapped the IE2-Ubc9-interacting region to residues 989 to 1037 of IE2. The interaction was found to be of functional significance to IE2, as Ubc9 overexpression significantly repressed promoter activation by IE2. The C93S Ubc9 mutant exhibited a similar effect on IE2, indicating that the E2 SUMO-conjugating function of Ubc9 is not required for its repressive action on IE2. No consensus sumoylation sites or evidence of IE2 conjugation to SUMO could be demonstrated under in vivo or in vitro conditions. Moreover, expression levels and nuclear localization of IE2 were not altered by Ubc9 overexpression, suggesting that Ubc9's repressive function likely occurs at the transcriptional complex level. Overall, our results indicate that Ubc9 influences IE2's function and provide new information on the complex interactions that occur between herpesviruses and the sumoylation pathway.

scholarly journals Interaction with a Host Ubiquitin-Conjugating Enzyme Is Required for the Pathogenicity of a Geminiviral DNA β Satellite

2009 ◽  
Vol 22 (6) ◽  
pp. 737-746 ◽  
Author(s):  
Omid Eini ◽  
Satish Dogra ◽  
Luke A. Selth ◽  
Ian B. Dry ◽  
John W. Randles ◽  
...  
Keyword(s):  
Single Gene ◽  
Bimolecular Fluorescence Complementation ◽  
Cotton Leaf ◽  
Specific Symptom ◽  
Ubiquitin Conjugating Enzyme ◽  
Ubiquitin Proteasome ◽  
Ubiquitin Conjugating Enzymes ◽  
Conjugating Enzyme

DNA β is a single-stranded satellite DNA which encodes a single gene, βC1. To better understand the role of βC1 in the pathogenicity of DNA β, a yeast two-hybrid screen of a tomato cDNA library was carried out using βC1 from Cotton leaf curl Multan virus (CLCuMV) DNA β as the bait. A ubiquitin-conjugating enzyme, designated SlUBC3, which functionally complemented a yeast mutant deficient in ubiquitin-conjugating enzymes was identified. The authenticity and specificity of the interaction between βC1 and SlUBC3 was confirmed both in vivo, using a bimolecular fluorescence complementation assay, and in vitro, using a protein-binding assay. Analysis of deletion mutants of the βC1 protein showed that a myristoylation-like motif is required both for its interaction with SlUBC3 and the induction of DNA-β-specific symptoms in host plants. The level of polyubiquitinated proteins in transgenic tobacco plants expressing βC1 was found to be reduced compared with wild-type plants. These results are consistent with the hypothesis that interaction of βC1 with SlUBC3 is required for DNA-β-specific symptom induction, and that this is possibly due to downregulation of the host ubiquitin proteasome pathway.

scholarly journals Structure-Function Analysis of Yeast mRNA Cap Methyltransferase and High-Copy Suppression of Conditional Mutants by AdoMet Synthase and the Ubiquitin Conjugating Enzyme Cdc34p

Genetics ◽  
2000 ◽  
Vol 155 (4) ◽  
pp. 1561-1576
Author(s):  
Beate Schwer ◽  
Nayanendu Saha ◽  
Xiangdong Mao ◽  
Hsiao-Wang Chen ◽  
Stewart Shuman
Keyword(s):  
Function Analysis ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Mrna Levels ◽  
Ubiquitin Conjugating Enzyme ◽  
Ubiquitin Conjugation ◽  
Yeast Genes ◽  
Conjugating Enzyme

Abstract Here we present a genetic analysis of the yeast cap-methylating enzyme Abd1p. To identify individual amino acids required for Abd1p function, we introduced alanine mutations at 35 positions of the 436-amino acid yeast protein. Two new recessive lethal mutations, F256A and Y330A, were identified. Alleles F256L and Y256L were viable, suggesting that hydrophobic residues at these positions sufficed for Abd1p function. Conservative mutations of Asp-178 established that an acidic moiety is essential at this position (i.e., D178E was viable whereas D178N was not). Phe-256, Tyr-330, and Asp-178 are conserved in all known cellular cap methyltransferases. We isolated temperature-sensitive abd1 alleles and found that abd1-ts cells display a rapid shut-off of protein synthesis upon shift to the restrictive temperature, without wholesale reduction in steady-state mRNA levels. These in vivo results are consistent with classical biochemical studies showing a requirement for the cap methyl group in cap-dependent translation. We explored the issue of how cap methylation might be regulated in vivo by conducting a genetic screen for high-copy suppressors of the ts growth defect of abd1 mutants. The identification of the yeast genes SAM2 and SAM1, which encode AdoMet synthase, as abd1 suppressors suggests that Abd1p function can be modulated by changes in the concentration of its substrate AdoMet. We also identified the ubiquitin conjugating enzyme Cdc34p as a high-copy abd1 suppressor. We show that mutations of Cdc34p that affect its ubiquitin conjugation activity or its capacity to interact with the E3-SCF complex abrogate its abd1 suppressor function. Moreover, the growth defect of abd1 mutants is exacerbated by cdc34-2. These findings suggest a novel role for Cdc34p in gene expression and engender a model whereby cap methylation or cap utilization is negatively regulated by a factor that is degraded when Cdc34p is overexpressed.

scholarly journals Phosphorylation of the Mitotic Regulator Protein Hec1 by Nek2 Kinase Is Essential for Faithful Chromosome Segregation

2002 ◽  
Vol 277 (51) ◽  
pp. 49408-49416 ◽  
Author(s):  
Yumay Chen ◽  
Daniel J. Riley ◽  
Lei Zheng ◽  
Phang-Lang Chen ◽  
Wen-Hwa Lee
Keyword(s):  
Chromosome Segregation ◽  
Critical Role ◽  
Coiled Coil ◽  
Serine Phosphorylation ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Chromosomal Segregation ◽  
M Phase

Hec1 (highlyexpressed incancer) plays essential roles in chromosome segregation by interacting through its coiled-coil domains with several proteins that modulate the G2/M phase. Hec1 localizes to kinetochores, and its inactivation either by genetic deletion or antibody neutralization leads to severe and lethal chromosomal segregation errors, indicating that Hec1 plays a critical role in chromosome segregation. The mechanisms by which Hec1 is regulated, however, are not known. Here we show that human Hec1 is a serine phosphoprotein and that it binds specifically to the mitotic regulatory kinase Nek2 during G2/M. Nek2 phosphorylates Hec1 on serine residue 165, bothin vitroandin vivo. Yeast cells are viable without scNek2/Kin3, a close structural homolog of Nek2 that binds to both human and yeast Hec1. When the same yeasts carry an scNek2/Kin3 (D55G) or Nek2 (E38G) mutation to mimic a similar temperature-sensitivenimamutation inAspergillus, their growth is arrested at the nonpermissive temperature, because the scNek2/Kin3 (D55G) mutant binds to Hec1 but fails to phosphorylate it. Whereas wild-type human Hec1 rescues lethality resulting from deletion of Hec1 inSaccharomyces cerevesiae, a human Hec1 mutant or yeast Hec1 mutant changing Ser165to Ala or yeast Hec1 mutant changing Ser201to Ala does not. Mutations changing the same Ser residues to Glu, to mimic the negative charge created by phosphorylation, partially rescue lethality but result in a high incidence of errors in chromosomal segregation. These results suggest that cell cycle-regulated serine phosphorylation of Hec1 by Nek2 is essential for faithful chromosome segregation.

scholarly journals Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (9) ◽  
pp. 4084-4092 ◽  
Author(s):  
P C McCabe ◽  
H Haubruck ◽  
P Polakis ◽  
F McCormick ◽  
M A Innis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Bound States ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Amino Terminal ◽  
Loop Region

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

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