Tim18p, a New Subunit of the TIM22 Complex That Mediates Insertion of Imported Proteins into the Yeast Mitochondrial Inner Membrane

Abstract

Import of carrier proteins from the cytoplasm into the mitochondrial inner membrane of yeast is mediated by a distinct system consisting of two soluble 70-kDa protein complexes in the intermembrane space and a 300-kDa complex in the inner membrane, the TIM22 complex. The TIM22 complex contains the peripheral subunits Tim9p, Tim10p, and Tim12p and the integral membrane subunits Tim22p and Tim54p. We identify here an additional subunit, an 18-kDa integral membrane protein termed Tim18p. This protein is made as a 21.9-kDa precursor which is imported into mitochondria and processed to its mature form. When mitochondria are gently solubilized, Tim18p comigrates with the other subunits of the TIM22 complex on nondenaturing gels and is coimmunoprecipitated with Tim54p and Tim12p. Tim18p does not cofractionate with the TIM23 complex upon immunoprecipitation or nondenaturing gel electrophoresis. Deletion of Tim18p decreases the growth rate of yeast cells by a factor of two and is synthetically lethal with temperature-sensitive mutations in Tim9p or Tim10p. It also impairs the import of several precursor proteins into isolated mitochondria, and lowers the apparent mass of the TIM22 complex. We suggest that Tim18p functions in the assembly and stabilization of the TIM22 complex but does not directly participate in protein insertion into the inner membrane.

ACKNOWLEDGMENTS

C. M. Koehler and M. P. Murphy contributed equally to this paper.

We are grateful to Nikolaus Pfanner for his generous gift of the plasmid carrying the TIM17 gene and to Paul Jeno for assistance with the electrospray mass spectrometry and peptide sequencing.

We thank the following agencies for their support: the Swiss National Science Foundation (to G.S.), the Human Frontier Science Program Organization (to G.S.), the European Economic Union (to G.S.), the F. Louis Jeantet Foundation (to G.S.), the Damon Runyon-Walter Winchell Cancer Research Foundation (to C.M.K.), the U.S. National Science Foundation (to C.M.K.), and the European Molecular Biology Organisation (to E.O.).