Genetic analysis of complex interactions among components of the mitochondrial import motor and translocon in Saccharomyces cerevisiae.
A highly conserved, Hsp70-based, import motor, which is associated with the translocase on the matrix side of the inner mitochondrial membrane, is critical for protein translocation into the matrix. Hsp70 is tethered to the translocon via interaction with Tim44. Pam18, the J-protein co-chaperone, and Pam16, a structurally related protein with which Pam18 forms a heterodimer, are also critical components of the motor. Their N termini are important for the heterodimer's translocon association, with Pam18's and Pam16's N termini interacting in the intermembrane space and the matrix, respectively. Here, using the model organism Saccharomyces cerevisiae, we report the identification of an N-terminal segment of Tim44, important for association of Pam16 with the translocon. We also report that higher amounts of Pam17, a nonessential motor component, are found associated with the translocon in both PAM16 and TIM44 mutants that affect their interaction with one another. These TIM44 and PAM16 mutations are also synthetically lethal with a deletion of PAM17. In contrast, a deletion of PAM17 has little, or no genetic interaction with a PAM18 mutation that affects translocon association of the Pam16:Pam18 heterodimer, suggesting a second role for the Pam16:Tim44 interaction. A similar pattern of genetic interactions and enhanced Pam17 translocon association was observed in the absence of the C terminus of Tim17, a core component of the translocon. We suggest the Pam16:Tim44 interaction may play two roles: (1) tethering the Pam16:Pam18 heterodimer to the translocon and (2) positioning the import motor for efficient engagement with the translocating polypeptide along with Tim17 and Pam17.
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53726, USA.
Brenda A
BA
Schilke
Masaya
M
Hayashi
Elizabeth A
EA
Craig
D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000378:metabolism; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D010641:Phenotype; D017354:Point Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D018840:HSP70 Heat-Shock Proteins; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; C484526:Pam16 protein, S cerevisiae; C090869:SSA3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C091191:TIM17 protein, S cerevisiae; C083081:TIM23 protein, S cerevisiae; C077016:TIM44 protein, S cerevisiae