Stable restoration of pyruvate dehydrogenase complex in E1-defective human lymphoblastoid cells: evidence that three C-terminal amino acids of E1 alpha are essential for the structural integrity of heterotetrameric E1.

In an attempt to restore pyruvate dehydrogenase complex (PDHC), expression vectors carrying wildtype E1 alpha cDNA (pRAWT) or 1162ins-mutant (pRA1162) were introduced into human lymphoblastoid cells which had a 4-bp insertion after nucleotide 1162 (1162ins) of E1 alpha cDNA, 28% of normal PDHC activity, and undetectable levels of both E1 alpha and E1 beta proteins. The amount of E1 alpha mRNA transcribed from the introduced cDNA was approximately 25 times greater than that transcribed from the endogenous gene. The PDHC activity of pRAWT-transformed cells increased to the normal level whereas this activity increased to 55% of the control in pRA1162-transformed cells. Mitochondria from pRAWT-transformed cells contained normal amounts of both the E1 alpha and the E1 beta subunits. These results suggest that the three C-terminal amino acids of E1 alpha, which were absent from 1162ins-mutant protein, may be important for the structural integrity of E1 and that a large amount of normal subunit, compared to the endogenous mutant enzyme, must be expressed to restore a multienzyme complex.