The role of Tim9p in the assembly of the TIM22 import complexes.
Tim9p is located in the soluble 70-kDa Tim9p-Tim10p complex and the 300-kDa membrane complex in the mitochondrial TIM22 protein import system, which mediates the import of inner membrane proteins. From a collection of temperature-sensitive mutants, we have analyzed two in detail. tim9-3 contained two mutations and tim9-19 contained one mutation, all located near the 'twin CX3C' motif that is conserved in the small Tim proteins. As a result, the import components in the tim9-3 mutant mitochondria were severely reduced and assembled into complexes of aberrant sizes. Protein import was severely reduced and Tim9p and Tim10p binding to in vitro imported ADP/ATP carrier was impaired. In the tim9-19 mutant mitochondria, the 300-kDa membrane complex was assembled, although the soluble 70-kDa Tim9p-Tim10p complex was not detectable. Protein import was decreased only two-fold. When coexpressed in Escherichia coli, tim9-19 and TIM10 proteins failed to assemble into a 70-kDa complex. Our findings suggest that residues near the 'twin CX3C' motif are important for the assembly of Tim9p in both the Tim9p-Tim10p complex and the 300-kDa membrane complex.
Leuenberger
Danielle
D
Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095-1569, USA.
Curran
Sean P
SP
Wong
David
D
Koehler
Carla M
CM
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C102549:TIM22 protein, S cerevisiae; C116366:Tim9 protein, S cerevisiae