Sequential deletion of C-terminal amino acids of the E(1)alpha component of the pyruvate dehydrogenase (PDH) complex leads to reduced steady-state levels of functional E(1)alpha(2)beta(2) tetramers: implications for patients with PDH deficiency.
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-E(1)alpha-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 E(1)alpha in greater detail. We constructed and expressed a series of PDH-E(1)alpha deletion mutants in a cell line with zero PDH complex activity due to a null E(1)alpha 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 E(1)alpha 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 E(1)alpha(2)beta(2)heterotetramer might depend on the intactness of the PDH-E(1)alpha C-terminus. In addition, we compared the somatic and the testis-specific isoforms of E(1)alphaand concluded that they are biochemically equivalent.
Seyda
A
A
Department of Biochemistry, University of Toronto, and Metabolism Research Programme, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada.
McEachern
G
G
Haas
R
R
Robinson
B H
BH
D000328:Adult; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D017353:Gene Deletion; D006801:Humans; D008297:Male; D008929:Mitochondria, Heart / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids; D030481:Pyruvate Dehydrogenase (Lipoamide); D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics* / Q000378:metabolism*; D015325:Pyruvate Dehydrogenase Complex Deficiency Disease / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013737:Testis / Q000378:metabolism
D018076:DNA, Complementary; D011768:Pyruvate Dehydrogenase Complex; D012333:RNA, Messenger; D030481:Pyruvate Dehydrogenase (Lipoamide); C111305:pyruvate dehydrogenase E1 beta subunit; C065640:pyruvate dehydrogenase E1alpha subunit