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Isolation of a full-length complementary DNA coding for human E1 alpha subunit of the pyruvate dehydrogenase complex
L De Meirleir1, N MacKay, A M Lam Hon Wah
1Department of Pediatrics and Biochemistry, University of Toronto, Ontario, Canada.
The Journal of Biological Chemistry
|February 5, 1988
Summary
Researchers isolated a human pyruvate dehydrogenase E1 cDNA clone, revealing a corrected protein sequence with mitochondrial targeting signals and phosphorylation sites similar to bovine E1 alpha. This finding refines understanding of human pyruvate metabolism.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Pyruvate dehydrogenase E1 (PDHE1) is a key mitochondrial enzyme in cellular respiration.
- Previous characterization of human PDHE1 alpha subunit cDNA had potential sequence discrepancies.
Purpose of the Study:
- To isolate and characterize a cDNA clone for the human pyruvate dehydrogenase E1 alpha subunit.
- To determine the complete nucleotide sequence and predict the amino acid sequence of human PDHE1.
- To identify potential regulatory sites and compare the human sequence to homologous proteins.
Main Methods:
- Screening of a lambda gt11 expression library using specific antibodies and oligonucleotide probes.
- Nucleotide sequence analysis of the isolated cDNA clone.
- Northern blot analysis of human fibroblast mRNA.
Main Results:
- A 1.5-kilobase cDNA clone for human PDHE1 alpha subunit was successfully isolated.
- Sequence analysis revealed a 72-nucleotide 5'-untranslated region, a 1170-nucleotide translated region, and a 223-nucleotide 3'-untranslated region with a poly(A) tail.
- The predicted mature protein consists of 362 amino acids, including a 29-amino acid leader sequence for mitochondrial import and three serine phosphorylation sites, identical to the bovine E1 alpha subunit.
- A frameslip mutation was identified, leading to an amended protein sequence compared to previous reports, confirmed by restriction enzyme analysis.
- Northern blot analysis detected a 1.8-kilobase mRNA and a minor 4.4-kilobase band in human fibroblasts.
Conclusions:
- The isolated cDNA clone provides an accurate sequence for the human pyruvate dehydrogenase E1 alpha subunit.
- The predicted protein structure includes a mitochondrial targeting signal and conserved phosphorylation sites, crucial for enzyme regulation.
- The amended sequence corrects previous discrepancies and enhances the understanding of human pyruvate metabolism and its regulation.