RESUMO
We determined that a mutation in the nucleoporin gene NUP170 leads to defects in chromosome transmission fidelity (ctf) and kinetochore integrity in Saccharomyces cerevisiae. A ctf mutant strain, termed s141, shows a transcription readthrough phenotype and stabilizes a dicentric chromosome fragment in two assays for kinetochore integrity. Previously, these assays led to the identification of two essential kinetochore components, Ctf13p and Ctf14p. Thus, s141 represents another ctf mutant involved in the maintenance of kinetochore integrity. We cloned and mapped the gene complementing the ctf mutation of s141 and showed that it is identical to the S. cerevisiae NUP170 gene. A deletion strain of NUP170 (nup170 Delta::HIS3) has a Ctf(-) phenotype similar to the s141 mutant (nup170-141) and also exhibits a kinetochore integrity defect. We identified a second nucleoporin, NUP157, a homologue of NUP170, as a suppressor of the Ctf(-) phenotype of nup170-141 and nup170 Delta::HIS3 strains. However, a deletion of NUP157 or several other nucleoporins did not affect chromosome segregation. Our data suggest that NUP170 encodes a specialized nucleoporin with a unique role in chromosome segregation and possibly kinetochore function.
Assuntos
Segregação de Cromossomos , Proteínas Fúngicas/fisiologia , Proteínas de Membrana/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares , Proteínas Nucleares/fisiologia , Proteínas de Saccharomyces cerevisiae , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Cinetocoros , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutagênese , Membrana Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
The mitochondrial inner membrane contains two separate translocons: one required for the translocation of matrix-targeted proteins (the Tim23p-Tim17p complex) and one for the insertion of polytopic proteins into the mitochondrial inner membrane (the Tim54p-Tim22p complex). To identify new members of the Tim54p-Tim22p complex, we screened for high-copy suppressors of the temperature-sensitive tim54-1 mutant. We identified a new gene, TIM18, that encodes an integral protein of the inner membrane. The following genetic and biochemical observations suggest that the Tim18 protein is part of the Tim54p-Tim22p complex in the inner membrane: multiple copies of TIM18 suppress the tim54-1 growth defect; the tim18::HIS3 disruption is synthetically lethal with tim54-1; Tim54p and Tim22p can be coimmune precipitated with the Tim18 protein; and Tim18p, along with Tim54p and Tim22p, is detected in an approximately 300-kDa complex after blue native electrophoresis. We propose that Tim18p is a new component of the Tim54p-Tim22p machinery that facilitates insertion of polytopic proteins into the mitochondrial inner membrane.
Assuntos
Antiporters , Proteínas de Transporte/metabolismo , Proteínas Fúngicas/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Transporte Biológico , Proteínas de Transporte/genética , Divisão Celular , Proteínas Fúngicas/genética , Hidroliases/genética , Hidroliases/metabolismo , Proteínas de Membrana/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Dados de Sequência Molecular , Coelhos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genéticaRESUMO
We have identified a new protein, Tim54p, located in the yeast mitochondrial inner membrane. Tim54p is an essential import component, required for the insertion of at least two polytopic proteins into the inner membrane, but not for the translocation of precursors into the matrix. Several observations suggest that Tim54p and Tim22p are part of a protein complex in the inner membrane distinct from the previously characterized Tim23p-Tim17p complex. First, multiple copies of the TIM22 gene, but not TIM23 or TIM17, suppress the growth defect of a tim54-1 temperature-sensitive mutant. Second, Tim22p can be coprecipitated with Tim54p from detergent-solubilized mitochondria, but Tim54p and Tim22p do not interact with either Tim23p or Tim17p. Finally, the tim54-1 mutation destabilizes the Tim22 protein, but not Tim23p or Tim17p. Our results support the idea that the mitochondrial inner membrane carries two independent import complexes: one required for the translocation of proteins across the inner membrane (Tim23p-Tim17p), and the other required for the insertion of proteins into the inner membrane (Tim54p-Tim22p).
