A complex of Cox4 and mitochondrial Hsp70 plays an important role in the assembly of the cytochrome c oxidase

Abstract

The biogenesis of Cox4 is unknown. Cox4, mtHsp70, and Mge1 form a complex that promotes the assembly of cytochrome c oxidase. In the absence of the mature cytochrome c oxidase, Cox4 arrests at the chaperone complex. This complex delivers Cox4 into the assembly line of complex IV when needed.

Mitochondrial Heat Shock Protein (Hsp) 70 and Hsp10 Cooperate in the Formation of Hsp60 Complexes

“…Yeast Strains and Growth Conditions-The yeast wild-type strains YPH499, YPH499 arg4⌬, and JK9 -3d (the wild type for hsp10ts and hsp60ts); the wild-type strain for ssc1-42; and the mutant strains mtHsp70 His , ssc1-42, hsp10ts, and hsp60ts have been described before (39,44,54). For SILAC-based mass spectrometric analysis of mtHsp70 His purification, a kanMX4 cassette was integrated into the ARG4 locus by homologous recombination in the yeast strain expressing mtHsp70 His .…”

“…Isolation of Mitochondria and in Vitro Protein Import Assays-Mitochondria were isolated by differential centrifugation following a published procedure (39). Mitochondria were stored in SE buffer (10 mM MOPS/KOH (pH 7.2), 1 mM EDTA, and 250 mM sucrose) in a protein concentration of 10 mg/ml at Ϫ80°C until use.…”

“…To generate 35 S-labeled precursor proteins, we used cell-free in vitro translation on the basis of rabbit reticulocyte lysate in the presence of 35 S-labeled methionine (Promega). Standard import reactions were performed following established assays (39,55). In brief, 35 S-labeled precursors (5-10% of the reaction volume) were incubated with isolated mitochondria at 25°C in import buffer (3% (w/v) BSA, 250 mM sucrose, 5 mM methionine, 80 mM KCl, 5 mM MgCl 2 , 10 mM MOPS/KOH (pH 7.2), and 2 mM KH 2 PO 4 ).…”

“…When the import reaction was studied by blue native electrophoresis, mitochondria were washed with SE buffer and solubilized under nondenaturing conditions using the non-ionic detergent digitonin to 1% (w/v) final concentration in lysis buffer (20 mM Tris-HCl (pH 7.4), 50 mM NaCl, 10% (v/v) glycerol, and 0.1 mM EDTA). (39). Mitochondria were isolated following the standard procedure (39).…”

“…Zinc finger motif protein of 17 kDa (Zim17, also termed mtHsp70 escort protein 1 (Hep1)) supports the folding and function of the chaperone (32)(33)(34)(35)(36)(37). Furthermore, mtHsp70 interacts with Mss51 and Cox4 to promote the biogenesis of the cytochrome c oxidase (complex IV of the respiratory chain) (38,39). MtHsp70 cooperates with other chaperone systems to maintain protein homeostasis.…”