RESUMO
SOL1, the founding member of the S. cerevisiae SOL family, was previously identified as a multi-copy suppressor of the los1 defect in tRNA-mediated nonsense suppression. Here we report that the four-member SOL family is not essential and that individual family members appear to have distinct functions. SOL1-SOL4 are homologous to genes encoding 6-phosphogluconolactonase (6Pgl) involved in the pentose phosphate pathway. Both Sol3p and Sol4p affect this activity. However, Sol4p does not act as a los1 multi-copy suppressor. In contrast, neither Sol1p nor Sol2p, both of which correct the los1 defect in nonsense suppression, possess detectable 6Pgl activity. Rather, Sol1p and Sol2p appear to function in tRNA nuclear export as sol1 and sol2 mutants possess elevated levels of nuclear tRNA. Members of the Sol protein family appear to have different subcellular distributions. Thus, Sol3p and Sol4p likely function in carbohydrate metabolism, while Sol1p and Sol2p appear to have roles in tRNA function and nuclear export, thereby defining an unusual protein family whose individual members are biochemically distinct and spatially dispersed.
Assuntos
Metabolismo dos Carboidratos , Núcleo Celular/metabolismo , Família Multigênica/genética , RNA de Transferência/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Núcleo Celular/fisiologia , Análise por Conglomerados , Biologia Computacional , Primers do DNA , Imunofluorescência , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Plasmídeos/genética , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Although yeast RNA polymerase III (Pol III) and the auxiliary factors TFIIIC and TFIIIB are well characterized, the mechanisms of class III gene regulation are poorly understood. Previous studies identified MAF1, a gene that affects tRNA suppressor efficiency and interacts genetically with Pol III. We show here that tRNA levels are elevated in maf1 mutant cells. In keeping with the higher levels of tRNA observed in vivo, the in vitro rate of Pol III RNA synthesis is significantly increased in maf1 cell extracts. Mutations in the RPC160 gene encoding the largest subunit of Pol III which reduce tRNA levels were identified as suppressors of the maf1 growth defect. Interestingly, Maf1p is located in the nucleus and coimmunopurifies with epitope-tagged RNA Pol III. These results indicate that Maf1p acts as a negative effector of Pol III synthesis. This potential regulator of Pol III transcription is likely conserved since orthologs of Maf1p are present in other eukaryotes, including humans.
Assuntos
Proteínas Fúngicas/fisiologia , RNA Polimerase III/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Animais , Northern Blotting , Núcleo Celular/metabolismo , Epitopos , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Humanos , Immunoblotting , Microscopia de Fluorescência , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Fenótipo , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , RNA/metabolismo , RNA Polimerase III/química , RNA Ribossômico/metabolismo , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Temperatura , Fator de Transcrição TFIIIB , Fatores de Transcrição/química , Fatores de Transcrição TFIII/química , Transcrição GênicaRESUMO
Reports of nuclear tRNA aminoacylation and its role in tRNA nuclear export (Lund and Dahlberg, 1998; Sarkar et al., 1999; Grosshans et al., 20001) have led to the prediction that there should be nuclear pools of aminoacyl-tRNA synthetases. We report that in budding yeast there are nuclear pools of tyrosyl-tRNA synthetase, Tys1p. By sequence alignments we predicted a Tys1p nuclear localization sequence and showed it to be sufficient for nuclear location of a passenger protein. Mutations of this nuclear localization sequence in endogenous Tys1p reduce nuclear Tys1p pools, indicating that the motif is also important for nucleus location. The mutations do not significantly affect catalytic activity, but they do cause defects in export of tRNAs to the cytosol. Despite export defects, the cells are viable, indicating that nuclear tRNA aminoacylation is not required for all tRNA nuclear export paths. Because the tRNA nuclear exportin, Los1p, is also unessential, we tested whether tRNA aminoacylation and Los1p operate in alternative tRNA nuclear export paths. No genetic interactions between aminoacyl-tRNA synthetases and Los1p were detected, indicating that tRNA nuclear aminoacylation and Los1p operate in the same export pathway or there are more than two pathways for tRNA nuclear export.
Assuntos
Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Núcleo Celular/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares , RNA de Transferência/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Tirosina-tRNA Ligase/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Fracionamento Celular , Núcleo Celular/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Reporter/genética , Teste de Complementação Genética , Humanos , Immunoblotting , Hibridização In Situ , Modelos Biológicos , Dados de Sequência Molecular , Sinais de Localização Nuclear , Aminoacil-RNA de Transferência/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Alinhamento de Sequência , Tirosina-tRNA Ligase/químicaRESUMO
Sorting isozymes are encoded by single genes, but the encoded proteins are distributed to multiple subcellular compartments. We surveyed the predicted protein sequences of several nucleic acid interacting sorting isozymes from the eukaryotic taxonomic domain and compared them with their homologs in the archaeal and eubacterial domains. Here, we summarize the data showing that the eukaryotic sorting isozymes often possess sequences not present in the archaeal and eubacterial counterparts and that the additional sequences can act to target the eukaryotic proteins to their appropriate subcellular locations. Therefore, we have named these protein domains ADEPTs (Additional Domains for Eukaryotic Protein Targeting). Identification of additional domains by phylogenetic comparisons should be generally useful for locating candidate sequences important for subcellular distribution of eukaryotic proteins.
Assuntos
Archaea/enzimologia , Proteínas/química , Frações Subcelulares/enzimologia , Alinhamento de SequênciaRESUMO
Nucleus/cytosol exchange requires a GTPase, Ran. In yeast Rna1p is the GTPase activating protein for Ran (RanGAP) and Prp20p is the Ran GDP/GTP exchange factor (GEF). RanGAP is primarily cytosolic and GEF is nuclear. Their subcellular distributions led to the prediction that Ran-GTP hydrolysis takes place solely in the cytosol and GDP/GTP exchange solely in the nucleus. Current models propose that the Ran-GTP/Ran-GDP gradient across the nuclear membrane determines the direction of exchange. We provide three lines of evidence that Rna1p enters and leaves the nuclear interior. (1) Rna1p possesses leucine-rich nuclear export sequences (NES) that are able to relocate a passenger karyophilic protein to the cytosol; alterations of consensus residues re-establish nuclear location. (2) Rna1p possesses other sequences that function as a novel nuclear localization sequence able to deliver a passenger cytosolic protein to the nucleus. (3) Endogenous Rna1p location is dependent upon Xpo1p/Crm1p, the yeast exportin for leucine-rich NES-containing proteins. The data support the hypothesis that Rna1p exists on both sides of the nuclear membrane, perhaps regulating the Ran-GTP/Ran-GDP gradient, participating in a complete RanGTPase nuclear cycle or serving a novel function.
Assuntos
Proteínas de Ligação ao GTP/análise , Proteínas de Ligação ao GTP/genética , Proteínas Ativadoras de GTPase , Proteínas Nucleares/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Imunofluorescência , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/metabolismo , Dados de Sequência Molecular , Sinais de Localização Nuclear , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Alinhamento de Sequência , Proteína ran de Ligação ao GTPRESUMO
MOD5 encodes a tRNA modification activity located in three subcellular compartments. Alternative translation initiation generates Mod5p-I, located in the mitochondria and the cytosol, and Mod5p-II, located in the cytosol and nucleus. Here we study the nucleus/cytosol distribution of overexpressed Mod5p-II. Nuclear Mod5p-II appears concentrated in the nucleolus, perhaps indicating that the nuclear pool may have a different biological role than the cytoplasmic and mitochondrial pools. Mod5p contains three motifs resembling bipartite-like nuclear localization sequences (NLSs), but only one is sufficient to locate a passenger protein to the nucleus. Mutations of basic residues of this motif cumulatively contribute to a cytosolic location for the fusion proteins. These alterations also cause decreased nuclear pools of endogenous Mod5p-II. Depletion of nuclear Mod5p-II does not affect tRNATyr function. Despite the NLS, most Mod5p is cytosolic. We assessed whether Mod5p sequences cause a karyophilic reporter to be located in the cytosol. By this assay, Mod5p may contain more than one region that functions as cytoplasmic retention and/or nuclear export sequences. Thus, distribution of Mod5p results from the presence/absence of mitochondrial targeting information and sequences antagonistic for nuclear and cytosolic locations. Mod5p is highly conserved; sequences responsible for subcellular distribution appear to reside in "accessory" motifs missing from prokaryotic counterparts.
Assuntos
Alquil e Aril Transferases , Isoenzimas/análise , Proteínas/análise , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Animais , Transporte Biológico , Núcleo Celular/enzimologia , Citoplasma , Citosol/enzimologia , Humanos , Isoenzimas/genética , Dados de Sequência Molecular , Sinais de Localização Nuclear , Proteínas/genética , Saccharomyces cerevisiae/genética , Frações SubcelularesRESUMO
The last step of tRNA splicing in the yeast Saccharomyces cerevisiae is catalyzed by an NAD-dependent 2'-phosphotransferase, which transfers the splice junction 2'-phosphate from ligated tRNA to NAD to produce ADP-ribose 1"-2" cyclic phosphate. We have purified the phosphotransferase about 28,000-fold from yeast extracts and cloned its structural gene by reverse genetics. Expression of this gene (TPT1) in yeast or in Escherichia coli results in overproduction of 2'-phosphotransferase activity in extracts. Tpt1 protein is essential for vegetative growth in yeast, as demonstrated by gene disruption experiments. No obvious binding motifs are found within the protein. Several candidate homologs in other organisms are identified by searches of the data base, the strongest of which is in Schizosaccharomyces pombe.
Assuntos
Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Splicing de RNA/genética , RNA Fúngico/genética , RNA de Transferência/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Dados de Sequência Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Alinhamento de SequênciaRESUMO
To understand the role of Los1p in pre-tRNA splicing, we sought los1 multicopy suppressors. We found SOL1 that suppresses both point and null LOS1 mutations. Since, when fused to the Ga14p DNA-binding domain, Los1p activates transcription, we tested whether Los1p regulates SOL1. We found that las1 mutants have depleted levels of SOL1 mRNA and Sol1p. Thus, LOS1 appears to positively regulate SOL1. SOL1 belongs to a multigene family with at least two additional members, SOL2 and SOL3. Sol proteins have extensive similarity to an unusual group of glucose-6-phosphate dehydrogenases. As the similarities are restricted to areas separate from the catalytic domain, these G6PDs may have more than one function. The SOL family appears to be unessential since cells with a triple disruption of all three SOL genes are viable. SOL gene disruptions negatively affect tRNA-mediated nonsense suppression and the severity increases with the number of mutant SOL genes. However, tRNA levels do not vary with either multicopy SOL genes or with SOL disruptions. Therefore, the Sol proteins affect tRNA expression/ function at steps other than transcription or splicing. We propose that LOS1 regulates gene products involved in tRNA expression/function as well as pre-tRNA splicing.
Assuntos
Proteínas de Drosophila , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Proteínas de Insetos , Splicing de RNA/genética , RNA Fúngico/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Calpaína , Hormônios de Inseto/genética , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , RNA de Transferência/genética , Supressão GenéticaRESUMO
The Saccharomyces cerevisiae genes, RRP1 and SRD1, are involved in processing rRNA precursor species to mature rRNAs. We reported previously that the rrp1-1 mutation caused temperature-sensitive lethality, hypersensitivity to aminoglycoside antibiotics, and defective processing of 27S pre-rRNA to 25S and 5.8S mature rRNAs. A second-site suppressor of the rrp1-1 mutation, srd1, corrects all three rrp1 mutant phenotypes. In order to learn more about the roles of the SRD1 and RRP1 genes in rRNA processing, we cloned and characterized the SRD1 gene. We identified an ORF, YCR18C, that complements srd1-2 suppression of rrp1-1. The DNA is physically located at the region of chromosome III where SRD1 has been genetically mapped. SRD1 encodes a putative 225 amino acid, 26 kDa protein containing a C2/C2 zinc finger motif that is also found in some transcription regulators and the eIF-2 beta translation initiating factors. The similarity of SRD1 to transcription regulators led us to test the model that srd1 mutations suppress rrp1 defects by altering the level of the RRP1 transcript. However, we found that SRD1 has no detectable effect on the steady state levels of RRP1 mRNA. We describe alternative models to explain the role of Srd1p in pre-rRNA processing.
Assuntos
Proteínas Fúngicas/genética , Genes Fúngicos , Processamento Pós-Transcricional do RNA , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Dedos de Zinco/genética , Sequência de Aminoácidos , Animais , Northern Blotting , Clonagem Molecular , Teste de Complementação Genética , Humanos , Dados de Sequência Molecular , Plasmídeos , Precursores de RNA/metabolismo , Mapeamento por Restrição , Supressão Genética , TemperaturaRESUMO
Mutations of the Saccharomyces cerevisiae LOS1 gene cause the accumulation of end matured intron-containing pre-tRNAs at elevated temperatures. In an effort to decipher the role of the LOS1 protein in pre-tRNA splicing, we have analyzed the LOS1 gene and its protein product. The LOS1 gene is located on the left arm of chromosome XI and the order of genes in this area of the chromosome is .... URA1 ... SAC1 TRP3 UBA1 STE6 LOS1 .... FAS1..... The LOS1 open reading frame encodes a putative protein of 1100 amino acids that shows no significant homology to other genes. The LOS1 open reading frame was tagged with the influenza virus hemagglutinin epitope recognized by the 12CA5 antibody. The 12CA5 antibody recognizes an epitope-tagged protein of the size predicted by the LOS1 open reading frame. Using this antibody for indirect immunofluorescence and cell fractionation studies we show that the LOS1 protein is located in nuclei. Los1p cannot be extracted from nuclei by treatment with nucleases, salts, or Triton X-100. This insolubility suggests that Los1p is a component of the nucleoskeleton. We propose that LOS1 mutations may affect pre-tRNA processing via alteration of the nuclear matrix.
Assuntos
Cromossomos Fúngicos , Genes Fúngicos , Proteínas Nucleares/biossíntese , Precursores de RNA/metabolismo , Splicing de RNA , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Núcleo Celular/metabolismo , Mapeamento Cromossômico , Clonagem Molecular , Escherichia coli/genética , Íntrons , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Matriz Nuclear/metabolismo , Proteínas Nucleares/genética , Oligodesoxirribonucleotídeos , Plasmídeos , Mapeamento por Restrição , Saccharomyces cerevisiae/genéticaRESUMO
STP1 is an unessential yeast gene involved in the removal of intervening sequences from some, but not all, families of intervening sequence-containing pre-tRNAs. Previously, we proposed that STP1 might encode a product that generates pre-tRNA conformations efficiently recognized by tRNA-splicing endonuclease. To test the predictions of this model, we have undertaken a molecular analysis of the STP1 gene and its products. The STP1 locus is located on chromosome IV close to at least two other genes involved in RNA splicing: PRP3 and SPP41. The STP1 open reading frame (ORF) could encode a peptide of 64,827 Da; however, inspection of putative transcriptional and translational regulatory signals and mapping of the 5' ends of mRNA provide evidence that translation of the STP1 ORF usually initiates at a second AUG to generate a protein of 58,081 Da. The STP1 ORF contains three putative zinc fingers. The first of these closely resembles both the DNA transcription factor consensus and the Xenopus laevis p43 RNA-binding protein consensus. The third motif more closely resembles the fingers found in spliceosomal proteins. Employing antisera to the endogenous STP1 protein and to STP1-LacZ fusion proteins, we show that the STP1 protein is localized to nuclei. The presence of zinc finger motifs and the nuclear location of the STP1 protein support the model that this gene product is involved directly in pre-tRNA splicing.
Assuntos
Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Splicing de RNA , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fatores de Transcrição , Dedos de Zinco , Sequência de Aminoácidos , Sequência de Bases , Compartimento Celular , Clonagem Molecular , Sequência Consenso , DNA Fúngico/genética , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Biossíntese de Proteínas , Mapeamento por Restrição , Alinhamento de SequênciaRESUMO
A complex locus on human chromosome 1 brings together sequences homologous to a G protein and two components of the RNA processing machinery of eukaryotic cells. Specifically, the seventh intron of the human Gi3 alpha gene contains a fusion of a partial snRNP E protein pseudogene to a variant U6 snRNA gene. The novel U6 sequence contains nine point mutations and a one nucleotide deletion relative to the major U6 genes from humans. Unlike all other vertebrate U6 genes characterized to date, the variant U6 gene is efficiently transcribed by RNA polymerase III even in the absence of all natural flanking sequences. The union of elements from the signal transduction pathway and the RNA processing machinery suggests the possibility of functional interplay.
Assuntos
Proteínas de Ligação ao GTP/genética , Regiões Promotoras Genéticas , RNA Nuclear Pequeno/genética , Ribonucleoproteínas/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos Par 1 , Humanos , Íntrons , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Pseudogenes , Ribonucleoproteínas Nucleares Pequenas , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais , Transcrição GênicaRESUMO
Sequence data from three previously-uncharacterized members of the snRNP E protein multigene family suggest that each is a non-transcribed processed pseudogene, even though one clone has the potential to code for a full-length protein with greater than 90% similarity to previously-characterized E protein cDNAs. Each of the newly-analyzed family members is without introns, contains a tract of polyadenylic acid residues, and is flanked by short direct repeats. In addition, the three sequences all contain point mutations that distinguish them from the E protein coding sequence. Seven point mutations are common to the three sequences described here and to two previously-described E protein pseudogenes. Although all of these mutations are transitions, only 5 of 7 could have been generated by deamination of methylated cytosines in inactive genes. Thus, the common mutations in the pseudogenes suggest an origin other than the expressed gene that we have described. Allelic variants for two of the pseudogenes were detected and repetitive elements are located near four of the five E protein pseudogenes that have been characterized.
Assuntos
Família Multigênica , Mutação , Pseudogenes , Ribonucleoproteínas/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA , Humanos , Dados de Sequência Molecular , Mapeamento por Restrição , Ribonucleoproteínas Nucleares PequenasRESUMO
Small nuclear ribonucleoproteins (snRNPs), which are composed of various U RNAs and several proteins, are components of the mRNA splicing apparatus. The snRNP protein E is encoded by a multigene family which consists of a single expressed gene and several processed pseudogenes. We have used somatic cell hybridization, in situ hybridization, and linkage analysis to both physically and genetically map the expressed E protein gene to human chromosome 1q25-43, with the most probable location being band 1q32. In addition to the snRNP E protein gene, two other snRNP components--the U1 RNA true multigene family and a group of class I U1 pseudogenes--are located on human chromosome 1.
Assuntos
Cromossomos Humanos Par 1 , Ligação Genética , Família Multigênica , Ribonucleoproteínas/genética , Animais , Bandeamento Cromossômico , Mapeamento Cromossômico , Genes , Humanos , Células Híbridas , Camundongos , Pseudogenes , Mapeamento por Restrição , Ribonucleoproteínas Nucleares Pequenas , Homologia de Sequência do Ácido NucleicoRESUMO
The human small nuclear ribonucleoprotein E protein is an 11,000-dalton basic protein which is an integral component of several small nuclear ribonucleoprotein complexes involved in RNA processing reactions. Sequence analysis of the E protein multigene family reveals that at least one gene for this component of the RNA splicing machinery is interrupted by four introns. The exons of this gene are identical to two cDNA clones isolated from independent tissue sources and span approximately 9 kilobase pairs. Primer extension data indicated the presence of two major transcription start sites. The upstream region of the small nuclear ribonucleoprotein E protein gene does not contain TATA or CCAAT sequences within 175 nucleotides of the transcription start sites. However, the proximal upstream region does contain several similarities to the promoter regions of both snRNA genes and vertebrate ribosomal protein genes.
Assuntos
Genes , Íntrons , Regiões Promotoras Genéticas , Ribonucleoproteínas/genética , Proteínas Ribossômicas/genética , Sequência de Aminoácidos , Sequência de Bases , Éxons , Humanos , Dados de Sequência Molecular , Conformação Proteica , Pseudogenes , Splicing de RNA , Mapeamento por Restrição , Ribonucleoproteínas Nucleares Pequenas , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
The snRNP E protein is one of four "core" proteins associated with the snRNAs of the U family (U1,U2,U4,U5, and U6). Screening of a human teratoma cDNA library with a partial cDNA for a human autoimmune antigen resulted in the isolation of a cDNA clone containing the entire coding region of this snRNP core protein. Comparison of the 5' end of this cDNA with the sequences of two processed pseudogenes and primer extension data suggest that the cDNA is nearly full length. The longest open reading frame in this clone codes for a basic 92 amino acid protein which is in perfect agreement with amino acid sequence data obtained from purified E protein. The predicted sequence of this protein reveals no extensive similarity to other snRNP proteins, but contains regions of similarity to a eukaryotic ribosomal protein.
Assuntos
Genes , Ribonucleoproteínas/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA de Neoplasias/genética , Humanos , Dados de Sequência Molecular , Conformação Proteica , Mapeamento por Restrição , Ribonucleoproteínas Nucleares Pequenas , Teratoma , Células Tumorais Cultivadas/metabolismoRESUMO
cDNAs coding for a plasma membrane Ca2+ pump were isolated from a human teratoma library and sequenced. The translated sequence contained 1,220 amino acids with a calculated molecular weight of 134,683. All regions of functional importance known from other ion-transporting ATPases could be identified. The translated sequence also contained, near the carboxyl terminus, the calmodulin-binding domain and two domains which are very rich in glutamic acid and aspartic acid. These two domains resemble calmodulin somewhat and one of them may play a role in the binding of Ca2+. The enzyme also contains domains rich in serine and threonine, one of which has a sequence matching those of good cAMP-dependent protein kinase substrates. The carboxyl-terminal region is important for regulation by calmodulin, proteolysis, and phosphorylation. Near the amino terminus are two domains which are very rich in lysine and glutamic acid, as well as two domains resembling EF hands, one of which also has some resemblance to calmodulin. Comparison of the cloned sequence with peptide sequences from the erythrocyte Ca2+ pump showed that the two proteins have a very high proportion of identical residues but are not 100% identical, indicating that they represent different isozymes.
Assuntos
ATPases Transportadoras de Cálcio/genética , Membrana Celular/enzimologia , Genes , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Enzimas de Restrição do DNA , DNA de Neoplasias/genética , Humanos , Dados de Sequência Molecular , Mapeamento de Nucleotídeos , Conformação Proteica , Especificidade da Espécie , TeratomaRESUMO
The complete amino acid sequence of the major clotting protein from the guinea pig seminal vesicle (SVP-1) has been determined by nucleotide sequencing of cDNA clones corresponding to the 3' terminus of an mRNA that codes for a protein precursor to SVP-1. The first 40 amino acids of the derived protein sequence are identical to those determined by N-terminal sequencing of SVP-1 isolated from the lumen of the seminal vesicle. This finding confirms that SVP-1 is cleaved from the C terminus of a larger precursor protein. The portion of the nucleotide sequence that codes for SVP-1 contains eight highly homologous but imperfect repeats of a 72-nucleotide domain. This repeated structure is also evident at the amino acid level. The consensus 24-amino acid repeat unit contains two lysine and three glutamine residues. Since the clotting of SVP-1 is known to involve the formation of gamma-glutamyl-epsilon-lysine crosslinks, it is likely that the 24-amino acid repeating unit is the unit of function of SVP-1.