Interaction of Tim23 with Tim50 Is essential for protein translocation by the mitochondrial TIM23 complex.
The TIM23 complex is the major translocase of the mitochondrial inner membrane responsible for the import of essentially all matrix proteins and a number of inner membrane proteins. Tim23 and Tim50, two essential proteins of the complex, expose conserved domains into the intermembrane space that interact with each other. Here, we describe in vitro reconstitution of this interaction using recombinantly expressed and purified intermembrane space domains of Tim50 and Tim23. We established two independent methods, chemical cross-linking and surface plasmon resonance, to track their interaction. In addition, we identified mutations in Tim23 that abolish its interaction with Tim50 in vitro. These mutations also destabilized the interaction between the two proteins in vivo, leading to defective import of preproteins via the TIM23 complex and to cell death at higher temperatures. This is the first study to describe the reconstitution of the Tim50-Tim23 interaction in vitro and to identify specific residues of Tim23 that are vital for the interaction with Tim50.
Gevorkyan-Airapetov
Lada
L
Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, 69778 Tel Aviv, Israel.
Zohary
Keren
K
Popov-Celeketic
Dusan
D
Mapa
Koyeli
K
Hell
Kai
K
Neupert
Walter
W
Azem
Abdussalam
A
Mokranjac
Dejana
D
D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000737:chemistry / Q000378:metabolism; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C083081:TIM23 protein, S cerevisiae; C469615:TIM50 protein, S cerevisiae