title: The cytosol-synthesized subunit II (Cox2) precursor with the point mutation W56R is correctly processed in yeast mitochondria to rescue cytochrome oxidase.
abstract: Deletion of the yeast mitochondrial gene COX2 encoding subunit 2 (Cox2) of cytochrome c oxidase (CcO) results in loss of respiration (Δcox2 strain). Supekova et al. (2010) [1] transformed a Δcox2 strain with a vector expressing Cox2 with a mitochondrial targeting sequence (MTS) and the point mutation W56R (Cox2(W56R)), restoring respiratory growth. Here, the CcO carrying the allotopically-expressed Cox2(W56R) was characterized. Yeast mitochondria from the wild-type (WT) and the Δcox2+Cox2(W56R) strains were subjected to Blue Native electrophoresis. In-gel activity of CcO and spectroscopic quantitation of cytochromes revealed that only 60% of CcO is present in the complemented strain, and that less CcO is found associated in supercomplexes as compared to WT. CcOs from the WT and the mutant exhibited similar subunit composition, although activity was 20-25% lower in the enzyme containing Cox2(W56R) than in the one with Cox2(WT). Tandem mass spectrometry confirmed that W(56) was substituted by R(56) in Cox2(W56R). In addition, Cox2(W56R) exhibited the same N-terminus than Cox2(WT), indicating that the MTS of Oxa1 and the leader sequence of 15 residues were removed from Cox2(W56R) during maturation. Thus, Cox2(W56R) is identical to Cox2(WT) except for the point mutation W56R. Mitochondrial Cox1 synthesis is strongly reduced in Δcox2 mutants, but the Cox2(W56R) complemented strain led to full restoration of Cox1 synthesis. We conclude that the cytosol-synthesized Cox2(W56R) follows a rate-limiting process of import, maturation or assembly that yields lower steady-state levels of CcO. Still, the allotopically-expressed Cox2(W56R) restores CcO activity and allows mitochondrial Cox1 synthesis to advance at WT levels.
authors.affiliation: Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México D.F., Mexico.
authors.forename: Valentín
authors.initials: V
authors.lastname: Cruz-Torres
authors.forename: Miriam
authors.initials: M
authors.lastname: Vázquez-Acevedo
authors.forename: Rodolfo
authors.initials: R
authors.lastname: García-Villegas
authors.forename: Xochitl
authors.initials: X
authors.lastname: Pérez-Martínez
authors.forename: Guillermo
authors.initials: G
authors.lastname: Mendoza-Hernández
authors.forename: Diego
authors.initials: D
authors.lastname: González-Halphen
mesh_terms: D000595:Amino Acid Sequence; D019069:Cell Respiration / Q000502:physiology; D003593:Cytoplasm / Q000201:enzymology*; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007118:Immunoassay; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D063347:Native Polyacrylamide Gel Electrophoresis; D010100:Oxygen / Q000378:metabolism*; D017354:Point Mutation / Q000235:genetics*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins; C117108:cytochrome C oxidase subunit II; C526453:Cox1 protein, S cerevisiae; D003576:Electron Transport Complex IV; D010100:Oxygen