TY - JOUR
T1 - Interaction of Tim23 with Tim50 is essential for protein translocation by the mitochondrial TIM23 complex
AU - Gevorkyan-Airapetov, Lada
AU - Zohary, Keren
AU - Popov-Čeleketić, Dušan
AU - Mapa, Koyeli
AU - Hell, Kai
AU - Neupert, Walter
AU - Azem, Abdussalam
AU - Mokranjac, Dejana
PY - 2009/2/20
Y1 - 2009/2/20
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=64149111105&partnerID=8YFLogxK
U2 - 10.1074/jbc.M807041200
DO - 10.1074/jbc.M807041200
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AN - SCOPUS:64149111105
SN - 0021-9258
VL - 284
SP - 4865
EP - 4872
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 8
ER -