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Investigation of the Interacting Partners of Yeast Coq6: A Component of the Multienzyme Complex Required for Coenzyme Q Biosynthesis
Author(s) -
Osonkie Odi,
Clarke Catherine F
Publication year - 2006
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.20.5.a952-c
Subject(s) - tandem affinity purification , biochemistry , saccharomyces cerevisiae , biosynthesis , biology , yeast , mutant , enzyme , gene , affinity chromatography
The COQ6 gene in Saccharomyces cerevisiae is predicted to encode a flavin‐dependent monooxygenase involved in Coenzyme Q (Q) biosynthesis, based on sequence similarity to known enzymes. Recent evidence indicates that the proteins required for Q biosynthesis in yeast form a multienzyme complex, which is defined by Coq3 and Coq4. To investigate the interacting partners of Coq6, a strain containing a tandem affinity purification (TAP) tag on Coq6 was analyzed. The TAP tag, which enables highly specific purification of the target protein, is an integrated ~22 kDa C‐terminal tag which consists of a calmodulin‐binding peptide, a TEV protease cleavage site and two IgG‐binding domains of Protein A. Growth curves indicate that the growth of the Coq6‐TAP strain on a non‐fermentable carbon source is superior to wild‐type, and immunoblot analyses reveal an enhanced expression of the Coq6 protein in the Coq6‐TAP strain, relative to wild‐type. Furthermore, blue native polyacrylamide gel electrophoresis analyses show that the native Coq6 protein is present in a ~180 kDa complex in both wild‐type and Coq6‐TAP strains, demonstrating that the TAP tag does not alter the oligomeric state of native Coq6 protein. The Coq6 protein was purified from the TAP‐tagged strain by tandem affinity purification, and subsequent immunoblot analyses reveal that Coq3 co‐purifies with Coq6. This indicates that a specific interaction exists between Coq3 and Coq6 in the polypeptide complex required for Q biosynthesis in yeast. This work was supported by National Institutes of Health Grant GM45952 (to C. F. Clarke).