Expression and functional characterization of human protein X variants in SV40-immortalized protein X-deficient and E2-deficient human skin fibroblasts.
To gain further insight into the nature and function of the domains of the human protein X (a pyruvate dehydrogenase complex component also known as the E3-binding protein), we expressed the wild-type as well as two artificially created variants, K37E and S422H, in SV40-immortalized protein X-deficient and E2-deficient human skin fibroblasts. The former mutant does not carry the lipoic acid moiety, the latter mutant was designed to investigate the possibility that protein X could exhibit an intrinsic acetyltransferase activity and use either its own catalytic center or the catalytic center of E2. Similar experiments have been performed in the past using the Saccharomyces cerevisiae expression system. However, lack of sequence similarity between the mammalian and the yeast protein X homologues suggests they are not biochemically equivalent. Mutant cells transfected with the wild-type gene for protein X produced a PDH complex that exhibited about 50% overall activity of the control cells. None of the expressed protein X variants had an effect on the specific activity of the PDH complex, suggesting that the human protein X plays a purely structural role in the functioning of the pyruvate dehydrogenase complex.
Seyda
A
A
Department of Biochemistry, University of Toronto, Ontario.
Robinson
B H
BH
D000595:Amino Acid Sequence; D001483:Base Sequence; D002461:Cell Line, Transformed; D017931:DNA Primers / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D014644:Genetic Variation; D006801:Humans; D010455:Peptides / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics*; D021122:Protein Subunits; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry* / Q000235:genetics*; D015325:Pyruvate Dehydrogenase Complex Deficiency Disease; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013539:Simian virus 40; D012867:Skin / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D005656:Fungal Proteins; C045867:PDHX protein, human; C489060:PDX1 protein, S cerevisiae; D010455:Peptides; D021122:Protein Subunits; D011768:Pyruvate Dehydrogenase Complex; D029701:Saccharomyces cerevisiae Proteins