Overexpression of the Coq8 kinase in Saccharomyces cerevisiae coq null mutants allows for accumulation of diagnostic intermediates of the coenzyme Q6 biosynthetic pathway.

Most of the Coq proteins involved in coenzyme Q (ubiquinone or Q) biosynthesis are interdependent within a multiprotein complex in the yeast Saccharomyces cerevisiae. Lack of only one Coq polypeptide, as in Δcoq strains, results in the degradation of several Coq proteins. Consequently, Δcoq strains accumulate the same early intermediate of the Q(6) biosynthetic pathway; this intermediate is therefore not informative about the deficient biosynthetic step in a particular Δcoq strain. In this work, we report that the overexpression of the protein Coq8 in Δcoq strains restores steady state levels of the unstable Coq proteins. Coq8 has been proposed to be a kinase, and we provide evidence that the kinase activity is essential for the stabilizing effect of Coq8 in the Δcoq strains. This stabilization results in the accumulation of several novel Q(6) biosynthetic intermediates. These Q intermediates identify chemical steps impaired in cells lacking Coq4 and Coq9 polypeptides, for which no function has been established to date. Several of the new intermediates contain a C4-amine and provide information on the deamination reaction that takes place when para-aminobenzoic acid is used as a ring precursor of Q(6). Finally, we used synthetic analogues of 4-hydroxybenzoic acid to bypass deficient biosynthetic steps, and we show here that 2,4-dihydroxybenzoic acid is able to restore Q(6) biosynthesis and respiratory growth in a Δcoq7 strain overexpressing Coq8. The overexpression of Coq8 and the use of 4-hydroxybenzoic acid analogues represent innovative tools to elucidate the Q biosynthetic pathway.

Affinity purification of soluble lysosomal proteins for mass spectrometric identification.

This chapter describes the process of production, purification, separation, and mass spectrometry identification of soluble lysosomal proteins. The rationale for purification of these proteins resides in their characteristic sugar, the mannose-6-phosphate (M6P), which allows an easy purification by affinity chromatography on immobilized M6P receptor (MPR). The secretion of M6P proteins (essentially soluble lysosomal proteins) from cells in culture is induced by adding a weak base in the culture medium. Secreted proteins are ammonium sulfate precipitated, dialyzed, and loaded onto the immobilized MPR column. After specific elution and collection of the M6P proteins, these are resolved by either bidimensional or monodimensional gel electrophoresis (designated as 2-DE or 1-DE, respectively). Mass spectrometry analysis is performed on spots excised from the 2-DE gel, or on discrete bands covering altogether the whole length of the 1-DE gel lane: these spots or bands are in-gel digested with trypsin and protein identification is obtained, thanks to peptide mass fingerprints [provided by analysis of the digests by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS)] or peptide amino acid sequences (provided by analysis of the digests by the coupling between liquid chromatography and tandem mass spectrometry, LC-MS/MS).