Selective over-synthesis and rapid turnover of mitochondrial protein components of respiratory complexes

Mammalian mitochondria assemble four complexes of the respiratory chain (RCI, III, IV and V) by combining 13 polypeptides synthesized within mitochondria on mitochondrial ribosomes (mitoribosomes) with over 70 polypeptides encoded in nuclear DNA, translated on cytoplasmic ribosomes and imported into mitochondria. We report that pulse-chase SILAC can also serve as a valuable approach to study RC assembly as it reveals considerable differences in the rates and efficiency of assembly of different complexes. While assembly of RCV, ATPase, was rapid with little excess synthesis of subunits, RCI, NADH dehydrogenase, assembly was far less efficient with dramatic over-synthesis of numerous proteins, particularly in the matrix exposed N- and Q- Domains. Subunits that do not engage in assembly are generally degraded within three hours. Differential assembly kinetics were also observed for individual complexes immunoprecipitated with complex-specific antibodies. Immunoprecipitation with an antibody that recognizes the ND1 subunit of RCI co-precipitated a number of proteins implicated in FeS cluster assembly as well as newly-synthesized UQCRFS1, the Rieske FeS protein in RCIII, reflecting a degree of coordination of RCI and RCIII assembly.