The Cbp3 protein of Saccharomyces cerevisiae is an enzyme-specific chaperone required for the assembly of ubiquinol-cytochrome c reductase of the mitochondrial respiratory chain. To gain preliminary insight into the role of Cbp3p during assembly, 29 independently isolated mutants were examined to define functional regions of the protein. Mutants were analyzed with respect to respiratory growth, ubiquinol-cytochrome c reductase assembly, and steady state amounts of enzyme subunits and Cbp3p. Three regions essential for Cbp3p activity were identified: regions 1 and 3 were required for Cbp3p function, while region 2 was necessary for protein stability. Mutation of Glu134 in region 1 (Cys124 through Ala140) impaired the ability of the Rieske FeS protein to assemble with the enzyme complex. Mutations targeted to region 3 (Gly223 through Asp229) primarily affected the 14 kDa subunit and cytochrome c(1) assembly. Gly223 was found especially sensitive to mutation and the introduction of charged residues at this site compromised Cbp3p functional activity. Region 2 (Leu167 through Pro175) overlapped the single hydrophobic domain of Cbp3p. Mutations within this area altered the association of Cbp3p with the mitochondrial membrane resulting in enhanced protein turnover. The role of the amino-terminus in Cbp3p activity was investigated using cbp3 deletion strains Delta12-23, Delta24-54, Delta56-96 and Delta12-96. All mutants were respiratory competent, indicating that residues 12-96 were not essential for Cbp3p function, stability or mitochondrial import. Analysis of carboxy-terminal deletion mutants demonstrated that the final 44 residues were not necessary for Cbp3p function; however, alterations in the secondary structure of the extreme carboxy-terminal 17 residues affected assembly protein activity.

• PMID: 11522252
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Journal Article | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, Non-P.H.S.

Authors

Shi G, Crivellone MD, Edderkaoui B

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