Characterization of the mitochondrial inner membrane translocase complex: the Tim23p hydrophobic domain interacts with Tim17p but not with other Tim23p molecules.
Tim23p is a mitochondrial inner membrane protein essential for the import of proteins from the cytosol. Tim23p contains an amino-terminal hydrophilic segment and a carboxyl-terminal hydrophobic domain (Tim23Cp). To study the functions and interactions of the two parts of Tim23p separately, we constructed tim23N, encoding only the hydrophilic region of Tim23p, and tim23C, encoding only the hydrophobic domain of Tim23p. Only the Tim23C protein is imported into mitochondria, indicating that the mitochondrial targeting information in Tim23p resides in its membrane spans or intervening loops. Tim23Cp, however, cannot substitute for full-length Tim23p, suggesting that the hydrophilic portion of Tim23p also performs an essential function in mitochondrial protein import. We found that overexpression of Tim23Cp is toxic to yeast cells that carry the tim23-1 mutation. Excess Tim23Cp causes Tim23-1p to disappear, leaving tim23-1 cells without a full-length version of the Tim23 protein. If Tim17p, another inner membrane import component, is overexpressed along with Tim23Cp, the toxicity of Tim23Cp is largely reversed and the Tim23-1 protein no longer disappears. In coimmunoprecipitations from solubilized mitochondria, Tim17p associates with the Tim23C protein. In addition, we show that Tim23p and Tim17p can be chemically cross-linked to each other in intact mitochondria. We conclude that the hydrophobic domain encoded by tim23C targets Tim23p to the mitochondria and mediates the direct interaction between Tim23p and Tim17p. In contrast, Tim23Cp cannot be coimmunoprecipitated with Tim23p, raising the possibility that the hydrophobic domain of Tim23p does not interact with other Tim23 molecules.
Ryan
K R
KR
Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Leung
R S
RS
Jensen
R E
RE
D002352:Carrier Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008928:Mitochondria / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D011485:Protein Binding; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins; D013367:Submitochondrial Particles / Q000378:metabolism
D002352:Carrier Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C091191:TIM17 protein, S cerevisiae; C083081:TIM23 protein, S cerevisiae