RESUMO
BST-2/tetherin is a cellular host factor capable of restricting the release of a variety of enveloped viruses, including HIV-1. Structurally, BST-2 consists of an N-terminal cytoplasmic domain, a transmembrane domain, an ectodomain, and a C-terminal membrane anchor. The BST-2 ectodomain encodes three cysteine residues in its N-terminal half, each of which can contribute to the formation of cysteine-linked dimers. We previously reported that any one of the three cysteine residues is sufficient to produce functional BST-2 dimers. Here we investigated the importance of cysteine positioning on the ectodomain for functional dimerization of BST-2. Starting with a cysteine-free monomeric form of BST-2, individual cysteine residues were reintroduced at various locations throughout the ectodomain. The resulting BST-2 variants were tested for expression, dimerization, surface presentation, and inhibition of HIV-1 virus release. We found significant flexibility in the positioning of cysteine residues, although the propensity to form cysteine-linked dimers generally decreased with increasing distance from the N terminus. Interestingly, all BST-2 variants, including the one lacking all three ectodomain cysteines, retained the ability to form non-covalent dimers, and all of the BST-2 variants were efficiently expressed at the cell surface. Importantly, not all BST-2 variants capable of forming cysteine-linked dimers were functional, suggesting that cysteine-linked dimerization of BST-2 is necessary but not sufficient for inhibiting virus release. Our results expose new structural constraints governing the functional dimerization of BST-2, a property essential to its role as a restriction factor tethering viruses to the host cell.
Assuntos
Antígenos CD/química , Cisteína/química , Multimerização Proteica , Sequência de Aminoácidos , Substituição de Aminoácidos , Antígenos CD/genética , Antígenos CD/metabolismo , Membrana Celular/metabolismo , Cisteína/genética , Cisteína/metabolismo , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Células HEK293 , HIV-1/fisiologia , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Transporte Proteico , Internalização do VírusRESUMO
BST-2/CD317/tetherin is a host factor that inhibits HIV-1 release and is counteracted by HIV-1 Vpu. Structural studies indicate that the BST-2 ectodomain assumes a coiled-coil conformation. Here we studied the role of the BST-2 ectodomain for tethering function. First, we addressed the importance of the length and structure of the ectodomain by adding or substituting heterologous coiled-coil or non-coiled-coil sequences. We found that extending or replacing the BST-2 ectodomain using non-coiled-coil sequences resulted in loss of BST-2 function. Doubling the size of the BST-2 ectodomain by insertion of a heterologous coiled-coil motif or substituting the BST-2 coiled-coil domain with a heterologous coiled-coil motif maintained tethering function. Reductions in the size of the BST-2 coiled-coil domain were tolerated as well. In fact, deletion of the C-terminal half of the BST-2 ectodomain, including a series of seven consecutive heptad motifs did not abolish tethering function. However, slight changes in the positioning of deletions affecting the relative placing of charged or hydrophobic residues on the helix severely impacted the functional properties of BST-2. Overall, we conclude that the size of the BST-2 ectodomain is highly flexible and can be reduced or extended as long as the positioning of residues important for the stability of the dimer interface is maintained.
Assuntos
Antígenos CD/química , Antígenos CD/metabolismo , Regulação para Baixo , Infecções por HIV/metabolismo , HIV-1/fisiologia , Vírion/fisiologia , Liberação de Vírus , Sequência de Aminoácidos , Antígenos CD/genética , Dimerização , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/genética , Células HeLa , Humanos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Vírion/genéticaRESUMO
BST-2/CD317/HM1.24/tetherin is a host factor that inhibits the release of HIV-1 and other enveloped viruses. Structurally, tetherin consists of an N-terminal transmembrane (TM) region, a central coiled coil motif, and a putative C-terminal glycosylphosphatidylinositol (GPI) anchor motif. A current working model proposes that BST-2 inhibits virus release by physically tethering viral particles to the cell surface via its TM motif and GPI anchor. Here we analyzed the functional importance of the C-terminal GPI anchor motif in BST-2. We replaced the GPI anchor motif in BST-2 with the TM regions of several surface markers and found that the TM motifs of CD40 and transferrin receptor, but not that of CD45, could functionally substitute for a GPI anchor in BST-2. Conversely, replacing the TM region of CD4 by the putative GPI anchor signal of human BST-2 resulted in proper membrane targeting and surface expression of the chimeric protein, indicating that the BST-2 GPI anchor signal can function as a bona fide TM region. In fact, attempts to demonstrate GPI anchor modification of human BST-2 by biochemical methods failed. Our results demonstrate that the putative C-terminal GPI anchor motif in human BST-2 fulfills the requirements of a bona fide TM motif, leading us to propose that human BST-2 may in fact contain a second TM segment rather than a GPI anchor.
Assuntos
Antígenos CD/metabolismo , Motivos de Aminoácidos , Antígenos CD/genética , Antígenos CD40/genética , Antígenos CD40/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Glicosilfosfatidilinositóis/genética , Glicosilfosfatidilinositóis/metabolismo , Células HeLa , Humanos , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
BST-2/CD317/tetherin is a host factor that inhibits the release of HIV-1 and other unrelated viruses. A current model proposes that BST-2 physically tethers virions to the surface of virus-producing cells. The HIV-1-encoded Vpu protein effectively antagonizes the activity of BST-2. How Vpu accomplishes this task remains unclear; however, it is known that Vpu has the ability to down-modulate BST-2 from the cell surface. Here we analyzed the effects of Vpu on BST-2 by performing a series of kinetic studies with HeLa, 293T, and CEMx174 cells. Our results indicate that the surface downregulation of BST-2 is not due to an accelerated internalization or reduced recycling of internalized BST-2 but instead is caused by interference with the resupply of newly synthesized BST-2 from within the cell. While our data confirm previous reports that the high-level expression of Vpu can cause the endoplasmic reticulum (ER)-associated degradation of BST-2, we found no evidence that Vpu targets endogenous BST-2 in the ER in the course of a viral infection. Instead, we found that Vpu acts in a post-ER compartment and increases the turnover of newly synthesized mature BST-2. Our observation that Vpu does not affect the recycling of BST-2 suggests that Vpu does not act directly at the cell surface but may interfere with the trafficking of newly synthesized BST-2 to the cell surface, resulting in the accelerated targeting of BST-2 to the lysosomal compartment for degradation.
Assuntos
Antígenos CD/metabolismo , HIV-1/imunologia , HIV-1/patogenicidade , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Antígenos CD/imunologia , Linhagem Celular , Regulação para Baixo , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Expressão Gênica , Proteínas do Vírus da Imunodeficiência Humana/imunologia , Humanos , Transporte Proteico , Proteínas Virais Reguladoras e Acessórias/imunologiaRESUMO
BST-2/CD317/HM1.24/tetherin is a B-cell antigen overexpressed on the surface of myeloma cell lines and on neoplastic plasma cells of patients with multiple myeloma. Antibodies to BST-2 are in clinical trial for the treatment of multiple myeloma and are considered for the treatment of solid tumors with high BST-2 antigen levels. Functionally, BST-2 restricts the secretion of retroviruses, including human immunodeficiency virus type 1, as well as members of the herpesvirus, filovirus, and arenavirus families, presumably by tethering nascent virions to the cell surface. Here we report that BST-2 antibody treatment facilitates virus release from BST-2(+) cells by interfering with the tethering activity of BST-2. BST-2 antibodies were unable to release already tethered virions and were most effective when added early during virus production. BST-2 antibody treatment did not affect BST-2 dimerization and did not reduce the cell surface expression of BST-2. Interestingly, BST-2 antibody treatment reduced the nonspecific shedding of BST-2 and limited the encapsidation of BST-2 into virions. Finally, flotation analyses indicate that BST-2 antibodies affect the distribution of BST-2 within membrane rafts. Our data suggest that BST-2 antibody treatment may enhance virus release by inducing a redistribution of BST-2 at the cell surface, thus preventing it from accumulating at the sites of virus budding.
Assuntos
Anticorpos/metabolismo , Antígenos CD/imunologia , HIV-1/fisiologia , HIV-2/fisiologia , Liberação de Vírus , Linhagem Celular , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/imunologia , HIV-1/crescimento & desenvolvimento , HIV-2/crescimento & desenvolvimento , HumanosRESUMO
HIV-1 Vpu enhances the release of virions from infected cells. Recent work identified Bst-2/CD317/tetherin as a host factor whose inhibitory activity on viral release is counteracted by Vpu. A current working model proposes that Bst-2 inhibits virus release by tethering viral particles to the cell surface. Here, we analyzed endogenous Bst-2 with respect to its effect on virus release from HeLa cells, T cells, and macrophages. We noted significant cell type-dependent variation in Bst-2 expression. Vpu caused a reduction in Bst-2 expression in transfected HeLa cells and long-term infected macrophages. However, Vpu expression did not result in cell surface down-modulation of Bst-2 or a reduction in intracellular Bst-2 expression in CEMx174 or H9 cells, yet virus replication in these cells was Vpu-responsive. Surprisingly, Bst-2 was undetectable in cell-free virions that were recovered from the surface of HeLa cells by physical shearing, suggesting that a tethering model may not explain all of the functional properties of Bst-2. Taken together we conclude that enhancement of virus release by Vpu does not, at least in CEMx174 and H9 cells, require cell surface down-modulation or intracellular depletion of Bst-2, nor does it entail exclusion of Bst-2 from viral particles.
Assuntos
Antígenos CD/fisiologia , Regulação para Baixo , HIV-1/fisiologia , Proteínas do Vírus da Imunodeficiência Humana/fisiologia , Glicoproteínas de Membrana/fisiologia , Proteínas Virais Reguladoras e Acessórias/fisiologia , Antígenos CD/metabolismo , Western Blotting , Linhagem Celular , Membrana Celular/metabolismo , Citometria de Fluxo , Proteínas Ligadas por GPI , Humanos , Soros Imunes , Glicoproteínas de Membrana/metabolismo , Vírion , Montagem de VírusRESUMO
CD317/Bst-2/tetherin is a host factor that restricts the release of human immunodeficiency virus type 1 (HIV-1) by trapping virions at the plasma membrane of certain producer cells. It is antagonized by the HIV-1 accessory protein Vpu. Previous light microscopy studies localized CD317 to the plasma membrane and the endosomal compartment and showed Vpu induced downregulation. In the present study, we performed quantitative immunoelectron microscopy of CD317 in cells producing wild-type or Vpu-defective HIV-1 and in control cells. Double-labeling experiments revealed that CD317 localizes to the plasma membrane, to early and recycling endosomes, and to the trans-Golgi network. CD317 largely relocated to endosomes upon HIV-1 infection, and this effect was partly counteracted by Vpu. Unexpectedly, CD317 was enriched in the membrane of viral buds and cell-associated and cell-free viruses compared to the respective plasma membrane, and this enrichment was independent of Vpu. These results suggest that the tethering activity of CD317 critically depends on its density at the cell surface and appears to be less affected by its density in the virion membrane.
Assuntos
Antígenos CD/análise , Membrana Celular/química , Regulação da Expressão Gênica , HIV-1/química , Interações Hospedeiro-Patógeno , Glicoproteínas de Membrana/análise , Linhagem Celular , Endossomos/química , Proteínas Ligadas por GPI , Deleção de Genes , Proteínas do Vírus da Imunodeficiência Humana/deficiência , Proteínas do Vírus da Imunodeficiência Humana/fisiologia , Humanos , Microscopia Imunoeletrônica , Proteínas Virais Reguladoras e Acessórias/deficiência , Proteínas Virais Reguladoras e Acessórias/fisiologia , Rede trans-Golgi/químicaRESUMO
BACKGROUND: The Human Immunodeficiency virus type 1 (HIV-1) Vpu protein enhances virus release from infected cells and induces proteasomal degradation of CD4. Recent work identified BST-2/CD317 as a host factor that inhibits HIV-1 virus release in a Vpu sensitive manner. A current working model proposes that BST-2 inhibits virus release by tethering viral particles to the cell surface thereby triggering their subsequent endocytosis. RESULTS: Here we defined structural properties of BST-2 required for inhibition of virus release and for sensitivity to Vpu. We found that BST-2 is modified by N-linked glycosylation at two sites in the extracellular domain. However, N-linked glycosylation was not important for inhibition of HIV-1 virus release nor did it affect surface expression or sensitivity to Vpu. Rodent BST-2 was previously found to form cysteine-linked dimers. Analysis of single, double, or triple cysteine mutants revealed that any one of three cysteine residues present in the BST-2 extracellular domain was sufficient for BST-2 dimerization, for inhibition of virus release, and sensitivity to Vpu. In contrast, BST-2 lacking all three cysteines in its ectodomain was unable to inhibit release of wild type or Vpu-deficient HIV-1 virions. This defect was not caused by a gross defect in BST-2 trafficking as the mutant protein was expressed at the cell surface of transfected 293T cells and was down-modulated by Vpu similar to wild type BST-2. CONCLUSION: While BST-2 glycosylation was functionally irrelevant, formation of cysteine-linked dimers appeared to be important for inhibition of virus release. However lack of dimerization did not prevent surface expression or Vpu sensitivity of BST-2, suggesting Vpu sensitivity and inhibition of virus release are separable properties of BST-2.
Assuntos
Antígenos CD/imunologia , HIV-1/imunologia , HIV-1/fisiologia , Proteínas do Vírus da Imunodeficiência Humana/fisiologia , Glicoproteínas de Membrana/imunologia , Proteínas Virais Reguladoras e Acessórias/fisiologia , Replicação Viral , Substituição de Aminoácidos/genética , Antígenos CD/metabolismo , Linhagem Celular , Membrana Celular/química , Cisteína/genética , Dimerização , Dissulfetos , Proteínas Ligadas por GPI , Glicosilação , Humanos , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/metabolismo , Mutagênese Sítio-DirigidaRESUMO
HIV-1 Vif and Vpu are accessory factors involved in late stages of viral replication. Vif regulates viral infectivity by preventing virion incorporation of APOBEC3G and other members of the family of cytidine deaminases, while Vpu causes degradation of CD4 and promotes virus release by functionally inactivating the host factor BST-2. This chapter described techniques used for the characterization of Vif and Vpu and their functional interaction with host factors. Many of the techniques are, however, applicable to the functional analysis of other viral proteins.
Assuntos
HIV-1/metabolismo , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Aminação , Biocatálise , DNA Viral/genética , DNA Viral/metabolismo , Escherichia coli/genética , HIV-1/genética , HIV-1/fisiologia , Proteínas do Vírus da Imunodeficiência Humana/química , Proteínas do Vírus da Imunodeficiência Humana/genética , Proteínas do Vírus da Imunodeficiência Humana/isolamento & purificação , Humanos , Imunoprecipitação , Membranas Artificiais , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Estabilidade Proteica , DNA Polimerase Dirigida por RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transcrição Gênica , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/isolamento & purificação , Produtos do Gene vif do Vírus da Imunodeficiência Humana/biossíntese , Produtos do Gene vif do Vírus da Imunodeficiência Humana/química , Produtos do Gene vif do Vírus da Imunodeficiência Humana/genéticaRESUMO
Viruses face a variety of obstacles when infecting a new host. The past few years have brought exciting new insights into the function of restriction factors, which form part of the host's innate immune system. One of the most recently identified restriction factors is bone marrow stromal antigen 2 (BST-2)/tetherin. BST-2 is an interferon-inducible gene whose expression dramatically reduces the release of viruses from infected cells. This effect of BST-2 is not specific to human immunodeficiency virus but affects a broad range of enveloped viruses. Since the identification of BST-2 as a restriction factor in 2008, much progress has been made in understanding the molecular properties and functional characteristics of this host factor. The goal of this review was to provide an update on our current understanding of the role of BST-2 in regulating virus release and to discuss its role in controlling virus spread during productive infection with special emphasis on human immunodeficiency virus-1.
Assuntos
Antígenos CD/metabolismo , Regulação da Expressão Gênica , Interferons/metabolismo , Vírion/fisiologia , Liberação de Vírus , Replicação Viral , Animais , Antígenos CD/química , Antígenos CD/genética , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Imunidade Inata/efeitos dos fármacos , Interferons/farmacologia , Domínios e Motivos de Interação entre Proteínas , Vírion/efeitos dos fármacos , Liberação de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Vpu is a small integral membrane protein encoded by HIV-1 and some SIV isolates. The protein is known to induce degradation of the viral receptor molecule CD4 and to enhance the release of newly formed virions from the cell surface. Vpu accomplishes these two functions through two distinct mechanisms. In the case of CD4, Vpu acts as a molecular adaptor to connect CD4 to an E3 ubiquitin ligase complex resulting in CD4 degradation by cellular proteasomes. This requires signals located in Vpu's cytoplasmic domain. Enhancement of virus release on the other hand involves the neutralization of a cellular host factor, BST-2 (also known as CD317, HM1.24, or tetherin) and requires Vpu's TM domain. The current review discusses recent advances on the role of Vpu in controlling degradation of CD4 and in regulating virus release.
Assuntos
Antígenos CD/metabolismo , Antígenos CD4/metabolismo , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Antígenos CD/química , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/metabolismo , Interações Hospedeiro-Patógeno , Proteínas do Vírus da Imunodeficiência Humana/química , Humanos , Processamento de Proteína Pós-Traducional , Proteínas Virais Reguladoras e Acessórias/química , Vírion/metabolismoRESUMO
Isu, the scaffold protein on which Fe-S clusters are built in the mitochondrial matrix, plays a central role in the biogenesis of Fe-S cluster proteins. We report that the reduction in the activity of several components of the cluster biogenesis system, including the specialized Hsp70 Ssq1, causes a 15-20-fold up-regulation of Isu. This up-regulation results from changes at both the transcriptional and posttranslational level: an increase in ISU mRNA levels and in stability of ISU protein. Its biological importance is demonstrated by the fact that cells lacking Ssq1 grow poorly when Isu levels are prevented from rising above those found in wild-type cells. Of the biogenesis factors tested, Nfs1, the sulfur donor, was unique. Little increase in Isu levels occurred when Nfs1 was depleted. However, its presence was required for the up-regulation caused by reduction in activity of other components. Our results are consistent with the existence of a mechanism to increase the stability of Isu, and thus its level, that is dependent on the presence of the cysteine desulfurase Nfs1.
Assuntos
Regulação Fúngica da Expressão Gênica , Chaperonas Moleculares/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Choque Térmico HSP70 , Mitocôndrias/metabolismo , Proteínas Mitocondriais , Chaperonas Moleculares/genética , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/genética , Sulfurtransferases , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The development of lysine 2,3-aminomutase as a robust biocatalyst hinges on the development of an in vivo activation system to trigger catalysis. This is the first report to show that, in the absence of chemical reductants, lysine 2,3-aminomutase activity is dependent upon the presence of flavodoxin, ferredoxin, or flavodoxin-NADP(+) reductase.
Assuntos
Transferases Intramoleculares/metabolismo , Porphyromonas gingivalis/enzimologia , Catálise , Ativação Enzimática , Escherichia coli/genética , Ferredoxinas/genética , Ferredoxinas/metabolismo , Flavodoxina/genética , Flavodoxina/metabolismo , Transferases Intramoleculares/genética , Cinética , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Porphyromonas gingivalis/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Jac1p is a conserved, specialized J-protein that functions with Hsp70 in Fe-S cluster biogenesis in mitochondria of the yeast Saccharomyces cerevisiae. Although Jac1p as well as its specialized Hsp70 partner, Ssq1p, binds directly to the Fe-S cluster scaffold protein Isu, the Jac1p-Isu1p interaction is not well understood. Here we report that a C-terminal fragment of Jac1p lacking its J-domain is sufficient for interaction with Isu1p, and amino acid alterations in this domain affect interaction with Isu1p but not Ssq1p. In vivo, such JAC1 mutations had no obvious phenotypic effect. However, when present in combination with a mutation in SSQ1 that causes an alteration in the substrate binding cleft, growth was significantly compromised. Wild type Jac1p and Isu1p cooperatively stimulate the ATPase activity of Ssq1p. Jac1p mutant protein is only slightly compromised in this regard. Our in vivo and in vitro results indicate that independent interaction of Jac1p and the Isu client protein with Hsp70 is sufficient for robust growth under standard laboratory conditions. However, our results also support the idea that Isu protein can be "targeted" to Ssq1p after forming a complex with Jac1p. We propose that Isu protein targeting may be particularly important when environmental conditions place high demands on Fe-S cluster biogenesis or in organisms lacking specialized Hsp70s for Fe-S cluster biogenesis.
Assuntos
Proteínas Ferro-Enxofre/química , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas Mitocondriais , Dados de Sequência Molecular , Mutação , Peptídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
The neurodegenerative disease Friedreich's ataxia is caused by reduced levels of frataxin, a mitochondrial matrix protein. The in vivo role of frataxin is under debate. Frataxin, as well as its yeast homologue Yfh1, binds multiple iron atoms as an oligomer and has been proposed to function as a crucial iron-storage protein. We identified a mutant Yfh1 defective in iron-induced oligomerization. This mutant protein was able to replace functionally wild-type Yfh1, even when expressed at low levels, when mitochondrial iron levels were high and in mutant strains having deletions of genes that had synthetic growth defects with a YFH1 deletion. The ability of an oligomerization-deficient Yfh1 to function in vivo suggests that oligomerization, and thus oligomerization-induced iron storage, is not a critical function of Yfh1. Rather, the capacity of this oligomerization-deficient mutant to interact with the Isu protein suggests a more direct role of Yfh1 in iron-sulphur cluster biogenesis.
Assuntos
Proteínas de Ligação ao Ferro/fisiologia , Ferro/metabolismo , Sequência de Aminoácidos , Animais , Ânions , Escherichia coli/metabolismo , Deleção de Genes , Haploidia , Humanos , Ferro/química , Proteínas de Ligação ao Ferro/metabolismo , Proteínas Ferro-Enxofre/química , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Mutação , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae , FrataxinaRESUMO
The major Hsp70 of the mitochondrial matrix (Ssc1 in yeast) is critically important for the translocation of proteins from the cytosol, across the mitochondrial inner membrane, and into the matrix. Tim44, a peripheral inner membrane protein with limited sequence similarity to the J domain of J-type cochaperones, tethers Ssc1 to the import channel. Here we report that, unlike a J protein, Tim44 does not stimulate the ATPase activity of Ssc1, nor does it affect the stimulation by either a known mitochondrial J protein or a peptide substrate. Thus, we conclude that Tim44 does not function as a J protein cochaperone of Ssc1; rather, it tethers Ssc1 to the import channel through interactions independent of those critical for J protein function. However, a previously unstudied essential gene, PAM18, encodes an 18-kDa protein that contains a J domain and is localized to the mitochondrial inner membrane. Pam18 stimulates the ATPase activity of Ssc1; depletion of Pam18 in vivo disrupts import of proteins into the mitochondrial matrix. We propose that Pam18 is the J protein partner for Ssc1 at the import channel and is critical for Ssc1's function in protein import.