Altered phosphorylation state of branched-chain 2-oxo acid dehydrogenase in a branched-chain acyltransferase deficient human fibroblast cell line.

The abundance and phosphorylation state of the polypeptide constituents of the human branched-chain 2-oxo acid dehydrogenase complex were examined in mitochondria from normal and maple syrup urine disease (MSUD) fibroblasts. In normal fibroblast mitochondria two forms of the E1 alpha subunit were observed: non-phosphorylated (E1 alpha) and phosphorylated (E1 alpha-P). About 40-50% of E1 alpha was present as E1 alpha-P. The ability to quantitate the two forms of E1 alpha permitted examination of the association between decreased capacity of oxidize branched-chain 2-oxo acids and the phosphorylation state of E1 alpha. Changes in phosphorylation state of E1 alpha were observed in MSUD fibroblasts as compared to control cells. Of particular interest was the absence of E1 alpha-P in an MSUD fibroblast line which lacked the dihydrolipoyl acyltransferase (E2) subunit of the dehydrogenase complex. In two MSUD cell lines deficient in E1 alpha, the abundance of E1 alpha-P appeared to be preferentially reduced. A fourth MSUD cell line contained normal quantities of E3, E2 and both forms of the E1 alpha polypeptide. Our results indicate that alterations in the abundance of dehydrogenase complex polypeptides in MSUD fibroblasts may influence the phosphorylation state of the E1 alpha polypeptide. They demonstrate the potential for examining simultaneously mutations which affect both the catalytic and regulatory components of the dehydrogenase complex.