The Assembly Factor Pet117 Couples Heme a Synthase Activity to Cytochrome Oxidase Assembly

Nicholas G Taylor,Samantha Swenson,Nicholas J Harris,Edward M Germany,Jennifer L Fox,Oleh Khalimonchuk

doi:10.1074/jbc.m116.766980

Nicholas G Taylor, Samantha Swenson + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m116.766980

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Abstract

Heme a is an essential metalloporphyrin cofactor of the mitochondrial respiratory enzyme cytochrome c oxidase (CcO). Its synthesis from heme b requires several enzymes, including the evolutionarily conserved heme a synthase (Cox15). Oligomerization of Cox15 appears to be important for the process of heme a biosynthesis and transfer to maturing CcO. However, the details of this process remain elusive, and the roles of any additional CcO assembly factors that may be involved remain unclear. Here we report the systematic analysis of one such uncharacterized assembly factor, Pet117, and demonstrate in Saccharomyces cerevisiae that this evolutionarily conserved protein is necessary for Cox15 oligomerization and function. Pet117 is shown to reside in the mitochondrial matrix, where it is associated with the inner membrane. Pet117 functions at the later maturation stages of the core CcO subunit Cox1 that precede Cox1 hemylation. Pet117 also physically interacts with Cox15 and specifically mediates the stability of Cox15 oligomeric complexes. This Cox15-Pet117 interaction observed by co-immunoprecipitation persists in the absence of heme a synthase activity, is dependent upon Cox1 synthesis and early maturation steps, and is further dependent upon the presence of the matrix-exposed, unstructured linker region of Cox15 needed for Cox15 oligomerization, suggesting that this region mediates the interaction or that the interaction is lost when Cox15 is unable to oligomerize. Based on these findings, it was concluded that Pet117 mediates coupling of heme a synthesis to the CcO assembly process in eukaryotes.

Highlights

Heme a is an essential metalloporphyrin cofactor of the mitochondrial respiratory enzyme cytochrome c oxidase (CcO)
We show that Pet117 is a small, inner membrane (IM)-associated protein residing in the mitochondrial matrix that is required for the stable oligomerization of Cox15, suggesting that Pet117 aids in coupling heme a synthase activity to the process of CcO maturation
We found that Pet117 and Cox15 interact in the absence of the late stage CcO assembly factor Shy1, yet their interaction is severely diminished in the absence of the early CcO assembly factor Mss51

Summary

The abbreviations used are

CcO, cytochrome c oxidase; BN, blue native; IP, immunoprecipitation; IM, inner membrane. We show that Pet117 is a small, IM-associated protein residing in the mitochondrial matrix that is required for the stable oligomerization of Cox, suggesting that Pet117 aids in coupling heme a synthase activity to the process of CcO maturation. We found that Pet117 and Cox interact in the absence of the late stage CcO assembly factor Shy, yet their interaction is severely diminished in the absence of the early CcO assembly factor Mss. We found that Pet117 and Cox interact in the absence of the late stage CcO assembly factor Shy, yet their interaction is severely diminished in the absence of the early CcO assembly factor Mss51 These results suggest that Pet117 is an important assembly factor for linking the vital process of heme a biosynthesis to CcO biogenesis

Results

Discussion

Experimental Procedures