UNEQUAL SYNTHESIS AND DIFFERENTIAL DEGRADATION OF PROPIONYL COA CARBOXYLASE SUBUNITS IN CELLS FROM NORMAL AND PROPIONIC ACIDEMIA PATIENTS

  • 1 July 1989
    • journal article
    • research article
    • Vol. 45  (1) , 33-40
Abstract
We have characterized further the molecular basis of human inherited propionyl CoA carboxylase deficiency by measuring steady state levels of the mRNAs coding for the enzyme''s two protein subunits (.alpha. and .beta.) and by estimating initial synthesis and steady state levels of the protein subunits in skin fibroblasts from controls and affected patients. We studied cell lines from both major complementation groups (pccA and pccBC) corresponding, respectively, to defects in the carboxylase''s .alpha. and .beta. subunits. Analysis of pccA lines revealed the absence of .alpha. chain mRNA in three and an abnormally small .alpha.-mRNA in a fourth. Despite the presence of normal .beta.-mRNA in each of these pccA lines, there was complete absence of both .alpha. and .beta. protein subunits under steady state conditions, even though new synthesis and mitochondrial import of .beta. precursors was normal. Results in nine pccBC lines revealed normal .alpha. mRNA in each, while the amounts of .beta.-mRNA were distinctly reduced in every case. Correspondingly, .alpha. protein subunits were present in normal amounts at steady-state, but .beta. subunits were uniformly decreased. In addition, in six of the nine .beta. deficient cell lines, partially degraded .beta.-subunits were observed. To help interpret these results, synthesis and stability of carboxylase subunits were studied in intact HeLa cells using a pulse-chase protocol. Whereas .alpha. chains were stable over the four hour interval studied, .beta. chains - initially synthesized in large excess over .alpha. chains - were degraded rapidly reaching equivalence with .alpha. chains after two hours. From these results we conclude that: .beta. chain subunits are normally synthesized and imported into mitochondria in excess of .alpha. chains, but only that portion assembled with 7a-subunits escapes degradation; in pccA patients, the primary defect in .alpha. chain synthesis leads secondarily to degradation of normally synthesized .beta. chains; and, in some pccBC patients, mutant .beta. chains are intrinsically unstable. Finally, we posit that the differential rates of synthesis of .alpha. and .beta. chains account for the prior reported finding that individuals heterozygous for pccBC mutations have normal carboxylase activity in their cells.