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Abstract

The mitochondrial cytochromecoxidase assembles in the inner membrane from subunits of dual genetic origin. The assembly process of the enzyme is initiated by membrane insertion of the mitochondria-encoded Cox1 subunit. During complex maturation, transient assembly intermediates, consisting of structural subunits and specialized chaperone-like assembly factors, are formed. In addition, cofactors such as heme and copper have to be inserted into the nascent complex. To regulate the assembly process, the availability of Cox1 is under control of a regulatory feedback cycle, in which translation of the COX1 mRNA is stalled when assembly intermediates of Cox1 accumulate through inactivation of the translational activator Mss51. Here we have isolated a cytochromecoxidase assembly intermediate in preparatory scale fromcoa1Δmutant cells using Mss51 as a bait. We demonstrate that at this stage of assembly the complex has not yet incorporated the heme a cofactors. Using quantitative mass spectrometry, we defined the protein composition of the assembly intermediate and unexpectedly identified the putative methyltransferase Oms1 as a constituent. Our analyses show that Oms1 participates in cytochromecoxidase assembly by stabilizing newly synthesized Cox1.