THE MEMBRANE OF PEROXISOMES IN SACCHAROMYCES-CEREVISIAE IS IMPERMEABLE TO NAD(H) AND ACETYL-COA UNDER IN-VIVO CONDITIONS

We investigated how NADH generated during peroxisomal beta-oxidation i s reoxidized to NAD(+) and how the end product of beta-oxidation, acet yl-CoA, is transported from peroxisomes to mitochondria in Saccharomyc es cerevisiae. Disruption of the peroxisomal malate dehydrogenase 3 ge ne (MDH3) resulted in impaired beta-oxidation capacity as measured in intact cells, whereas beta-oxidation was perfectly normal in cell lysa tes, In addition, mdh3-disrupted cells were unable to grow on oleate w hereas growth on other non-fermentable carbon sources was normal, sugg esting that MDH3 is involved in the reoxidation of NADH generated duri ng fatty acid beta-oxidation rather than functioning as part of the gl yoxylate cycle. To study the transport of acetyl units from peroxisome s, we disrupted the peroxisomal citrate synthase gene (CIT2). The lack of phenotype of the cit2 mutant indicated the presence of an alternat ive pathway for transport of acetyl units, formed by the carnitine ace tyltransferase protein (YCAT). Disruption of both the CIT2 and YCAT ge ne blocked the beta-oxidation in intact cells, but not in lysates. Our data strongly suggest that the peroxisomal membrane is impermeable to NAD(H) and acetyl-CoA in vivo, and predict the existence of metabolit e carriers in the peroxisomal membrane to shuttle metabolites from per oxisomes to cytoplasm and vice versa.