Investigation of the Interacting Partners of Yeast Coq6: A Component of the Multienzyme Complex Required for Coenzyme Q Biosynthesis

Abstract

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).