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Characterization of proteins associated with a coenzyme Q biosynthetic complex in yeast mitochondria (605.19)
Author(s) -
Allan Christopher,
Johnson Jarrett,
Shirasaki Dyna,
Loo Joseph,
Clarke Catherine
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.605.19
Subject(s) - coenzyme q – cytochrome c reductase , biochemistry , biosynthesis , saccharomyces cerevisiae , biology , mitochondrion , mitochondrial matrix , mitochondrial respiratory chain , membrane protein , cofactor , yeast , chemistry , cytosol , cytochrome c , gene , enzyme , membrane
Coenzyme Q (Q) is a fully substituted benzoquinone lipid crucial to the respiratory electron transport chain where it transfers electrons from NADH and succinate to cytochrome c . In the yeast Saccharomyces cerevisiae Q synthesis requires eleven known mitochondrial proteins, Coq1p‐Coq9p, Yah1p, and Arh1p. Several of these proteins have been shown to exist in a high molecular weight complex localized to the matrix side of the inner mitochondrial membrane. The function of some of these proteins in Q biosynthesis is not well characterized and it is not known whether additional proteins are required. Additionally, the organization of the biosynthetic complex as well as the role of protein phosphorylation in Q biosynthesis is not fully understood. To better characterize the Q biosynthetic complex as well as to identify potentially novel associated proteins we generated chromosomally‐integrated tandem affinity tags for Coq3p and Coq9p which were analyzed through co‐precipitation of the dual‐tagged Coq proteins followed by proteomic analysis. This tag, termed the CNAP tag, allows for purification of the tagged proteins under native conditions, preserving any noncovalent protein‐protein interactions that may exist in a macromolecular complex. The presence of the tag on either Coq3p or Coq9p was shown not to disrupt steady‐state levels of the Coq proteins and not to impair Q biosynthesis. Using this tag we demonstrated that Coq3p and Coq9p co‐precipitate each other as well as Coq4p, Coq5p, Coq6p, and Coq7p. Proteomic analysis also identified potentially phosphorylated peptides in Coq4p and Coq6p, which have not previously been shown to be phosphorylated. Three additional uncharacterized candidate proteins were found in this analysis, two of which were shown to have impaired de novo Q biosynthesis.