Sequential deletion of C-terminal amino acids of the E₁α component of the pyruvate dehydrogenase (PDH) complex leads to reduced steady-state levels of functional E₁α₂β₂ tetramers: implications for patients with PDH deficiency

Abstract

Human pyruvate dehydrogenase (PDH) complex deficiency is an extremely heterogeneous disease in its presentation and clinical course. We have characterized novel mutations that affect the C-terminal portion of the PDH-E₁α-coding sequence. Although the molecular defects underlying these mutations are different, both effectively produce a stop codon prematurely three amino acids from the C-terminus. The clinical and biochemical consequences of these mutations are unusual in that the affected individuals are very long-term survivors with PDH complex deficiency despite having low (<20%) activity in skin fibroblasts. These findings prompted us to investigate the C-terminus of E₁α in greater detail. We constructed and expressed a series of PDH-E₁α deletion mutants in a cell line with zero PDH complex activity due to a null E₁α allele. Sequential deletion of the C-terminus by one, two, three and four amino acids resulted in PDH complex activities of 100, 60, 36 and 14%, respectively, compared with wild-type E₁α expressed in PDH complex-deficient cells. The immunodetectable protein was decreased by the same amount as the activity, suggesting that the stability and/or assembly of the E₁α₂β₂ heterotetramer might depend on the intactness of the PDH-E₁α C-terminus. In addition, we compared the somatic and the testis-specific isoforms of E₁αand concluded that they are biochemically equivalent.