The cytosol-synthesized subunit II (Cox2) precursor with the point mutation W56R is correctly processed in yeast mitochondria to rescue cytochrome oxidase

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• 作者：Valent¨ªn Cruz-Torres ; Miriam V¨¢zquez-Acevedo ; Rodolfo Garc¨ªa-Villegas ; Xochitl P¨¦rez-Mart¨ªnez ; Guillermo Mendoza-Hern¨¢ndez ; Diego Gonz¨¢lez-Halphen
• 关键词：Allotopic expression ; Mitochondrial targeting signals ; Subunit 2 of cytochrome c oxidase ; Cytochrome c oxidase biogenesis
• 刊名：Biochimica et Biophysica Acta (BBA)/Bioenergetics
• 出版年：2012
• 出版时间：December, 2012
• 年：2012
• 卷：1817
• 期：12
• 页码：2128-2139
• 全文大小：1431 K

文摘

Deletion of the yeast mitochondrial gene COX2 encoding subunit 2 (Cox2) of cytochrome c oxidase (CcO) results in loss of respiration (¦¤cox2 strain). Supekova et al. (2010) transformed a ¦¤cox2 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 ¦¤cox2 + 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 W₅₆ was substituted by R₅₆ 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 ¦¤cox2 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.