CORRECTION OF THE METABOLIC DEFECT IN PROPIONIC ACIDEMIA FIBROBLASTS BY MICROINJECTION OF A FULL-LENGTH CDNA OR RNA TRANSCRIPT ENCODING THEPROPIONYL-COA CARBOXYLASE BETA-SUBUNIT

Propionyl-CoA carboxylase (PCC) is a mitochondrial, biotin-dependent e nzyme, composed of an equal number of alpha and beta subunits, that fu nctions in the catabolism of branched-chain amino acids and other meta bolites. Mutations of the PCCA (alpha subunit) or PCCB (beta subunit) gene cause the inherited metabolic disease, propionic acidemia. We rep ort the cloning of a full-length cDNA encoding the beta subunit of hum an PCC. The open reading frame encodes a pre-beta polypeptide of 539 a mino acids (58,205 Da). The cDNA was introduced into the expression ve ctor, pRc/CMV, and microinjected into the nucleus or, as ribotranscrip ts, into the cytoplasm of fibroblast lines from patients with defects of the beta subunit. The restoration of function was monitored by auto radiography of PCC-dependent [C-14]- propionate incorporation into cel lular protein. These results confirm the completeness of the clone and demonstrate the capacity for beta subunits derived from the microinje cted cDNA or RNA to be transported into mitochondria and assembled wit h endogenously derived a subunits to form functional PCC. (C) 1994 Aca demic Press, Inc.