RESUMO
Phosphatidylethanolamine synthesis through the phosphatidylserine (PtdSer) decarboxylation pathway requires PtdSer transport from the endoplasmic reticulum or mitochondrial-associated membrane to the mitochondrial inner membrane in mammalian cells. The transport-dependent PtdSer decarboxylation in permeabilized Chinese hamster ovary (CHO) cells was enhanced by cytosolic factors from bovine brain. A cytosolic protein factor exhibiting this enhancing activity was purified, and its amino acid sequence was partially determined. The sequence was identical to part of the amino acid sequence of an EF-hand type calcium-binding protein, S100B. A His(6)-tagged recombinant CHO S100B protein was able to remarkably enhance the transport-dependent PtdSer decarboxylation in permeabilized CHO cells. Under the standard assay conditions for PtdSer decarboxylase, the recombinant S100B protein did not stimulate PtdSer decarboxylase activity and exhibited no PtdSer decarboxylase activity. These results implicated the S100B protein in the transport of PtdSer to the mitochondrial inner membrane.
Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Mitocôndrias/metabolismo , Fatores de Crescimento Neural/fisiologia , Fosfatidilserinas/metabolismo , Proteínas S100 , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico , Encéfalo/metabolismo , Células CHO , Cálcio/farmacologia , Proteínas de Ligação ao Cálcio/isolamento & purificação , Bovinos , Cricetinae , Citosol/química , Descarboxilação , Membranas Intracelulares/metabolismo , Dados de Sequência Molecular , Fatores de Crescimento Neural/isolamento & purificação , Permeabilidade , Proteínas Recombinantes/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100 , Homologia de Sequência de AminoácidosRESUMO
Phosphatidylglycerophosphate (PGP) synthase catalyses the committed step in the biosynthesis of phosphatidylglycerol and cardiolipin in mammalian cells. Recently we isolated a Chinese hamster ovary (CHO) PGS1 cDNA encoding PGP synthase. In the present study we purified this PGP synthase to near-homogeneity from the mitochondrial fraction of CHO-K1 cells; the final enzyme preparation gave a single 60 kDa protein on SDS/PAGE. Polyclonal antibodies raised against a recombinant CHO PGS1 protein cross-reacted with the purified 60 kDa protein and with CHO membrane proteins of 60 kDa and 62 kDa that increased after transfection with the PGS1 cDNA. The 60 and 62 kDa protein levels in a PGP synthase-defective mutant of CHO-K1 cells were markedly lower than those in CHO-K1 cells. These results indicated that the purified 60 kDa protein was PGP synthase encoded by the PGS1 gene. In addition we found that the purified PGP synthase had no PGP phosphatase activity, indicating that phosphatidylglycerol was produced from CDP-diacylglycerol through two steps catalysed by distinct enzymes, PGP synthase and PGP phosphatase.
Assuntos
Transferases (Outros Grupos de Fosfato Substituídos)/isolamento & purificação , Animais , Western Blotting , Células CHO , Catálise , Cromatografia Líquida , Cricetinae , Cricetulus , Eletroforese em Gel de Poliacrilamida , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Frações Subcelulares/enzimologia , Transferases (Outros Grupos de Fosfato Substituídos)/genéticaRESUMO
A CHO-K1 cell mutant with a specific decrease in cellular phosphatidylethanolamine (PE) level was isolated as a variant resistant to Ro09-0198, a PE-directed antibiotic peptide. The mutant was defective in the phosphatidylserine (PS) decarboxylation pathway for PE formation, in which PS produced in the endoplasmic reticulum is transported to mitochondria and then decarboxylated by an inner mitochondrial membrane enzyme, PS decarboxylase. Neither PS formation nor PS decarboxylase activity was reduced in the mutant, implying that the mutant is defective in some step of PS transport. The transport processes of phospholipids between the outer and inner mitochondrial membrane were analyzed by use of isolated mitochondria and two fluorescence-labeled phospholipid analogs, 1-palmitoyl-2-[N-[6(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]caproyl]-PS (C6-NBD-PS) and C6-NBD-phosphatidylcholine (C6-NBD-PC). On incubation with the CHO-K1 mitochondria, C6-NBD-PS was readily decarboxylated to C6-NBD-PE, suggesting that the PS analog was partitioned into the outer leaflet of mitochondria and then translocated to the inner mitochondrial membrane. The rate of decarboxylation of C6-NBD-PS in the mutant mitochondria was reduced to approximately 40% of that in the CHO-K1 mitochondria. The quantity of phospholipid analogs translocated from the outer leaflet of mitochondria into inner mitochondrial membranes was further examined by selective extraction of the analogs from the outer leaflet of mitochondria. In the mutant mitochondria, the translocation of C6-NBD-PS was significantly reduced, whereas the translocation of C6-NBD-PC was not affected. These results indicate that the mutant is defective in PS transport between the outer and inner mitochondrial membrane and provide genetic evidence for the existence of a specific mechanism for intramitochondrial transport of PS.
Assuntos
Mitocôndrias/metabolismo , Mutação , Fosfatidilserinas/metabolismo , Animais , Transporte Biológico , CDPdiacilglicerol-Serina O-Fosfatidiltransferase/metabolismo , Células CHO , Carboxiliases/metabolismo , Cricetinae , Cricetulus , Fosfatidiletanolaminas/biossínteseRESUMO
Phosphatidylserine (PtdSer) in Chinese hamster ovary (CHO) cells is synthesized through the action of PtdSer synthase (PSS) I and II, which catalyzes the exchange of L-serine with the base moiety of phosphatidylcholine and phosphatidylethanolamine, respectively. The PtdSer synthesis in a CHO cell mutant, PSA-3, which lacks PSS I but has normal PSS II activity, was almost completely inhibited by the addition of PtdSer to the culture medium, like that in the wild-type CHO-K1 cells. In contrast, the PtdSer synthesis in a PSS II-overproducing stable transformant of CHO-K1, K1/wt-pssB, was reduced by only 35% upon addition of PtdSer. The serine exchange activity in a membrane fraction of K1/wt-pssB cells was not inhibited by PtdSer at all, whereas those of PSA-3 and CHO-K1 cells were inhibited by >95%. These results indicated that PSS II activity in PSA-3 and CHO-K1 cells is inhibited by exogenous PtdSer and that overproduction of PSS II leads to the loss of normal control of PSS II activity by exogenous PtdSer. Although overproduced PSS II in K1/wt-pssB cells was not normally controlled by exogenous PtdSer, K1/wt-pssB cells cultivated without exogenous PtdSer exhibited a normal PtdSer biosynthetic rate similar to that in CHO-K1 cells. In contrast to K1/wt-pssB cells, another stable transformant of CHO-K1, K1/R97K-pssB, which overproduces R97K mutant PSS II, exhibited a approximately 4-fold higher PtdSer biosynthetic rate compared with that in CHO-K1 cells. These results suggested that for maintenance of a normal PtdSer biosynthetic rate, the activity of overproduced wild-type PSS II in K1/wt-pssB cells is depressed by an as yet unknown post-translational mechanisms other than those for the exogenous PtdSer-mediated inhibition and that Arg-97 of PSS II is critical for this depression of overproduced PSS II activity. When the cDNA-directed wild-type and R97K mutant PSS II activities were expressed at nonoverproduction levels in a PSS I- and PSS II-defective mutant of CHO-K1 cells, expression of the mutant PSS II activity but not that of the wild-type PSS II activity induced the PtdSer-resistant PtdSer biosynthesis. This suggested that Arg-97 of PSS II is critical also for the exogenous PtdSer-mediated inhibition of PSS II.
Assuntos
Transferases de Grupos Nitrogenados/metabolismo , Animais , Arginina , Células CHO , Cricetinae , Transferases de Grupos Nitrogenados/antagonistas & inibidores , Fosfatidilserinas/biossíntese , Fosfatidilserinas/farmacologia , Relação Estrutura-Atividade , TransfecçãoAssuntos
Fosfatidilserinas/metabolismo , Sequência de Aminoácidos , Animais , Células CHO/metabolismo , Carboxiliases/genética , Carboxiliases/fisiologia , Clonagem Molecular , Cricetinae , Dados de Sequência Molecular , Transferases de Grupos Nitrogenados/genética , Transferases de Grupos Nitrogenados/fisiologia , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/biossínteseRESUMO
Phosphatidylglycerophosphate (PGP) synthase catalyzes the first step in the cardiolipin (CL) branch of phospholipid biosynthesis in mammalian cells. In this study, we isolated a Chinese hamster ovary (CHO) cDNA encoding a putative protein similar in sequence to the yeast PGS1 gene product, PGP synthase. The gene for the isolated CHO cDNA was named PGS1. Expression of the CHO PGS1 cDNA in CHO-K1 cells and production of a recombinant CHO PGS1 protein with a N-terminal extension in Escherichia coli resulted in 15-fold and 90-fold increases of PGP synthase specific activity, respectively, establishing that CHO PGS1 encodes PGP synthase. A PGP synthase-defective CHO mutant, PGS-S, isolated previously (Ohtsuka, T., Nishijima, M., and Akamatsu, Y. (1993) J. Biol. Chem. 268, 22908-22913) exhibits striking reductions in biosynthetic rate and cellular content of phosphatidylglycerol (PG) and CL and shows mitochondrial morphological and functional abnormalities. The CHO PGS-S mutant transfected with the CHO PGS1 cDNA exhibited 620-fold and 7-fold higher PGP synthase activity than mutant PGS-S and wild type CHO-K1 cells, respectively, and had a normal cellular content and rate of biosynthesis of PG and CL. In contrast to mutant PGS-S, the transfectant had morphologically normal mitochondria. When the transfectant and mutant PGS-S cells were cultivated in a glucose-depleted medium, in which cellular energy production mainly depends on mitochondrial function, the transformant but not mutant PGS-S was capable of growth. These results demonstrated that the morphological and functional defects displayed by the PGS-S mutant are due directly to the reduced ability to make normal levels of PG and/or CL.
Assuntos
Mitocôndrias/enzimologia , Mitocôndrias/genética , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Cardiolipinas/biossíntese , Cricetinae , DNA Complementar/isolamento & purificação , Humanos , Microscopia Eletrônica , Mitocôndrias/ultraestrutura , Dados de Sequência Molecular , Mutação , Fosforilação Oxidativa , Fosfatidilgliceróis/biossíntese , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae , Alinhamento de Sequência , Transferases (Outros Grupos de Fosfato Substituídos)/biossínteseRESUMO
Phosphatidylserine (PS) in mammalian cells is synthesized through the exchange of free L-serine with the base moiety of phosphatidylcholine or phosphatidylethanolamine (PE). The serine base exchange in Chinese hamster ovary (CHO) cells is catalyzed by at least two enzymes, PS synthase (PSS) I and II. A PSS I-lacking mutant of CHO-K1 cells, PSA-3, which exhibits approximately 2-fold lower serine base exchange activity than CHO-K1, is defective in the conversion of phosphatidylcholine to PS but has the ability to convert PE to PS. The PSA-3 mutant requires exogenous PS or PE for cell growth. In the present study, from PSA-3 mutant cells, we isolated a mutant, named PSB-2, with a further decrease in the serine base exchange activity. The activity in the homogenate of PSB-2 mutant cells was approximately 10% that of PSA-3 mutant cells and approximately 5% that of CHO-K1 cells. The PSB-2 mutant exhibited an approximately 80% reduction in the PSS II mRNA level relative to that in PSA-3 mutant and CHO-K1 cells. These results showed that the PSB-2 mutant is defective in PSS II. Like the PSA-3 mutant, the PSB-2 mutant grew well in medium supplemented with PS. However, in the medium supplemented with PE, the PSB-2 mutant was incapable of growth, in contrast to the PSA-3 mutant. In the medium with exogenous PE, the PSB-2 mutant was defective in PS biosynthesis, whereas the PSA-3 mutant synthesized a normal amount of PS. A metabolic labeling experiment with exogenous [32P]PE revealed that the PSB-2 mutant was defective in the conversion of exogenous PE to PS. This defect and the growth and PS biosynthetic defects of the PSB-2 mutant cultivated with exogenous PE were complemented by the PSS II cDNA. In addition, the cDNA of the other PS synthase, PSS I, was shown not to complement the defect in the conversion of exogenous PE to PS of the PSB-2 mutant, implying that PSS I negligibly contributes to the conversion of PE to PS in CHO-K1 cells. These results indicated that PSS II is critical for the growth and PS biosynthesis of PSA-3 mutant cells cultivated with exogenous PE and suggested that most of the PS formation from PE in CHO-K1 cells is catalyzed by PSS II.
Assuntos
Transferases de Grupos Nitrogenados/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/biossíntese , Animais , Células CHO , Cricetinae , Cricetulus , DNA Complementar , Mutação , Transferases de Grupos Nitrogenados/genética , TransfecçãoRESUMO
Phosphatidylserine (PtdSer) synthesis in Chinese hamster ovary (CHO) cells occurs through the exchange of L-serine with the base moiety of phosphatidylcholine or phosphatidylethanolamine. The synthesis is depressed on the addition of PtdSer to the culture medium. A CHO cell mutant named mutant 29, whose PtdSer biosynthesis is highly resistant to this depression by exogenous PtdSer, has been isolated from CHO-K1 cells. In the present study, the PtdSer-resistant PtdSer biosynthesis in the mutant was traced to a point mutation in the PtdSer synthase I gene, pssA, resulting in the replacement of Arg-95 of the synthase by lysine. Introduction of the mutant pssA cDNA, but not the wild-type pssA cDNA, into CHO-K1 cells induced the PtdSer-resistant PtdSer biosynthesis. In a cell-free system, the serine base-exchange activity of the wild-type pssA-transfected cells was inhibited by PtdSer, but that of the mutant pssA-transfected cells was resistant to the inhibition. Like the mutant 29 cells, the mutant pssA-transfected cells grown without exogenous PtdSer exhibited an approximately 2-fold increase in the cellular PtdSer level compared with that in CHO-K1 cells, although the wild-type pssA-transfected cells did not exhibit such a significant increase. These results indicated that the inhibition of PtdSer synthase I by PtdSer is essential for the maintenance of a normal PtdSer level in CHO-K1 cells and that Arg-95 of the synthase is a crucial residue for the inhibition.
Assuntos
Transferases de Grupos Nitrogenados/metabolismo , Fosfatidilserinas/biossíntese , Fosfatidilserinas/farmacologia , Fosfolipídeos/metabolismo , Animais , Células CHO , Células Clonais , Cricetinae , Retroalimentação , Humanos , Cinética , Transferases de Grupos Nitrogenados/antagonistas & inibidores , Transferases de Grupos Nitrogenados/genética , Mutação Puntual , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Serina/metabolismo , TransfecçãoRESUMO
Phosphatidylserine (PtdSer) in mammalian cells is synthesized through an exchange of free L-serine for the base moiety of pre-existing phospholipids. Studies on PtdSer biosynthesis in Chinese hamster ovary (CHO) cells have suggested that the serine base-exchange is catalyzed by at least two different enzymes; one, named PtdSer synthase I (PSS I), uses phosphatidylcholine (PtdCho) and possibly phosphatidylethanolamine (PtdEtn) as phosphatidyl donors for the serine base-exchange, and the other, named PtdSer synthase II (PSS II), uses PtdEtn but not PtdCho as a phosphatidyl donor. Recently, cDNAs of the PSS I and II have been isolated from CHO-K1 cells. This review will briefly describe the current understanding of PtdSer synthases of mammalian cells, mainly CHO cells.
Assuntos
Isoenzimas/metabolismo , Transferases de Grupos Nitrogenados/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Clonagem Molecular , Cricetinae , Isoenzimas/química , Isoenzimas/genética , Dados de Sequência Molecular , Transferases de Grupos Nitrogenados/química , Transferases de Grupos Nitrogenados/genética , Conformação Proteica , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Cultured mammalian cell mutants defective in the biosynthesis of membrane phospholipids, although limited in number, are increasing our understanding of the molecular mechanisms underlying the biogenesis and the biological significance of membrane phospholipids in higher eukaryotes. This review summarizes the progress in the isolation and characterization of such mutants, focusing on those isolated from cultured Chinese hamster ovary (CHO) cells.
RESUMO
Phosphatidylserine (PtdSer) in mammalian cells is synthesized through the exchange of free L-serine for the polar head group (base) of preexisting phospholipid. We previously showed the presence of two different enzymes catalyzing the serine base exchange in Chinese hamster ovary (CHO) cells and isolated the cDNA of one of the enzymes, PtdSer synthase (PSS) I, which also catalyzes the exchange of the base moiety of phospholipid(s) for ethanolamine and choline. In this study, we cloned a CHO cDNA, designated as pssB, which encodes a protein exhibiting 32% amino acid sequence identity with CHO PSS I. Introduction of the pssB cDNA into CHO-K1 cells resulted in striking increases in both the serine and ethanolamine base exchange activities. In contrast to the PSS I cDNA, the pssB cDNA was incapable of increasing the choline base exchange activity. The expression of the pssB gene in Sf9 insect cells also results in striking increases in both serine and ethanolamine base exchange activities. The pssB cDNA was found to transform a PtdSer-auxotrophic PSS I-lacking mutant of CHO-K1 cells to PtdSer prototrophy. The PtdSer content of the resultant transformant grown without exogenous PtdSer for 2 days was 4-fold that of the mutant and similar to that of CHO-K1 cells, indicating that the pssB cDNA complemented the PtdSer biosynthetic defect of the PSS I-lacking mutant. These results suggested that the pssB cDNA encoded the second PtdSer synthase PSS II, which catalyzed the serine and ethanolamine base exchange, but not the choline base exchange.
Assuntos
Transferases de Grupos Nitrogenados/genética , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/biossíntese , Transferases/deficiência , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Clonagem Molecular , Cricetinae , Dados de Sequência Molecular , Mutação , Transferases de Grupos Nitrogenados/metabolismo , Fosfatidilcolinas/metabolismo , TransfecçãoRESUMO
Serine palmitoyltransferase (SPT; EC 2.3.1.50) catalyzes the initial step dedicated to sphingolipid biosynthesis and is thought to be a key enzyme for regulating cellular sphingolipid content. For SPT activity, the yeast Saccharomyces cerevisiae requires two genes, LCB1 and LCB2. We isolated mammalian LCB1 cDNA homologs from mouse and Chinese hamster ovary (CHO) cells and an LCB2 cDNA homolog from CHO cells. The mammalian LCB1 proteins are predicted to have about 35% amino acid identity to the yeast Lcb1 protein, whereas the CHO LCB2 protein is predicted to have about 40% amino acid identity to the yeast Lcb2 protein. Northern blot analysis of mRNA isolated from various mouse tissues revealed that the tissue distribution of both LCB1 and LCB2 messengers followed a similar pattern. Transfection of an SPT-defective CHO mutant strain with a CHO LCB1-expressing plasmid restored both SPT activity and de novo sphingolipid synthesis to the wild type levels, whereas transfection of the mutant strain with a CHO LCB2-expressing plasmid did not exhibit any recovery effects, indicating that the SPT defect in the mutant cells is specifically complemented by the CHO LCB1 homolog. Furthermore, when the SPT-defective mutant cells were transfected with a plasmid encoding a His6-tagged CHO LCB1 protein, SPT activity bound to a Ni2+-immobilized resin. These results indicate that the CHO LCB1 homolog encodes a component of SPT.
Assuntos
Aciltransferases/genética , Saccharomyces cerevisiae/genética , Esfingolipídeos/biossíntese , Aciltransferases/química , Aciltransferases/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Catálise , Clonagem Molecular , Cricetinae , DNA Complementar , Teste de Complementação Genética , Humanos , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae , Homologia de Sequência de Aminoácidos , Serina C-PalmitoiltransferaseRESUMO
In a previous study, we purified PAF-acetylhydrolase, which converts PAF to an inactive metabolite, lysoPAF, from peritoneal fluid of guinea pigs subjected to experimental endotoxin shock and found that this purified enzyme had similar biochemical properties to the plasma enzyme [Karasawa, K., Yato, M., Setaka, M., and Nojima, S. (1994) J. Biochem. 116, 374-379]. In this study, we isolated a homogeneous enzyme preparation from guinea pig plasma using a similar procedure. The molecular mass of this purified enzyme, as determined by SDS-PAGE was 58-63 kDa, larger than that (43 kDa) of the human enzyme. To elucidate the molecular structure of this enzyme and clarify its relationships with PAF-acetylhydrolases of other species, we isolated and sequenced a cDNA encoding this enzyme. Its cDNA contains an open reading frame encoding 436 amino acids and its predicted molecular mass (49 kDa) is lower than that of the native enzyme, suggesting that guinea pig plasma PAF-acetylhydrolase, unlike the human enzyme, is modified post-translationally, perhaps by glycosylation.
Assuntos
Escherichia coli/enzimologia , Fosfolipases A/biossíntese , Fosfolipases A/genética , Proteínas Recombinantes/biossíntese , 1-Alquil-2-acetilglicerofosfocolina Esterase , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Escherichia coli/genética , Cobaias , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta , Fosfolipases A/sangue , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
We have previously shown that a Chinese hamster ovary (CHO) cell mutant defective in phosphatidylserine synthase I recovers the enzyme activity on transfection with a pssA cDNA clone isolated from the parental CHO-K1. The resultant transfectant, CDT-1, exhibited about 20-fold higher specific activity of the enzyme in the membrane fraction than CHO-K1 cells. Polyclonal antibodies against two peptides of the predicted pssA product cross-reacted with a membrane protein having an apparent molecular mass of 42 kDa, which was overproduced in CDT-1 cells. By immunoprecipitation with the antibody, phosphatidylserine synthase I activity as well as the 42-kDa protein was eliminated from solubilized membrane proteins of CDT-1 cells. Both the enzyme activity and the 42-kDa protein of CHO-K1 cells were enriched in the mitochondria-associated membrane fraction and the microsome fraction, but neither was enriched in the mitochondria fraction or the cytosol fraction. These results suggest that the pssA gene encodes phosphatidylserine synthase I.
Assuntos
Proteínas de Membrana/metabolismo , Transferases de Grupos Nitrogenados , Transferases/metabolismo , Animais , Anticorpos , Células CHO , Cricetinae , Citosol/enzimologia , DNA Complementar , Immunoblotting , Cinética , Proteínas de Membrana/análise , Proteínas de Membrana/biossíntese , Microssomos/enzimologia , Mitocôndrias/enzimologia , Frações Subcelulares/enzimologia , Transfecção , Transferases/análise , Transferases/biossínteseRESUMO
We have isolated a full-length cDNA clone of the Chinese hamster ovary (CHO) pssC gene, which encodes mitochondrial phosphatidylserine decarboxylase. The cDNA clone is capable of increasing phosphatidylserine decarboxylase activity to 11-fold in CHO-K1 cells. The pssC gene product predicted from the cDNA sequence is composed of 409 amino acid residues. In an in vitro translation system coupled with in vitro transcription, the cDNA clone directs the formation of a protein with an apparent molecular mass of 46 kDa. In CHO-K1 cells, the cDNA clone leads to the production of two major peptides with apparent molecular masses of 38 and 34 kDa, as determined by Western blotting with an antibody raised against a recombinant pssC protein. When CHO-K1 cells transfected with the cDNA clone are labelled with [35S]methionine for a short period, proteins immunoprecipitated with the antibody lack radioactive 38 and 34 kDa peptides, but contain two radioactive peptides with apparent molecular masses of 46 and 42 kDa instead. The pssC gene product predicted from the cDNA sequence has, near its C-terminus, a unique Leu-Gly-Ser-Thr sequence which is known as a processing site for Escherichia coli phosphatidylserine decarboxylase. A mutant pssC cDNA clone, in which Ser378 in the conserved sequence is replaced by Ala, leads to overproduction of 46, 42 and 38 kDa peptides, but not a 34 kDa peptide. This mutant clone is incapable of increasing phosphatidylserine decarboxylase activity, in contrast to the wild-type clone. These results indicate that the processing at the Leu-Gly-Ser-Thr sequence is essential for formation of the active enzyme. Thus, the pssC gene product is converted into mature phosphatidylserine decarboxylase through multiple steps of post-translational processing.
Assuntos
Carboxiliases/metabolismo , Ovário/enzimologia , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Sequência de Bases , Carboxiliases/genética , Clonagem Molecular , Sequência Conservada , Cricetinae , Cricetulus , DNA Complementar , Feminino , Dados de Sequência Molecular , Peso Molecular , Reação em Cadeia da Polimerase , RNA/química , Frações Subcelulares/enzimologiaAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Capsídeo , Vesículas Revestidas/fisiologia , Proteínas de Ligação ao GTP , Proteínas/metabolismo , Fatores de Ribosilação do ADP , Acil Coenzima A , Animais , Transporte Biológico , Capsídeo/fisiologia , Vesículas Revestidas/metabolismo , Ciclofosfamida , Complexo de Golgi/metabolismo , Prednisona , VincristinaRESUMO
We have identified a novel gene, Translin, encoding a protein which specifically binds to consensus sequences at breakpoint junctions of chromosomal translocations in many cases of lymphoid malignancies. The encoded protein, Translin, is a previously undescribed type with no significant similarity to known proteins. In the native form, Translin polypeptides form a multimeric structure which is responsible for its DNA binding activity. Nuclear localization of Translin is limited to lymphoid cell lines, raising the intriguing possibility that nuclear transport of Translin is regulated in a physiologically significant way such that active nuclear transport is associated with the lymphoid specific process known as Ig/TCR gene rearrangement.
Assuntos
Proteínas de Ligação a DNA/genética , Recombinação Genética , Translocação Genética , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico , Núcleo Celular/genética , Núcleo Celular/metabolismo , Clonagem Molecular , Sequência Consenso , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Rearranjo Gênico , Humanos , Zíper de Leucina , Leucemia-Linfoma de Células T do Adulto/genética , Leucemia-Linfoma de Células T do Adulto/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Células Tumorais CultivadasRESUMO
Two new members (Sar1a and Sar1b) of the SAR1 gene family have been identified in mammalian cells. Using immunoelectron microscopy, Sar1 was found to be restricted to the transitional region where the protein was enriched 20-40-fold in vesicular carriers mediating ER to Golgi traffic. Biochemical analysis revealed that Sar1 was essential for an early step in vesicle budding. A Sar1-specific antibody potently inhibited export of vesicular stomatitis virus glycoprotein (VSV-G) from the ER in vitro. Consistent with the role of guanine nucleotide exchange in Sar1 function, a trans-dominant mutant (Sar1a[T39N]) with a preferential affinity for GDP also strongly inhibited vesicle budding from the ER. In contrast, Sar1 was not found to be required for the transport of VSV-G between sequential Golgi compartments, suggesting that components active in formation of vesicular carriers mediating ER to Golgi traffic may differ, at least in part, from those involved in intra-Golgi transport. The requirement for novel components at different stages of the secretory pathway may reflect the recently recognized differences in protein transport between the Golgi stacks as opposed to the selective sorting and concentration of protein during export from the ER.