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M Goebl

Publications and source records attributed to M Goebl.

At least 19 recordsLinked to original sources
PubMed1999

The abundance of cell cycle regulatory protein Cdc4p is controlled by interactions between its F box and Skp1p.

Posttranslational modification of a protein by ubiquitin usually results in rapid degradation of the ubiquitinated protein by the proteasome. The transfer of ubiquitin to substrate is a multistep process. Cdc4p is a component of a ubiquitin ligase that tethers the ubiquitin-conjugating enzyme Cdc34p to its substrates. Among the domains of Cdc4p that are crucial for function are the F-box, which links Cdc4p to Cdc53p through Skp1p, and the WD-40 repeats, which are required for binding the substrate for Cdc34p. In addition to Cdc4p, other F-box proteins, including Grr1p and Met30p, may similarly act together with Cdc53p and Skp1p to function as ubiquitin ligase complexes. Because the relative abundance of these complexes, known collectively as SCFs, is important for cell viability, we have sought evidence of mechanisms that modulate F-box protein regulation. Here we demonstrate that the abundance of Cdc4p is subject to control by a peptide segment that we term the R-motif (for "reduced abundance"). Furthermore, we show that binding of Skp1p to the F-box of Cdc4p inhibits R-motif-dependent degradation of Cdc4p. These results suggest a general model for control of SCF activities.

Binding Sites↗
PubMed1998

Modification of yeast Cdc53p by the ubiquitin-related protein rub1p affects function of the SCFCdc4 complex.

The RUB1/NEDD-8 family of ubiquitin-related genes is widely represented among eukaryotes. Here we report that Cdc53p in Saccharomyces cerevisiae, a member of the Cullin family of proteins, is stably modified by the covalent attachment of a single Rub1p molecule. Two genes have been identified that are required for Rub1p conjugation to Cdc53p. The first gene, designated ENR2, encodes a protein with sequence similarity to the amino-terminal half of the ubiquitin-activating enzyme. By analogy with Aos1p, we infer that Enr2p functions in a bipartite Rub1p-activating enzyme. The second gene is SKP1, shown previously to be required for some ubiquitin-conjugation events. A deletion allele of ENR2 is lethal with temperature-sensitive alleles of cdc34 and enhances the phenotypes of cdc4, cdc53, and skp1, strongly implying that Rub1p conjugation to Cdc53p is required for optimal assembly or function of the E3 complex SCFCdc4. Consistent with this model, both enr2delta and an allele of Cdc53p that is not Rub1p modified, render cells sensitive to alterations in the levels of Cdc4p, Cdc34p, and Cdc53p.

Amino Acid Sequence↗
PubMed1996

SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box.

We have identified the yeast and human homologs of the SKP1 gene as a suppressor of cdc4 mutants and as a cyclin F-binding protein. Skp1p indirectly binds cyclin A/Cdk2 through Skp2p, and directly binds Skp2p, cyclin F, and Cdc4p through a novel structural motif called the F-box. SKP1 is required for ubiquitin-mediated proteolysis of Cin2p, Clb5p, and the Cdk inhibitor Sic1p, and provides a link between these molecules and the proteolysis machinery. A large number of proteins contain the F-box motif and are thereby implicated in the ubiquitin pathway. Different skp1 mutants arrest cells in either G1 or G2, suggesting a connection between regulation of proteolysis in different stages of the cycle.

Amino Acid Sequence↗
PubMed1996

Identification of a positive regulator of the cell cycle ubiquitin-conjugating enzyme Cdc34 (Ubc3).

The Cdc34 (Ubc3) ubiquitin-conjugating enzyme from Saccharomyces cerevisiae plays an essential role in the progression of cells from the G1 to S phase of the cell division cycle. Using a high-copy suppression strategy, we have identified a yeast gene (UBS1) whose elevated expression suppresses the conditional cell cycle defects associated with cdc34 mutations. The UBS1 gene encodes a 32.2-kDa protein of previously unknown function and is identical in sequence to a genomic open reading frame on chromosome II (GenBank accession number Z36034). Several lines of evidence described here indicate that Ubs1 functions as a general positive regulator of Cdc34 activity. First, overexpression of UBS1 suppresses not only the cell proliferation and morphological defects associated with cdc34 mutants but also the inability of cdc34 mutant cells to degrade the general amino acid biosynthesis transcriptional regulator, Gcn4. Second, deletion of the UBS1 gene profoundly accentuates the cell cycle defect when placed in combination with a cdc34 temperature-sensitive allele. Finally, a comparison of the Ubs1 and Cdc34 polypeptide sequences reveals two noncontiguous regions of similarity, which, when projected onto the three-dimensional structure of a ubiquitin-conjugating enzyme, define a single region situated on its surface. While cdc34 mutations corresponding to substitutions outside this region are suppressed by UBS1 overexpression, Ubs1 fails to suppress amino acid substitutions made within this region. Taken together with other findings, the allele specificity exhibited by UBS1 expression suggests that Ubs1 regulates Cdc34 by interaction or modification.

Amino Acid Sequence↗
PubMed1994

The identification of a gene family in the Saccharomyces cerevisiae ergosterol biosynthesis pathway.

The Saccharomyces cerevisiae ERG24 gene, encoding sterol delta 14 reductase (Erg24p), was cloned by selecting strains carrying sequences on a 2 mu-based vector for resistance to the morpholine fungicide, fenpropimorph (Fp). Four distinct plasmid inserts which conferred Fp resistance (FpR) were recovered (plasmids pML99, pML100, pML101 and pM103). Although Fp is reported to inhibit activity of Erg24p and sterol delta 8-delta 7 isomerase (Erg2p; encoded by ERG2), none of the inserts had restriction maps resembling ERG2. In addition, a 2 mu plasmid overexpression of the ERG2 sequence did not produce FpR. Characterization studies were focused on plasmid pML100, because it was the only plasmid to confer FpR consistently when tested in a number of different genetic backgrounds. Tests with a panel of fungicides indicated that pML100 conferred significant resistance only to compounds (Fp, tridemorph, fenpropidin and azasterol) which have a shared site of action, Erg24p. An insertional disruption of pML100 resulted in an obligate anaerobic phenotype, indicating a lesion in sterol biosynthesis. Sterol analysis of the disrupted mutant demonstrated the accumulation of ignosterol, indicating a loss of Erg24p activity. A SphI-XbaI fragment of pML100 was sequenced, revealing the presence of an ORF encoding a 438-amino-acid protein, which is highly similar to those encoded by two previously reported yeast drug sensitivity genes, sts1+ (Schizosaccharomyces pombe) and YGL022 (S. cerevisiae). Analyses of these genes demonstrated that strains carrying disruptions of sts1+ or YGL022 have ergosterol biosynthesis defects in the enzyme, sterol C-24(28) reductase (Erg4p; encoded by ERG4).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

A novel gene, STT4, encodes a phosphatidylinositol 4-kinase in the PKC1 protein kinase pathway of Saccharomyces cerevisiae.

A staurosporine-sensitive mutation (stt1) in yeast has been found in the PKC1 gene that encodes a protein kinase C homologue (Yoshida, S., Ikeda, E., Uno, I., and Mitsuzawa, H. (1992) Mol. Gen. Genet. 231, 337-344). We report here another staurosporine-sensitive mutant, stt4, which shows very similar phenotypes to that of the stt1 mutant. The stt4 temperature-sensitive mutant arrests mostly in G2/M phase at 37 degrees C, and the stt4 deletion mutant shows an osmoremedial phenotype. Staurosporine sensitivity of the stt4 mutant was suppressed by overexpression of PKC1/STT1, indicating genetic interaction between stt4 and pkc1/stt1. The nucleotide sequence of STT4 predicts a hydrophilic protein composed of 1,900 amino acid residues, with 26% sequence identity to the yeast VPS34 gene product and 27% to the catalytic subunit of mammalian phosphatidylinositol (PI) 3-kinase, respectively. Cell homogenates of the stt4 deletion mutant show normal PI3-kinase activity but lack most of the PI4-kinase activity that is detected in the wild-type. We conclude that STT4 encodes a yeast PI4-kinase that functions in the PKC1 protein kinase pathway.

1-Phosphatidylinositol 4-Kinase↗
PubMed1993

Yeast bZip proteins mediate pleiotropic drug and metal resistance.

Saccharomyces cerevisiae contains a group of transcription factors related to mammalian c-Jun. This yeast Jun-family of proteins consists of GCN4, a regulator of genes involved in amino acid biosynthesis, and yAP-1, a factor conferring pleiotropic drug resistance when overexpressed. In the work described here, we show that a third member of the yeast Jun-family exists. This protein has been designated CAD1 and provides resistance to cadmium when present on a high-copy plasmid. CAD1 and yAP-1 are related in their amino-terminal DNA binding domains and can recognize the same DNA target site in vitro. Overproduction of CAD1 leads to transcriptional activation of an artificial reporter gene in delta yap1 cells. High level production of either CAD1 or yAP-1 causes cells to acquire a pleiotropic drug-resistant phenotype and to be able to tolerate normally toxic levels of iron chelators and zinc. Surprisingly, disruption of the CAD1 gene has no effect on the normal cellular resistance to cadmium but delta yap1 mutants are hypersensitive to this cytotoxic metal. The cadmium hypersensitivity of the delta yap1 mutant described here indicates that one major role of YAP1 in the yeast cell is to mediate resistance to this metal.

Amino Acid Sequence↗
PubMed1993

Localization of Drosophila retinal degeneration B, a membrane-associated phosphatidylinositol transfer protein.

The Drosophila retinal degeneration B (rdgB) mutation causes abnormal photoreceptor response and light-enhanced retinal degeneration. Immunoblots using polyclonal anti-rdgB serum showed that rdgB is a 160-kD membrane protein. The antiserum localized the rdgB protein in photoreceptors, antennae, and regions of the Drosophila brain, indicating that the rdgB protein functions in many sensory and neuronal cells. In photoreceptors, the protein localized adjacent to the rhabdomeres, in the vicinity of the subrhabdomeric cisternae. The rdgB protein's amino-terminal 281 residues are > 40% identical to the rat brain phosphatidylinositol transfer protein (PI-TP). A truncated rdgB protein, which contains only this amino-terminal domain, possesses a phosphatidylinositol transfer activity in vitro. The remaining 773 carboxyl terminal amino acids have additional functional domains. Nitrocellulose overlay experiments reveal that an acidic amino acid domain, adjacent to the PI transfer domain, binds 45Ca+2. Six hydrophobic segments are found in the middle of the putative translation product and likely function as membrane spanning domains. These results suggest that the rdgB protein, unlike the small soluble PI-TPs, is a membrane-associated PI-TP, which may be directly regulated by light-induced changes in intracellular calcium.

Alleles↗
PubMed1993

Molecular cloning of a human homologue of Drosophila heterochromatin protein HP1 using anti-centromere autoantibodies with anti-chromo specificity.

We have identified a novel autoantibody specificity in scleroderma that we term anti-chromo. These antibodies recognize several chromosomal antigens with apparent molecular mass of between 23 and 25 kDa, as determined by immunoblots. Anti-chromo autoantibodies occur in 10-15% of sera from patients with anti-centromere antibodies (ACA). We used anti-chromo antibodies to screen a human expression library and obtained cDNA clones encoding a 25 kDa chromosomal autoantigen. DNA sequence analysis reveals this protein to be a human homologue of HP1, a heterochromatin protein of Drosophila melanogaster. We designate our cloned protein HP1Hs alpha. Epitope mapping experiments using both human and Drosophila HP1 reveal that anti-chromo antibodies target a region at the amino terminus of the protein. This region contains a conserved motif, the chromo domain (or HP1/Pc box), first recognized by comparison of Drosophila HP1 with the Polycomb gene product. Both proteins are thought to play a role in creating chromatin structures in which gene expression is suppressed. Anti-chromo thus defines a novel type of autoantibody that recognizes a conserved structural motif found on a number of chromosomal proteins.

Amino Acid Sequence↗
PubMed1992

New human gene encoding a positive modulator of HIV Tat-mediated transactivation.

The human immunodeficiency virus-1 (HIV-1) protein Tat is a potent activator of virus gene expression. Tat functions through a sequence known as TAR, located immediately downstream of the transcription start site in the long terminal repeat. Several observations suggest that cellular factors cooperate with Tat in the overall transactivating process. We have isolated a human complementary DNA from the new gene MSS1, which may encode such a cellular factor, by transcomplementation of a yeast sgv1- mutant. The MSS1 protein shares 42% sequence identity with the human TBP-1 protein, which binds Tat in vitro and suppresses Tat-mediated transactivation in vivo (ref. 6). We report here that the levels of HIV activation by Tat correlate with endogenous levels of MSS1 messenger RNA. Furthermore, we provide evidence that expression of MSS1 enhances the Tat-mediated transactivation. Our results suggest that MSS1 has a key role in activation of HIV genes regulated by Tat.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1992

Yeast casein kinase I homologues: an essential gene pair.

We report the isolation of an essential pair of Saccharomyces cerevisiae genes that encode protein kinase homologues. The two genes were independently isolated as dosage-dependent suppressors. Increased dosage of YCK1 suppressed defects caused by reduced SNF1 protein kinase activity, and increased dosage of YCK2 relieved sensitivity of wild-type cells to salt stress. The two genes function identically in the two growth assays, and loss of function of either gene alone has no discernible effect on growth. However, loss of function of both genes results in inviability. The two predicted protein products share 77% overall amino acid identity and contain sequence elements conserved among protein kinases. Partial sequence obtained for rabbit casein kinase I shares 64% identity with the two yeast gene products. Moreover, an increase in casein kinase I activity is observed in extracts from cells overexpressing YCK2. Thus YCK1 and YCK2 appear to encode casein kinase I homologues.

Amino Acid Sequence↗
PubMed1992

A chimeric ubiquitin conjugating enzyme that combines the cell cycle properties of CDC34 (UBC3) and the DNA repair properties of RAD6 (UBC2): implications for the structure, function and evolution of the E2s.

The CDC34 (UBC3) protein from Saccharomyces cerevisiae has a 125 residue tail that contains a polyacidic region flanked on either side by sequences of mixed composition. We show that although a catalytic domain is essential for CDC34 activity, a major cell cycle determinant of this enzyme is found within a 74 residue segment of the tail that does not include the polyacidic stretch or downstream sequences. Transposition of the CDC34 tail onto the catalytic domain of a functionally unrelated E2 such as RAD6 (UBC2) results in a chimeric E2 that combines RAD6 and CDC34 activities within the same polypeptide. In addition to the tail, the cell cycle function exhibited by the chimera and CDC34 is probably dependent on a conserved region of the catalytic domain that is shared by both RAD6 and CDC34. Despite this similarity, the CDC34 catalytic domain cannot substitute for the DNA repair and growth functions of the RAD6 catalytic domain, indicating that although these domains are structurally related, sufficient differences exist to maintain their functional individuality. Expression of the CDC34 catalytic domain and tail as separate polypeptides are capable of only partial function; thus, while the tail displays autonomous structural characteristics, there is considerable advantage gained when both domains coexist within the same polypeptide. The ability of these and other derivatives to restore partial function to a cdc34 temperature-sensitive mutant but not to a disruption mutant suggests that interaction between two CDC34 polypeptides is a requirement of CDC34 activity. Based on this idea we propose a model that accounts for the initiating steps leading to multi-ubiquitin chain synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Fission yeast sts1+ gene encodes a protein similar to the chicken lamin B receptor and is implicated in pleiotropic drug-sensitivity, divalent cation-sensitivity, and osmoregulation.

The Schizosaccharomyces pombe sts1+ gene, identified by supersensitive mutations to a protein kinase inhibitor, staurosporine, was isolated by complementation by the use of a fission yeast genomic library. Nucleotide sequencing shows that the sts1+ gene encodes a 453 amino acid putative membrane-associated protein that is significantly similar (26% identity) to the chicken lamin B receptor. It is also highly related (53% identity) to a budding yeast ORF, YGL022. These three proteins contain a similar hydrophobicity pattern consisting of eight or nine putative transmembrane domains. By gene disruption we demonstrate that the sts1+ gene is not essential for viability. These disruptants exhibit pleiotropic defects, such as cold-sensitivity for growth and at the permissive temperature, a supersensitivity to divalent cations and several unrelated drugs including staurosporine, caffeine, chloramphenicol, sorbitol, and SDS. Disruption of the sts1+ gene does not lead to a sensitivity to thiabendazole or hydroxyurea.

Amino Acid Sequence↗
PubMed1992

Yeast KRE2 defines a new gene family encoding probable secretory proteins, and is required for the correct N-glycosylation of proteins.

We have cloned, sequenced and disrupted the KRE2 gene of Saccharomyces cerevisiae, identified by killer-resistant mutants with a defective cell wall receptor for the toxin. The KRE2 gene is close to PHO8 on chromosome 4, and encodes a predicted 49-kD protein, Kre2p, that probably enters the secretory pathway. Haploid cells carrying a disruption of the KRE2 locus grow more slowly than wild-type cells at 30 degrees, and fail to grow at 37 degrees. At 30 degrees, kre2 mutants showed altered N-linked glycosylation of proteins, as the average size of N-linked outer chains was reduced. We identified two other genes, YUR1 on chromosome 10, and KTR1 on chromosome 15, whose predicted products share 36% identity with Kre2p over more than 300 amino acid residues. Yur1p has an N-terminal signal sequence like Kre2p, while Ktr1p has a predicted topology consistent with a type 2 membrane protein. In all cases the conserved regions of these proteins appear to be on the lumenal side of secretory compartments, suggesting related function. KRE2, KTR1 and YUR1 define a new yeast gene family.

Amino Acid Sequence↗
PubMed1991

The medium chains of the mammalian clathrin-associated proteins have a homolog in yeast.

We have cloned and sequenced mouse brain AP47, the medium chain of the trans-Golgi network clathrin-associated protein complex AP-1. The predicted protein sequence of AP47 is closely related to rat and calf brain AP50, the corresponding medium chain of the plasma-membrane clathrin-associated protein complex AP-2. We have also identified in the yeast genome an open reading frame encoding a protein of previously unknown function. Referred to here as YAP54, its predicted protein sequence displays a striking homology to AP47. We therefore propose that Yap54 is the medium chain subunit of a putative AP-1 complex in yeast. From the analyses of the optimized sequence alignments of AP47, AP50 and Yap54p, we suggest a model for the domain organization of the medium chains.

Adaptor Protein Complex 2↗
PubMed1991

DNA-binding properties of the product of the testis-determining gene and a related protein.

THE upstream region of the human glyceraldehyde-3-phosphate dehydrogenase gene contains an insulin-response element (IRE-A) responsible for insulin-dependent transcription of the gene. The open reading frame of a rat complementary DNA encoding a protein (IRE-ABP) that binds to this sequence contains an HMG box motif that is 67% identical to the mouse candidate gene for the testis-determining factor SRY, and 98% identical to the mouse SRY-like gene, a4. Here we report that IRE-ABP and SRY bind to IRE-A DNA with comparable specificity in a DNase-I footprinting assay. Two females with sex reversal were found to have a single amino-acid substitution in the HMG box domain of SRY at position 3 and 7, respectively. SRY derivatives containing corresponding mutations do not make contact with IRE-A DNA. These results are direct evidence that mouse SRY-like proteins are sequence-specific DNA-binding proteins and identify two amino acids critical to this interaction. Moreover, IRE-A is a candidate SRY-response element.

Amino Acid Sequence↗