RESUMO
Recent studies in human cells and in the yeast Yarrowia lipolytica have shown that peroxisomes consist of numerous structurally distinct subcompartments that differ in their import competency for various proteins and are related through a time-ordered conversion of one subcompartment to another. Our studies have implicated the fusion of small peroxisomal precursors as an early event in the multistep assembly of peroxisomes operating in Y. lipolytica. Newly discovered unexpected roles for peroxisomes in specific developmental programs have expanded the remarkable plasticity of peroxisomal functions. Here, we highlight recent discoveries on the highly dynamic nature of peroxisome assembly and function and suggest questions for future research in these areas.
Assuntos
Peroxissomos/fisiologia , Animais , Humanos , Fusão de Membrana , Transporte Proteico , Saccharomycetales/fisiologiaRESUMO
We have identified and purified six subforms of peroxisomes, designated P1 to P6, from the yeast, Yarrowia lipolytica. An analysis of trafficking of peroxisomal proteins in vivo suggests the existence of a multistep peroxisome assembly pathway in Y. lipolytica. This pathway operates by conversion of peroxisomal subforms in the direction P1, P2-->P3-->P4-->P5-->P6 and involves the import of various peroxisomal proteins into distinct vesicular intermediates. We have also reconstituted in vitro the fusion of the earliest intermediates in the pathway, small peroxisomal vesicles P1 and P2. Their fusion leads to the formation of a larger and more dense peroxisomal vesicle, P3. Fusion of P1 and P2 in vitro requires cytosol and ATP hydrolysis and is inhibited by antibodies to two membrane-associated ATPases of the AAA family, Pex1p and Pex6p. We provide evidence that the fusion in vitro of P1 and P2 peroxisomes reconstructs an actual early step in the peroxisome assembly pathway operating in vivo in Y. lipolytica.
Assuntos
Membranas Intracelulares/fisiologia , Fusão de Membrana/fisiologia , Peroxissomos/fisiologia , Saccharomycetales/fisiologia , Proteínas de Transporte Vesicular , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/metabolismo , Sequência de Bases , Proteínas de Transporte/metabolismo , DNA Complementar , Dados de Sequência Molecular , Proteínas Sensíveis a N-Etilmaleimida , Saccharomycetales/metabolismoRESUMO
Two AAA family ATPases, NSF and p97, have been implicated in membrane fusion during assembly and inheritance of organelles of the secretory pathway. We have now investigated the roles of AAA ATPases in membrane fusion during assembly of the peroxisome, an organelle outside the classical secretory system. Here, we show that peroxisomal membrane fusion in the yeast Yarrowia lipolytica requires two AAA ATPases, Pex1p and Pex6p. Release of membrane- associated Pex1p and Pex6p drives the asymmetric priming of two fusion partners. The next step, peroxisome docking, requires release of Pex1p from one partner. Subsequent fusion of the peroxisomal membranes is independent of both Pex1p and Pex6p.
Assuntos
Adenosina Trifosfatases/metabolismo , Membranas Intracelulares/fisiologia , Fusão de Membrana/fisiologia , Peroxissomos/fisiologia , ATPases Associadas a Diversas Atividades Celulares , Modelos Biológicos , Esferoplastos/fisiologia , Leveduras/enzimologia , Leveduras/fisiologiaRESUMO
Pex mutants of the yeast Yarrowia lipolytica are defective in peroxisome assembly. The mutant strain pex16-1 lacks morphologically recognizable peroxisomes. Most peroxisomal proteins are mislocalized to a subcellular fraction enriched for cytosol in pex16 strains, but a subset of peroxisomal proteins is localized at, or near, wild-type levels to a fraction typically enriched for peroxisomes. The PEX16 gene was isolated by functional complementation of the pex16-1 strain and encodes a protein, Pex16p, of 391 amino acids (44,479 D). Pex16p has no known homologues. Pex16p is a peripheral protein located at the matrix face of the peroxisomal membrane. Substitution of the carboxylterminal tripeptide Ser-Thr-Leu, which is similar to the consensus sequence of peroxisomal targeting signal 1, does not affect targeting of Pex16p to peroxisomes. Pex16p is synthesized in wild-type cells grown in glucose-containing media, and its levels are modestly increased by growth of cells in oleic acid-containing medium. Overexpression of the PEX16 gene in oleic acid- grown Y. lipolytica leads to the appearance of a small number of enlarged peroxisomes, which contain the normal complement of peroxisomal proteins at levels approaching those of wild-type peroxisomes.
Assuntos
Proteínas Fúngicas/genética , Proteínas de Membrana/genética , Microcorpos , Saccharomycetales/genética , Leveduras/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Ácidos Carboxílicos , DNA Fúngico , Matriz Extracelular/metabolismo , Proteínas Fúngicas/metabolismo , Expressão Gênica , Genes Fúngicos , Cobaias , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Ácido Oleico/farmacologia , Coelhos , Saccharomycetales/crescimento & desenvolvimento , Saccharomycetales/metabolismo , Saccharomycetales/ultraestrutura , Leveduras/crescimento & desenvolvimento , Leveduras/metabolismo , Leveduras/ultraestruturaRESUMO
Free and membrane-bound polyribosomes were separated from liver homogenates and characterized by electron microscopy. Using the wheat germ cell-free translation system, total translation products of poly A+RNA extracted from free polyribosomes (poly A+RNAf) showed some correlation to total liver cytosol proteins. In contrast, translation products of poly A+RNA from membrane-bound polyribosomes (poly A+RNAmb) showed some similarity to rat serum. Antibody to purified rat serum albumin immunoprecipitated from only the translation products of poly A+RNAmb a single polypeptide of mol wt 68,000. i.e., 3,000 greater than secreted serum albumin. In contrast, antibody to detergent-extracted cytochrome b5 immunoprecipitated from only the translation products of poly A+RNAf a single polypeptide of mol wt 17,500, identical to that of microsomal cytochrome b5. A consideration of the known properties of cytochrome b5 is consistent with an exclusive site of synthesis on free ribosomes.
Assuntos
Polirribossomos/metabolismo , Albuminas/biossíntese , Animais , Fracionamento Celular , Citocromos , Citocromos b5 , Microssomos Hepáticos , Peso Molecular , Biossíntese de Proteínas , RNA/metabolismo , RatosRESUMO
This report describes the microinjection of a purified peroxisomal protein, alcohol oxidase, from Pichia pastoris into mammalian tissue culture cells and the subsequent transport of this protein into vesicular structures. Transport was into membrane-enclosed vesicles as judged by digitonin-permeabilization experiments. The transport was time and temperature dependent. Vesicles containing alcohol oxidase could be detected as long as 6 d after injection. Coinjection of synthetic peptides containing a consensus carboxyterminal tripeptide peroxisomal targeting signal resulted in abolition of alcohol oxidase transport into vesicles in all cell lines examined. Double-label experiments indicated that, although some of the alcohol oxidase was transported into vesicles that contained other peroxisomal proteins, the bulk of the alcohol oxidase did not appear to be transported to preexisting peroxisomes. While the inhibition of transport of alcohol oxidase by peptides containing the peroxisomal targeting signal suggests a competition for some limiting component of the machinery involved in the sorting of proteins into peroxisomes, the organelles into which the majority of the protein is targeted appear to be unusual and distinct from endogenous peroxisomes by several criteria. Microinjected alcohol oxidase was transported into vesicles in normal fibroblasts and also in cell lines derived from patients with Zellweger syndrome, which are unable to transport proteins containing the ser-lys-leu-COOH peroxisomal targeting signal into peroxisomes (Walton et al., 1992). The implications of this result for the mechanism of peroxisomal protein transport are discussed.
Assuntos
Oxirredutases do Álcool/metabolismo , Microcorpos/metabolismo , Pichia/enzimologia , Células 3T3 , Oxirredutases do Álcool/química , Sequência de Aminoácidos , Animais , Transporte Biológico , Linhagem Celular , Imunofluorescência , Humanos , Membranas Intracelulares/metabolismo , Cinética , Camundongos , Microinjeções , Dados de Sequência Molecular , TemperaturaRESUMO
This report describes the involvement of 70-kD heat-shock proteins (hsp70) in the import of proteins into mammalian peroxisomes. Employing a microinjection-based assay (Walton, P. A., S. J. Gould, J. R. Feramisco, and S. Subramani. 1992. Mol. Cell Biol. 12:531-541), we demonstrate that proteins of the hsp70 family were associated with proteins being imported into the peroxisomal matrix. Import of peroxisomal proteins could be inhibited by coinjection of antibodies directed against the constitutive hsp70 proteins (hsp73). In a permeabilized-cell assay (Wendland and Subramani. 1993. J. Cell Biol. 120:675-685), antibodies directed against hsp70 proteins were shown to inhibit peroxisomal protein import. Inhibition could be overcome by the addition of exogenous hsp70 proteins. Purified rat liver peroxisomes were shown to have associated hsp70 proteins. The amount of associated hsp70 was increased under conditions of peroxisomal proliferation. Furthermore, proteinase protection assays indicated that the hsp70 molecules were located on the outside of the peroxisomal membrane. Finally, the process of heat-shocking cells resulted in a considerable delay in the import of peroxisomal proteins. Taken together, these results indicate that heat-shock proteins of the cytoplasmic hsp70 family are involved in the import of peroxisomal proteins.
Assuntos
Proteínas de Choque Térmico/metabolismo , Microcorpos/metabolismo , Animais , Transporte Biológico , Western Blotting , Linhagem Celular , Endopeptidase K , Imunofluorescência , Proteínas de Choque Térmico/química , Temperatura Alta , Humanos , Fígado/metabolismo , Ratos , Serina Endopeptidases/farmacologiaRESUMO
Pex mutants are defective in peroxisome assembly. In the pex20-1 mutant strain of the yeast Yarrowia lipolytica, the peroxisomal matrix protein thiolase is mislocalized exclusively to the cytosol, whereas the import of other peroxisomal proteins is unaffected. The PEX20 gene was isolated by functional complementation of the pex20-1 strain and encodes a protein, Pex20p, of 424 amino acids (47,274 D). Despite its role in the peroxisomal import of thiolase, which is targeted by an amino-terminal peroxisomal targeting signal-2 (PTS2), Pex20p does not exhibit homology to Pex7p, which acts as the PTS2 receptor. Pex20p is mostly cytosolic, whereas 4-8% is associated with high-speed (200,000 g) pelletable peroxisomes. In the wild-type strain, all newly synthesized thiolase is associated with Pex20p in a heterotetrameric complex composed of two polypeptide chains of each protein. This association is independent of PTS2. Pex20p is required for both the oligomerization of thiolase in the cytosol and its targeting to the peroxisome. Our data suggest that monomeric Pex20p binds newly synthesized monomeric thiolase in the cytosol and promotes the formation of a heterotetrameric complex of these two proteins, which could further bind to the peroxisomal membrane. Translocation of the thiolase homodimer into the peroxisomal matrix would release Pex20p monomers back to the cytosol, thereby permitting a new cycle of binding-oligomerization-targeting-release for Pex20p and thiolase.
Assuntos
Acetil-CoA C-Acetiltransferase/metabolismo , Ascomicetos/metabolismo , Proteínas Fúngicas/metabolismo , Acetil-CoA C-Acetiltransferase/química , Acil-CoA Oxidase , Sequência de Aminoácidos , Ascomicetos/genética , Ascomicetos/ultraestrutura , Sequência de Bases , Transporte Biológico Ativo , Clonagem Molecular , Citosol/metabolismo , DNA Fúngico/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Isocitrato Liase/química , Isocitrato Liase/metabolismo , Substâncias Macromoleculares , Microcorpos/metabolismo , Microscopia Eletrônica , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Oxirredutases/química , Oxirredutases/metabolismo , Conformação ProteicaRESUMO
Pay mutants of the yeast Yarrowia lipolytica fail to assemble functional peroxisomes. One mutant strain, pay32-1, has abnormally small peroxisomes that are often found in clusters surrounded by membraneous material. The functionally complementing gene PAY32 encodes a protein, Pay32p, of 598 amino acids (66,733 D) that is a member of the tetratricopeptide repeat family. Pay32p is intraperoxisomal. In wild-type peroxisomes, Pay32p is associated primarily with the inner surface of the peroxisomal membrane, but approximately 30% of Pay32p is localized to the peroxisomal matrix. The majority of Pay32p in the matrix is complexed with two polypeptides of 62 and 64 kD recognized by antibodies to SKL (peroxisomal targeting signal-1). In contrast, in peroxisomes of the pay32-1 mutant, Pay32p is localized exclusively to the matrix and forms no complex. Biochemical characterization of the mutants pay32-1 and pay32-KO (a PAY32 gene disruption strain) showed that Pay32p is a component of the peroxisomal translocation machinery. Mutations in the PAY32 gene prevent the translocation of most peroxisome-bound proteins into the peroxisomal matrix. These proteins, including the 62-kD anti-SKL-reactive polypeptide, are trapped in the peroxisomal membrane at an intermediate stage of translocation in pay32 mutants. Our results suggest that there are at least two distinct translocation machineries involved in the import of proteins into peroxisomes.
Assuntos
Proteínas Fúngicas/genética , Proteínas de Membrana/genética , Microcorpos/química , Saccharomycetales/metabolismo , Leveduras/metabolismo , Sequência de Aminoácidos , Especificidade de Anticorpos , Sequência de Bases , Transporte Biológico/fisiologia , Clonagem Molecular , Proteínas Fúngicas/metabolismo , Genes Fúngicos/fisiologia , Imuno-Histoquímica , Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Microcorpos/fisiologia , Microscopia Eletrônica , Dados de Sequência Molecular , Mutação/fisiologia , Peptídeos/imunologia , Saccharomycetales/genética , Saccharomycetales/ultraestrutura , Frações Subcelulares/metabolismo , Leveduras/genética , Leveduras/ultraestruturaRESUMO
The distribution of galactosyl transferase was studied using trans and cis Golgi fractions isolated by a modification of the Ehrenreich et al. procedure (1973. J. Cell Biol. 59:45-72) as well as an intact Golgi fraction isolated by a new one-step procedure. Two methods of assay were used. The first method analyzed the ability of Golgi fractions to transfer galactose (from uridine diphosphogalactose [UDP-gal] substrate) to the defined exogenous acceptor ovomucoid. The second method assessed the transfer of galactose from UDP-gal substrate to endogenous acceptors (endogenous glycosylation). The trans Golgi fraction (Golgi light) was highly active by the first method but revealed only low activity by the second method. Golgi fractions enriched in central and cis elements (the Golgi intermediate, heavy and especially the intact Golgi fraction) were highly active in both methods of assay. The endogenous glycosylation approach was validated by gel fluorography of the endogenous acceptors. For all Golgi fractions, transfer of galactose was revealed to secretory glycopeptides. It is concluded that galactosyl transferase activity in vivo occurs primarily in central and cis Golgi elements but not trans Golgi vesicles.
Assuntos
Galactosiltransferases/metabolismo , Complexo de Golgi/fisiologia , Animais , Fracionamento Celular/métodos , Glicoproteínas/metabolismo , Complexo de Golgi/ultraestrutura , Ovomucina/metabolismo , Ratos , Especificidade por SubstratoRESUMO
We investigated the site of synthesis of two abundant proteins in clofibrate-induced rat hepatic peroxisomes. RNA was extracted from free and membrane-bound polysomes, heated to improve translational efficiency, and translated in the mRNA-dependent, reticulocyte-lysate-cell-free, protein-synthesizing system. The peroxisomal acyl-CoA oxidase and enoyl-CoA hydratase-beta-hydroxyacyl-CoA dehydrogenase 35S-translation products were isolated immunochemically, analyzed by SDS PAGE and fluorography, and quantitated by densitometric scanning. The RNAs coding for these two peroxisomal proteins were found predominantly on free polysomes, and the translation products co-migrated with the mature proteins. As in normal rat liver, preproalbumin and catalase were synthesized mainly by membrane-bound and by free polysomes, respectively. mRNAs for a number of minor 35S-translation products also retained by the anti-peroxisomal immunoadsorbent were similarly found on free polysomes. These results, together with previous data, allow the generalization that the content proteins of rat liver peroxisomes are synthesized on free polysomes, and the data imply a posttranslational packaging mechanism for these major content proteins.
Assuntos
Clofibrato/farmacologia , Enoil-CoA Hidratase/genética , Hidroliases/genética , Fígado/enzimologia , Microcorpos/enzimologia , Oxirredutases/genética , Polirribossomos/enzimologia , Acil-CoA Oxidase , Animais , Enoil-CoA Hidratase/biossíntese , Cinética , Fígado/efeitos dos fármacos , Masculino , Microcorpos/efeitos dos fármacos , Peso Molecular , Oxirredutases/biossíntese , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos F344 , Reticulócitos/metabolismoRESUMO
When purified Golgi fractions were incubated with UDP-[3H]galactose in the absence of Triton-X-100, radioactivity was incorporated into an endogenous lipid and several peptide acceptors. Electron microscope analysis of Golgi fractions incubated in the endogenous galactosyl transferase assay medium revealed extensive fusion of Golgi saccules. Systematic removal of constituents in the galactosyl transferase assay medium showed enhanced (minus beta-mercaptoethanol) or reduced (minus ATP, minus sodium cacodylate buffer or minus MnCl2) fusion of Golgi membranes compared to the complete medium, Stereologic analysis revealed a correlation between membrane fusion and galactosyl transferase activity (r = 0.99, P less than 0.001). Electron microscope radioautography was carried out after incubation of Golgi fractions with UDP-[3H]galactose. Silver grains were not observed over trans elements of Golgi but were revealed mainly over large fused saccules with the number of silver grains being proportionate to membrane fusion (r = 0.92, P less than 0.001). Bilayer destabilization at points of Golgi membrane fusion may act to translocate galactose across the Golgi membrane and thereby provide a fusion regulated substrate for terminal glycosylation.
Assuntos
Glicolipídeos/metabolismo , Glicoproteínas/metabolismo , Complexo de Golgi/fisiologia , Fusão de Membrana , Proteínas de Membrana/metabolismo , Animais , Transporte Biológico , Sistema Livre de Células , Galactose/metabolismo , Galactosiltransferases/metabolismo , Fígado/ultraestrutura , Lipídeos de Membrana/metabolismo , RatosRESUMO
Mature Arabidopsis seeds are enriched in storage proteins and lipids, but lack starch. In the shrunken seed 1 (sse1) mutant, however, starch is favored over proteins and lipids as the major storage compound. SSE1 has 26 percent identity with Pex16p in Yarrowia lipolytica and complements pex16 mutants defective in the formation of peroxisomes and the transportation of plasma membrane- and cell wall-associated proteins. In Arabidopsis maturing seeds, SSE1 is required for protein and oil body biogenesis, both of which are endoplasmic reticulum-dependent. Starch accumulation in sse1 suggests that starch formation is a default storage deposition pathway.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/metabolismo , Proteínas Fúngicas , Organelas/metabolismo , Proteínas de Plantas/fisiologia , Sementes/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/ultraestrutura , Expressão Gênica , Teste de Complementação Genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Microcorpos/metabolismo , Microcorpos/ultraestrutura , Microscopia Eletrônica , Dados de Sequência Molecular , Mutação , Organelas/ultraestrutura , Peroxinas , Fenótipo , Óleos de Plantas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomycetales/química , Saccharomycetales/genética , Saccharomycetales/metabolismo , Sementes/ultraestrutura , Amido/metabolismoRESUMO
Mutations in the SEC238 and SRP54 genes of the yeast Yarrowia lipolytica not only cause temperature-sensitive defects in the exit of the precursor form of alkaline extracellular protease and of other secretory proteins from the endoplasmic reticulum and in protein secretion but also lead to temperature-sensitive growth in oleic acid-containing medium, the metabolism of which requires the assembly of functionally intact peroxisomes. The sec238A and srp54KO mutations at the restrictive temperature significantly reduce the size and number of peroxisomes, affect the import of peroxisomal matrix and membrane proteins into the organelle, and significantly delay, but do not prevent, the exit of two peroxisomal membrane proteins, Pex2p and Pex16p, from the endoplasmic reticulum en route to the peroxisomal membrane. Mutations in the PEX1 and PEX6 genes, which encode members of the AAA family of N-ethylmaleimide-sensitive fusion protein-like ATPases, not only affect the exit of precursor forms of secretory proteins from the endoplasmic reticulum but also prevent the exit of the peroxisomal membrane proteins Pex2p and Pex16p from the endoplasmic reticulum and cause the accumulation of an extensive network of endoplasmic reticulum membranes. None of the peroxisomal matrix proteins tested associated with the endoplasmic reticulum in sec238A, srp54KO, pex1-1, and pex6KO mutant cells. Our data provide evidence that the endoplasmic reticulum is required for peroxisome biogenesis and suggest that in Y. lipolytica, the trafficking of some membrane proteins, but not matrix proteins, to the peroxisome occurs via the endoplasmic reticulum, results in their glycosylation within the lumen of the endoplasmic reticulum, does not involve transport through the Golgi, and requires the products encoded by the SEC238, SRP54, PEX1, and PEX6 genes.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/metabolismo , Microcorpos/metabolismo , Saccharomycetales/genética , ATPases Associadas a Diversas Atividades Celulares , Transporte Biológico/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilação , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Ácido Oleico/farmacologia , Fator 2 da Biogênese de Peroxissomos , Saccharomycetales/ultraestrutura , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/metabolismo , TemperaturaRESUMO
We have identified and characterized mutants of the yeast Yarrowia lipolytica that are deficient in protein secretion, in the ability to undergo dimorphic transition from the yeast to the mycelial form, and in peroxisome biogenesis. Mutations in the SEC238, SRP54, PEX1, PEX2, PEX6, and PEX9 genes affect protein secretion, prevent the exit of the precursor form of alkaline extracellular protease from the endoplasmic reticulum, and compromise peroxisome biogenesis. The mutants sec238A, srp54KO, pex2KO, pex6KO, and pex9KO are also deficient in the dimorphic transition from the yeast to the mycelial form and are affected in the export of only plasma membrane and cell wall-associated proteins specific for the mycelial form. Mutations in the SEC238, SRP54, PEX1, and PEX6 genes prevent or significantly delay the exit of two peroxisomal membrane proteins, Pex2p and Pex16p, from the endoplasmic reticulum en route to the peroxisomal membrane. Mutations in the PEX5, PEX16, and PEX17 genes, which have previously been shown to be essential for peroxisome biogenesis, affect the export of plasma membrane and cell wall-associated proteins specific for the mycelial form but do not impair exit from the endoplasmic reticulum of either Pex2p and Pex16p or of proteins destined for secretion. Biochemical analyses of these mutants provide evidence for the existence of four distinct secretory pathways that serve to deliver proteins for secretion, plasma membrane and cell wall synthesis during yeast and mycelial modes of growth, and peroxisome biogenesis. At least two of these secretory pathways, which are involved in the export of proteins to the external medium and in the delivery of proteins for assembly of the peroxisomal membrane, diverge at the level of the endoplasmic reticulum.
Assuntos
Proteínas Fúngicas/metabolismo , Microcorpos/metabolismo , Saccharomycetales/metabolismo , Animais , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Modelos Biológicos , Mutagênese , Endopeptidase Neutra Reguladora de Fosfato PHEX , Precursores de Proteínas/imunologia , Precursores de Proteínas/metabolismo , Proteínas/metabolismo , Coelhos , Saccharomycetales/genética , Saccharomycetales/crescimento & desenvolvimento , Serina Endopeptidases/imunologia , Serina Endopeptidases/metabolismoRESUMO
PEX genes encode peroxins, which are required for the biogenesis of peroxisomes. The Yarrowia lipolytica PEX17 gene encodes the peroxin Pex17p, which is 671 amino acids in length and has a predicted molecular mass of 75,588 Da. Pex17p is peripherally associated with the peroxisomal membrane. The carboxyl-terminal tripeptide, Gly-Thr-Leu, of Pex17p is not necessary for its targeting to peroxisomes. Synthesis of Pex17p is low in cells grown in glucose-containing medium and increases after the cells are shifted to oleic acid-containing medium. Cells of the pex17-1 mutant, the original mutant strain, and the pex17-KA mutant, a strain in which most of the PEX17 gene is deleted, fail to form normal peroxisomes but instead contain numerous large, multimembraned structures. The import of peroxisomal matrix proteins in these mutants is selectively impaired. This selective import is not a function of the nature of the peroxisomal targeting signal. We suggest a regulatory role for Pex17p in the import of a subset of matrix proteins into peroxisomes.
Assuntos
Proteínas de Transporte/genética , Proteínas da Matriz Extracelular/metabolismo , Proteínas Fúngicas/genética , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras , Microcorpos/metabolismo , Saccharomycetales/genética , Leveduras/genética , Sequência de Aminoácidos , Sequência de Bases , Centrifugação com Gradiente de Concentração , Clonagem Molecular , Proteínas Fúngicas/metabolismo , Dados de Sequência Molecular , Peso Molecular , Mutagênese , RNA Mensageiro/metabolismo , Saccharomycetales/metabolismo , Leveduras/metabolismoRESUMO
PEX genes encode peroxins, which are proteins required for peroxisome assembly. The PEX19 gene of the yeast Yarrowia lipolytica was isolated by functional complementation of the oleic acid-nonutilizing strain pex19-1 and encodes Pex19p, a protein of 324 amino acids (34,822 Da). Subcellular fractionation and immunofluorescence microscopy showed Pex19p to be localized primarily to peroxisomes. Pex19p is detected in cells grown in glucose-containing medium, and its levels are not increased by incubation of cells in oleic acid-containing medium, the metabolism of which requires intact peroxisomes. pex19 cells preferentially mislocalize peroxisomal matrix proteins and the peripheral intraperoxisomal membrane peroxin Pex16p to the cytosol, although small amounts of these proteins could be reproducibly localized to a subcellular fraction enriched for peroxisomes. In contrast, the peroxisomal integral membrane protein Pex2p exhibits greatly reduced levels in pex19 cells compared with its levels in wild-type cells. Importantly, pex19 cells were shown by electron microscopy to contain structures that resemble wild-type peroxisomes in regards to size, shape, number, and electron density. Subcellular fractionation and isopycnic density gradient centrifugation confirmed the presence of vesicular structures in pex19 mutant strains that were similar in density to wild-type peroxisomes and that contained profiles of peroxisomal matrix and membrane proteins that are similar to, yet distinct from, those of wild-type peroxisomes. Because peroxisomal structures form in pex19 cells, Pex19p apparently does not function as a peroxisomal membrane protein receptor in Y. lipolytica. Our results are consistent with a role for Y. lipolytica Pex19p in stabilizing the peroxisomal membrane.
Assuntos
Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas de Membrana/genética , Peroxissomos/metabolismo , Yarrowia/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Transporte Biológico , Citosol/metabolismo , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Humanos , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutagênese , Ácido Oleico/farmacologia , Prenilação de Proteína , Homologia de Sequência de Aminoácidos , Yarrowia/efeitos dos fármacos , Yarrowia/isolamento & purificaçãoRESUMO
pex mutants are defective in peroxisome assembly. The mutant strain pex23-1 of the yeast Yarrowia lipolytica lacks morphologically recognizable peroxisomes and mislocalizes all peroxisomal matrix proteins investigated preferentially to the cytosol. pex23 strains accumulate vesicular structures containing both peroxisomal matrix and membrane proteins. The PEX23 gene was isolated by functional complementation of the pex23-1 strain and encodes a protein, Pex23p, of 418 amino acids (47,588 Da). Pex23p exhibits high sequence similarity to two hypothetical proteins of the yeast Saccharomyces cerevisiae. Pex23p is an integral membrane protein of peroxisomes that is completely, or nearly completely, sequestered from the cytosol. Pex23p is detected at low levels in cells grown in medium containing glucose, and its levels are significantly increased by growth in medium containing oleic acid, the metabolism of which requires intact peroxisomes.
Assuntos
Proteínas Fúngicas/genética , Proteínas de Membrana/genética , Saccharomycetales/genética , Sequência de Aminoácidos , Sequência de Bases , Meios de Cultura , Citosol/metabolismo , DNA Complementar , Proteínas Fúngicas/biossíntese , Genes Fúngicos , Humanos , Membranas Intracelulares/metabolismo , Proteínas de Membrana/biossíntese , Dados de Sequência Molecular , Mutagênese , Ácido Oleico , Peroxissomos/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Saccharomycetales/crescimento & desenvolvimento , Homologia de Sequência de AminoácidosRESUMO
Peroxisome proliferators, which are structurally diverse nonmutagenic agents, induce hepatocarcinogenesis in rats and mice. Exposure to these xenobiotics leads to a rapid and coordinated transcriptional activation of the genes for the peroxisomal beta-oxidation enzyme system pathway in the liver. We have previously identified a peroxisome proliferator-responsive element in the 5'-flanking region of the rat peroxisomal hydratase/dehydrogenase (PBE) gene, the second enzyme in the beta-oxidation pathway. The peroxisome proliferator-responsive element in the PBE gene was shown to direct the induction of a luciferase reporter gene in vitro. We have now used this 3.2-kilobase 5'-flanking region of the PBE gene fused to the coding region of luciferase to generate transgenic mice. Three independent lines of transgenic mice expressed luciferase in response to ciprofibrate, a peroxisome proliferator. The induction of luciferase is specific to the liver; this agrees with the tissue-specific induction of PBE. Two other hypolipidemic drugs, nafenopin and Wy-14,643, were also capable of inducing luciferase activity in the liver. This study suggests that the PBE upstream element can be used to direct and modulate the expression of cloned genes by changing the levels of peroxisome proliferators. Also, the PBE-luciferase transgenic mouse should be an excellent model system for screening xenobiotics for potential peroxisome proliferator property.
Assuntos
3-Hidroxiacil-CoA Desidrogenases/genética , Enoil-CoA Hidratase/genética , Genes Reporter/genética , Fígado/enzimologia , Luciferases/metabolismo , Camundongos Transgênicos/genética , Microcorpos/efeitos dos fármacos , Animais , Ácido Clofíbrico/análogos & derivados , Ácido Clofíbrico/farmacologia , Dietilexilftalato/farmacologia , Ácidos Fíbricos , Luciferases/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Nafenopina/farmacologia , Pirimidinas/farmacologiaRESUMO
A cDNA library, complementary to mRNAs of alkane-grown Candida tropicalis, was screened by differential DNA dot-blot hybridization with [32P]cDNA reverse-transcribed from mRNA of alkane-grown cells or from cells in which peroxisome formation was repressed by growth on glucose. 9% of the library encodes alkane-induced sequences. The cell-free translation products of eight hybrid-selected mRNAs were characterized by SDS-polyacrylamide gel electrophoresis and fluorography: most of them are probably peroxisomal proteins. Among these, a catalase clone was identified by immunoprecipitation of the translation product with anti-catalase. The clone was sequenced: the inferred amino acid sequence is homologous to the carboxytermini of mammalian and Saccharomyces cerevisiae catalases. C. tropicalis catalase mRNA is 1.7-1.8 kb long by Northern analysis, of which 1.5-1.6 kb is required to code for the 57 kDa polypeptide. Catalase mRNA (assayed by dot-blot hybridization) is strikingly induced in C. tropicalis by growth on alkanes, suggesting that peroxisome induction is transcriptionally regulated. This sublibrary of alkane-induced, mostly peroxisomal clones, together with a recently developed cell-free peroxisome protein import assay, will permit investigation of the targeting of proteins to peroxisomes.