Biosynthesis of precursor forms of the pyruvate dehydrogenase complex and mitochondrial phosphate transporter in Saccharomyces cerevisiae

Abstract

BIOCHEMICAL SOCIETY TRANSACTIONSplexes generates an extra thiol in the dithiolane ring, which is then susceptible to a stable modification with maleimides, resulting in loss of catalytic function (Brown & Perham, 1976).PDC and O G D C were purified from fresh ox heart (De Marcucci et ul.. 1985). T h e activities of both these complexes were not affected by incubation at room temperature with 0.5 mM-N-ethylmaleimide for 1 h. Addition of. respectively, 2 mwpyruvate and 2 m~-2-oxoglutarate resulted in rapid, irreversible inhibition complete within 10 min. Use o f N-ethyl-[ 2,3-"C]maleimide in this reaction and resolution of the inhibited complexes by SDS/polyacrylamide-gel electrophoresis and fluorography confirmed that the lipoate acetyltransferase and component X subunits of PDC and the lipoate succinyltransferase of OGDC were modified by the maleimide. However, when PDC was preincubated at room temperature with pyruvate before addition of Ncthylmaleimide, there was a time-dependent protection against inhibition of overall activity by the maleimide. After SO min incubation of PDC with pyruvate, addition of Ncthylmaleimide could only produce approx. 10% inhibition o f activity. Prior addition of acetyl-CoA to NADH-reduced PDC also prevented the inhibitory effect of N-ethylmaleimide but more rapidly, with 90% protection occurring 1 0 min after addition of acetyl-CoA. Incubation of PDC with either substrate in the absence of N-ethylmaleimide had no effect on ovcrall activity. There was greatly reduced incorporation of N-ethyl [ 2,3-1'C]maleimide into E 2 and component X subunits if PDC was preincubated with pyruvate or acetyl-CoA. These results suggested that prolonged incubation of PDC with pyruvate in the absence of acetyl-CoA or with acctyl-CoA plus N A D H led to the second thiol on the E 2 lipoyl group becoming unamenable to modification with N-ethylmaleimidc. When PDC was incubated with 12-1JC]pyruvate it was found that acquisition of resistance to N-ethylmaleimide inhibition over 60 rnin was accompanied by a slow increase in ["C]acetyl group incorporation into the complex after the initial rapid phase. Table 1 shows that incorporation of [ lJC]acetyI groups from [2-"C]pyruvate into both E2 and component X was approximately doubled in the absence of N-ethylmaleimide. One explanation of these observations is that a prolonged incubation with acetylating substrate under the conditions employed produced S", Sx-(diacetylj-lipoyl groups. T h e formation of Sh, Sx-(diacetyljlipoamide by mammalian PDC has been detected previously by '?C-n.m.r. spectroscopy ( O C o n n o r et al., 1982). Another interpretation is that acetylation of a second lipoate residue permitted interaction of two acetyl-lipoyl groups such that neither might react with N-ethylmaleimide, perhaps by Table 1. Iticmprutiori o~/Z-"('ll?vrirviite inlo I S titid X siihirriits PDC was incubated with [2-"C]pyruvate in the absence or presence of 0.5 mM-N-cthylmaleimidc (NEM). After 30 rnin incubation, approx. 22 pg of protein was resolved on a 10'%, (w/v)...