CoQ biosynthesis regulation in yeast through a phosphorylation cycle of Coq7p

Abstract

In the last years it has been suggested that Q6 biosynthesis in yeast is produced through a multienzymatic complex (Hsu, et al. 2000, Biochem. Biophys. Acta; Gin and Clarke 2005, J Biol Chem). The lack of precursor accumulation in coq null mutant strains and the accumulation of a common early precursor (hexaprenyl hydroxy benzoic acid, HHB) supports this idea. On the contrary, several point mutants in coq genes such COQ5 and COQ7 accumulate its own precursor. It suggests a double function for the encoded proteins: catalytical and structural (Baba et al. 2004, J Biol Chem; Padilla et al. 2004, J. Biol. Chem.). Several studies performed by size exclusion chromatography or BN-PAGE have demonstrated that COQ proteins can be components of protein complexes with different sizes and composition (Marbois et al. 2005, J. Biol. Chem.). The protein Coq7p seem to be a key element for the Q6 biosynthetic complex assembly or its regulation. The substrate of Coq7p, DMQ6, is largely accumulated in wild type strains during the growth in fermentable and non-fermentable carbons sources (Padilla et al. 2008, Cell Mol Life Sci). The over-expression of a possible regulatory protein, Coq8p, induces the accumulation of DMQ6 in coq7 mutants (Padilla, et al. 2008, Cell Mol Life Sci). Coq8p is a putative kinase (ADCK2 family) that indirectly is required for Coq3p phosphorylation (Tauche et al. 2008, FEM Yeast Res). Our initial hypothesis is that phosphorylation play a important role on Q6 biosynthesis. Given that Coq7p show three putative sites for Ser/Thr phosphorylation, we have analyzed its participation on Coq7p functions. The Coq7p protein was phosphorylated in vitro by PKA and PKC while the change of putative phosphoaminocids for alanine removes the phosphorylation signal. When Coq7p V5-tagged protein was expressed in null coq7 mutants, we have detected that Coqp7 is a phosphoprotein in vivo, after purification by IP and 2D IEF/SDS-PAGE and staining with the fluorochrome Pro-Q Diamond. That detection was abolished after the phosphoaminoacids removal in mutant versions of Coq7p. We have obtained evidences of the relationship of Coq7p phosphorylation and the metabolic state of the cells. V5-tagged Coq7p was expressed in a null coq7 strain and phosphorylation state was analyzed after the growth in several carbon sources, non-fermentables and several glucose concentrations. Coq7p phosphorylation was increased in high glucose levels and decrease significantly at low glucose level or in non-fermentable carbon sources. These data correlate with a phosphorylated state under non-respiratory growth and our hypothesis is consider this state as a low Q6 biosynthesis state. This idea was demonstrated after performs a functional complementation of null coq7 strain with versions of Coq7p without phosphoaminoacids (loss-of-function, pL) and a version with negatively charged aminoacids instead (gain-of-function, pG). All versions (pL or pG) growth in non-fermentable carbon sources but differs significantly in the levels of Q6. In pL versions, Q6 was increased between 160-250% while in pG versions was decreased to 50-60% compared to the wild type strain. In general, Q6 variations induce changes on the physiological functions of Q6, electron transport, antioxidant functions and even was affected the chronological life span. It was demonstrated in vitro as in vivo that the protein Coq7p is a phosphoprotein. The regulation by phosphorylation modify the levels of Q6 produced that fit with the changes of phosphorylation state produced in different carbon sources.