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1.
Genetic screening of Saccharomyces cerevisiae mutants defective in Ca2+ homeostasis identified cls2, which exhibits a specific Ca2+-sensitive growth phenotype. We describe here the CLS2 gene and a multicopy suppressor (named BCL21, for bypass of CLS2) of the cls2 mutation. The CLS2 gene encodes a polypeptide of 410 amino acid residues, and its hydropathy profile indicates that the predicted Cls2 protein (Cls2p) contains ten putative membrane spanning regions. Immunofluorescent staining of the yeast cells expressing epitopetagged Cls2p suggests that Cls2p is localized to endoplasmatic reticulum (ER) membrane. A cls2 disruption strain is viable, but shows a Ca2+-sensitive phenotype like the original cls2 mutants. BCL21 suppresses the cls2 disruption mutation, indicating that the multicopy suppression does not require the Cls2p. Suppression of cls2 was observed even after introduction of a singlecopy plasmid harboring BCL21. The BCL21 gene encodes a protein of 382 amino acid residues and is identical to the SUR1 gene. sur1 was originally isolated as a suppressor of rvs161, which has reduced viability in nutrient starvation conditions. Possible mechanisms of the multicopy suppression are discussed. 相似文献
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GIGANTEA: a circadian clock-controlled gene that regulates photoperiodic flowering in Arabidopsis and encodes a protein with several possible membrane-spanning domains. 总被引:19,自引:0,他引:19
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S Fowler K Lee H Onouchi A Samach K Richardson B Morris G Coupland J Putterill 《The EMBO journal》1999,18(17):4679-4688
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A DBL-homologous region of the yeast CLS4/CDC24 gene product is important for Ca(2+)-modulated bud assembly. 总被引:7,自引:0,他引:7
S Miyamoto Y Ohya Y Sano S Sakaguchi H Iida Y Anraku 《Biochemical and biophysical research communications》1991,181(2):604-610
The CLS4/CDC24 is essential for the budding process of the yeast Saccharomyces cerevisiae. Disruption of the CLS4/CDC24 gene is lethal, and expression of the CLS4 product under the control of the GAL1 promoter is sufficient for cellular growth. The CLS4 product is detected in yeast cell lysate with an apparent molecular mass of 93 kD (854 amino acid residues) and shows homology with the human DBL oncogene product. Temperature-sensitive cdc24-1 mutation is located in the N-terminal portion of the protein whereas Ca(2+)-sensitive cls4-1 mutation is present after the DBL-homologous region (amino acid residues 281-518) near the putative Ca(2+)-binding site. Mutations within the DBL-homologous region are responsible for the Ca(2+)-sensitive phenotype. Thus the CLS4 gene product seems to have several functional domains within the molecule essential for bud assembly. 相似文献
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Furuya T Okura M Ruiz FA Scott DA Docampo R 《The Journal of biological chemistry》2001,276(35):32437-32445
Intracellular Ca(2+) in Trypanosoma cruzi is mainly located in an acidic compartment named the acidocalcisome, which among other pumps and exchangers possesses a plasma membrane-type Ca(2+)-ATPase. Evidence for an endoplasmic reticulum-located Ca(2+) uptake has been more elusive and based on indirect results. Here we report the cloning and sequencing of a gene encoding a sarcoplasmic-endoplasmic reticulum-type Ca(2+)-ATPase from T. cruzi. The protein (TcSCA) predicted from the nucleotide sequence of the gene has 1006 amino acids and a molecular mass of 109.7 kDa. Several sequence motifs found in sarcoplasmic-endoplasmic reticulum-type Ca(2+)-ATPases were present in TcSCA. Expression of TcSCA in yeast mutants deficient in the Golgi and vacuolar Ca(2+) pumps (pmr1 pmc1 cnb 1) restored growth on EGTA. Membranes were isolated from the pmr1 pmc1 cnb1 mutant transformed with TcSCA, and it was found that the TcSCA polypeptide formed a Ca(2+)-dependent and hydroxylamine-sensitive (32)P-labeled phosphoprotein of 110 kDa in the presence of [gamma-(32)P]ATP. Cyclopiazonic acid, but not thapsigargin, blocked this phosphoprotein formation. Transgenic parasites expressing constructs of TcSCA with green fluorescent protein exhibited co-localization of TcSCA with the endoplasmic reticulum proteins BiP and calreticulin. An endoplasmic reticulum location was also found in amastigotes and trypomastigotes using a polyclonal antibody against a COOH-terminal region of the protein. The ability of TcSCA to restore growth of mutant pmr1 pmc1 cnb 1 on medium containing Mn(2+) suggests that TcSCA may also regulate Mn(2+) homeostasis by pumping Mn(2+) into the endoplasmic reticulum of T. cruzi. 相似文献
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The yeast EUG1 gene encodes an endoplasmic reticulum protein that is functionally related to protein disulfide isomerase. 总被引:4,自引:0,他引:4
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The product of the EUG1 gene of Saccharomyces cerevisiae is a soluble endoplasmic reticulum protein with homology to both the mammalian protein disulfide isomerase (PDI) and the yeast PDI homolog encoded by the essential PDI1 gene. Deletion or overexpression of EUG1 causes no growth defects under a variety of conditions. EUG1 mRNA and protein levels are dramatically increased in response to the accumulation of native or unglycosylated proteins in the endoplasmic reticulum. Overexpression of the EUG1 gene allows yeast cells to grow in the absence of the PDI1 gene product. Depletion of the PDI1 protein in Saccharomyces cerevisiae causes a soluble vacuolar glycoprotein to accumulate in its endoplasmic reticulum form, and this phenotype is only partially relieved by the overexpression of EUG1. Taken together, our results indicate that PDI1 and EUG1 encode functionally related proteins that are likely to be involved in interacting with nascent polypeptides in the yeast endoplasmic reticulum. 相似文献
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The ACA4 gene of Arabidopsis encodes a vacuolar membrane calcium pump that improves salt tolerance in yeast 总被引:13,自引:0,他引:13
Several lines of evidence suggest that regulation of intracellular Ca(2+) levels is crucial for adaptation of plants to environmental stress. We have cloned and characterized Arabidopsis auto-inhibited Ca(2+)-ATPase, isoform 4 (ACA4), a calmodulin-regulated Ca(2+)-ATPase. Confocal laser scanning data of a green fluorescent protein-tagged version of ACA4 as well as western-blot analysis of microsomal fractions obtained from two-phase partitioning and Suc density gradient centrifugation suggest that ACA4 is localized to small vacuoles. The N terminus of ACA4 contains an auto-inhibitory domain with a binding site for calmodulin as demonstrated through calmodulin-binding studies and complementation experiments using the calcium transport yeast mutant K616. ACA4 and PMC1, the yeast vacuolar Ca(2+)-ATPase, conferred protection against osmotic stress such as high NaCl, KCl, and mannitol when expressed in the K616 strain. An N-terminally modified form of ACA4 specifically conferred increased NaCl tolerance, whereas full-length ATPase had less effect. 相似文献
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A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection 总被引:91,自引:0,他引:91
Murine type C ecotropic retrovirus infection is initiated by virus envelope binding to a membrane receptor expressed on mouse cells. We have identified a cDNA clone that may encode for this receptor through a strategy combining gene transfer of mouse NIH 3T3 DNA into nonpermissive human EJ cells, selection of EJ clones that have acquired susceptibility to infection by retrovirus vectors containing drug resistance genes, and identification of the putative receptor cDNA clone through linkage to a mouse repetitive DNA sequence. Human EJ cells that express the cDNA acquire a million-fold increase in MuLV infectivity. The predicted 622 amino acid sequence of the putative receptor protein is extremely hydrophobic; 14 potential membrane-spanning domains have been identified. A computer-based search of sequence data banks did not identify a protein with significant similarity to the putative receptor. We conclude that a novel membrane protein determines susceptibility to ecotropic MuLV infection by binding and/or fusion with the virus envelope. 相似文献
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A new lymphocyte-specific gene which encodes a putative Ca2+-binding protein is not expressed in transformed T lymphocyte lines 总被引:4,自引:0,他引:4
J Jongstra G F Tidmarsh J Jongstra-Bilen M M Davis 《Journal of immunology (Baltimore, Md. : 1950)》1988,141(11):3999-4004
The LSP1 gene is a new lymphocyte-specific gene which is expressed in normal mouse B and T lymphocytes and in transformed B cells but not (or in much smaller amounts) in nine T lymphoma lines tested. No LSP1 mRNA is found in myeloid cells or in liver, kidney, or heart tissue. Inspection of the predicted LSP1 protein sequence reveals the presence of two putative Ca2+-binding domains in the LSP1 protein. Southern blotting analysis of genomic DNA from mouse liver suggests that the LSP1 gene is present as one copy per haploid genome. Similar analysis of genomic DNA extracted from three transformed B cell lines and five transformed T cell lines shows that the absence of LSP1 mRNA in T cell lines is not due to deletion or gross rearrangements of the LSP1 locus. With the use of the mouse LSP1 cDNA as a probe we can detect a cross-hybridizing RNA species in four normal human functional T cell lines but not in three transformed human T cell lines. This suggests that at least part of the DNA sequence and the expression pattern of the LSP1 gene is conserved between mouse and man. These conserved features, together with the particular expression pattern and the protein sequence homologies, suggest that the LSP1 protein is involved in a Ca2+-dependent aspect of normal T cell growth. 相似文献
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A cDNA clone, called CLB1, was isolated from a cDNA library from tomato (Lycopersicon esculentum) and characterized. The CLB1 cDNA contains an open reading frame of 1518 bp, and encodes a putative protein of 506 amino acids with a predicted molecular mass of 54 633 Da. The deduced CLB1 amino acid sequence contains a domain that exhibits from 26% to 37% identity with the Ca2+-dependent lipid-binding domains of cytosolic phospholipase A2, protein kinase C, Rabphilin-3A, and Synaptotagmin I of animals. Southern blot analysis indicates that the CLB1 gene belongs to a small gene family in the tomato genome. The CLB1 mRNA is preferentially expressed in fruit tissues. 相似文献
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A gene that encodes a protein consisting solely of zinc finger domains is preferentially expressed in transformed mouse cells. 总被引:6,自引:3,他引:3
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We describe the cloning and characterization of the mouse MOK-2 gene, a new member of the Krüppel family of zinc finger proteins. Sequencing of both cDNA and genomic clones showed that the predicted MOK-2 protein consists of seven zinc finger domains with only five additional amino acids. The finger domains of MOK-2 are highly homologous to one another but not to those of other zinc finger proteins. MOK-2 is preferentially expressed in transformed cell lines, brain tissue, and testis tissue. Its possible role in cellular transformation is discussed. 相似文献
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The cell cycle control gene cdc2+ of fission yeast encodes a protein kinase potentially regulated by phosphorylation 总被引:75,自引:0,他引:75
The cdc2+ gene function has an important role in controlling the commitment of the fission yeast cell to the mitotic cycle and the timing of mitosis. We have raised antibodies against the cdc2+ protein using synthetic peptides and have demonstrated that it is a 34 kd phosphoprotein with protein kinase activity. The protein level and phosphorylation state remain unchanged during the mitotic cycle of rapidly growing cells. When cells cease to proliferate and arrest in G1 the protein becomes dephosphorylated and loses protein kinase activity. Exit from the mitotic cycle and entry into stationary phase may be controlled in part by modulation of the cdc2 protein kinase activity by changes in its phosphorylation state. 相似文献
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Holmes AR McNab R Millsap KW Rohde M Hammerschmidt S Mawdsley JL Jenkinson HF 《Molecular microbiology》2001,41(6):1395-1408
Streptococcus pneumoniae colonizes the nasopharynx in up to 40% of healthy subjects, and is a leading cause of middle ear infections (otitis media), meningitis and pneumonia. Pneumococci adhere to glycosidic receptors on epithelial cells and to immobilized fibronectin, but the bacterial adhesins mediating these reactions are largely uncharacterized. In this report we describe a novel pneumococcal protein PavA, which binds fibronectin and is associated with pneumococcal adhesion and virulence. The pavA gene, present in 64 independent isolates of S. pneumoniae tested, encodes a 551 amino acid residue polypeptide with 67% identical amino acid sequence to Fbp54 protein in Streptococcus pyogenes. PavA localized to the pneumococcal cell outer surface, as demonstrated by immunoelectron microscopy, despite lack of conventional secretory or cell-surface anchorage signals within the primary sequence. Full-length recombinant PavA polypeptide bound to immobilized human fibronectin in preference to fluid-phase fibronectin, in a heparin-sensitive interaction, and blocked binding of wild-type pneumococcal cells to fibronectin. However, a C-terminally truncated PavA' polypeptide (362 aa residues) failed to bind fibronectin or block pneumococcal cell adhesion. Expression of pavA in Enterococcus faecalis JH2-2 conferred > sixfold increased cell adhesion levels to fibronectin over control JH2-2 cells. Isogenic mutants of S. pneumoniae, either abrogated in PavA expression or producing a 42 kDa C-terminally truncated protein, showed up to 50% reduced binding to immobilized fibronectin. Inactivation of pavA had no effects on growth rate, cell morphology, cell-surface physico-chemical properties, production of pneumolysin, autolysin, or surface proteins PspA and PsaA. Isogenic pavA mutants of encapsulated S. pneumoniae D39 were approximately 104-fold attenuated in virulence in the mouse sepsis model. These results provide evidence that PavA fibronectin-binding protein plays a direct role in the pathogenesis of pneumococcal infections. 相似文献
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Characterization of near-native excited states of a protein provides insights into various biological functions such as co-operativity, protein-ligand, and protein-protein interactions. In the present study, we investigated the ruggedness of the native state of EhCaBP using nonlinear temperature dependence of backbone amide-proton chemical shifts. EhCaBP is a two-domain EF-hand calcium sensor protein consisting of two EF-hands in each domain and binds four Ca2+ ions. It has been observed that approximately 30% of the residues in the protein access alternative conformations. Theoretical modeling suggested that these low-energy excited states are within 2-3 kcal/mol from the native state. Further, it is interesting to note that the residues accessing alternative conformations are more dominated in the C-terminal domain compared with its N-terminal counterpart suggesting that the former is more rugged in its native state. These distinct characteristics of N- and C-terminal domains of a calcium sensor protein belonging to the super family of calmodulin would have implications for domain dependent Ca2+ signaling pathways. 相似文献
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The Rhizobium nodulation gene nodO encodes a Ca2(+)-binding protein that is exported without N-terminal cleavage and is homologous to haemolysin and related proteins. 总被引:23,自引:2,他引:21
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Nodulation and host-specific recognition of legumes such as peas and Vicia spp. are encoded by the nodulation (nod) genes of Rhizobium leguminosarum biovar viciae. One of these genes, nodO, has been shown to encode an exported protein that contains a multiple tandem repeat of a nine amino acid domain. This domain was found to be homologous to repeated sequences in a group of bacterial exported proteins that includes haemolysin, cyclolysin, leukotoxin and two proteases. These proteins are secreted by a mechanism that does not involve an N-terminal signal peptide. The NodO protein is present in the growth medium of Rhizobium bacteria induced for nod gene expression, and partial protein sequencing of the purified protein showed that there is no N-terminal cleavage of the exported protein. It has been suggested that the internally repeated domain of haemolysin may be involved in Ca2(+)-mediated binding to erythrocytes and we show that the NodO protein can bind 45Ca2+. It is proposed that the NodO protein may interact directly with plant root cells in a Ca2(+)-dependent way, thereby mediating an early stage in the recognition that occurs between Rhizobium and its host legume. 相似文献