Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1993 Feb 15;90(4):1252–1256. doi: 10.1073/pnas.90.4.1252

Characterization of PDH beta 1, the structural gene for the pyruvate dehydrogenase beta subunit from Saccharomyces cerevisiae.

S G Miran 1, J E Lawson 1, L J Reed 1
PMCID: PMC45850  PMID: 8433986

Abstract

The gene encoding the pyruvate dehydrogenase (PDH) beta subunit (E1 beta) of the PDH complex from Saccharomyces cerevisiae has been cloned, sequenced, disrupted, and expressed. Two overlapping DNA fragments were generated from a yeast genomic DNA library by the polymerase chain reaction with synthetic oligonucleotide primers based on amino acid sequences of the yeast and human E1 beta subunits. The DNA fragments were subcloned and sequenced. The composite sequence has an open reading frame of 1098 nucleotides encoding a putative presequence of 33 amino acid residues and a mature protein of 333 residues with a calculated M(r) = 36,486. Yeast and human E1 beta exhibit 62% sequence identity. The size of the mRNA is approximately 1.5 kilobases. Hybridization analysis showed that the E1 beta gene (PDH beta 1) is localized to chromosome II. Disruption of PDH beta 1 is not lethal under vegetative growth conditions. The null mutant transformed with PDH beta 1 on a unit-copy plasmid produced mature E1 beta and a functional PDH complex.

Full text

PDF
1252

Images in this article

1254Image
on p.1254
1255Image
on p.1255
1255Image
on p.1255

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barrera C. R., Namihira G., Hamilton L., Munk P., Eley M. H., Linn T. C., Reed L. J. -Keto acid dehydrogenase complexes. XVI. Studies on the subunit structure of the pyruvate dehydrogenase complexes from bovine kidney and heart. Arch Biochem Biophys. 1972 Feb;148(2):343–358. doi: 10.1016/0003-9861(72)90152-x. [DOI] [PubMed] [Google Scholar]
  2. Behal R. H., Browning K. S., Reed L. J. Nucleotide and deduced amino acid sequence of the alpha subunit of yeast pyruvate dehydrogenase. Biochem Biophys Res Commun. 1989 Oct 31;164(2):941–946. doi: 10.1016/0006-291x(89)91549-0. [DOI] [PubMed] [Google Scholar]
  3. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  4. Daum G., Böhni P. C., Schatz G. Import of proteins into mitochondria. Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. J Biol Chem. 1982 Nov 10;257(21):13028–13033. [PubMed] [Google Scholar]
  5. Emr S. D., Vassarotti A., Garrett J., Geller B. L., Takeda M., Douglas M. G. The amino terminus of the yeast F1-ATPase beta-subunit precursor functions as a mitochondrial import signal. J Cell Biol. 1986 Feb;102(2):523–533. doi: 10.1083/jcb.102.2.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gopalakrishnan S., Rahmatullah M., Radke G. A., Powers-Greenwood S., Roche T. E. Role of protein X in the function of the mammalian pyruvate dehydrogenase complex. Biochem Biophys Res Commun. 1989 Apr 28;160(2):715–721. doi: 10.1016/0006-291x(89)92492-3. [DOI] [PubMed] [Google Scholar]
  7. Hawkins C. F., Borges A., Perham R. N. Cloning and sequence analysis of the genes encoding the alpha and beta subunits of the E1 component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. Eur J Biochem. 1990 Jul 31;191(2):337–346. doi: 10.1111/j.1432-1033.1990.tb19128.x. [DOI] [PubMed] [Google Scholar]
  8. Ho L., Patel M. S. Cloning and cDNA sequence of the beta-subunit component of human pyruvate dehydrogenase complex. Gene. 1990 Feb 14;86(2):297–302. doi: 10.1016/0378-1119(90)90294-2. [DOI] [PubMed] [Google Scholar]
  9. Isola N. R., Harn H. J., Cooper D. L. Screening recombinant DNA libraries: a rapid and efficient method for isolating cDNA clones utilizing the PCR. Biotechniques. 1991 Nov;11(5):580–582. [PubMed] [Google Scholar]
  10. Kroczek R. A., Siebert E. Optimization of northern analysis by vacuum-blotting, RNA-transfer visualization, and ultraviolet fixation. Anal Biochem. 1990 Jan;184(1):90–95. doi: 10.1016/0003-2697(90)90017-4. [DOI] [PubMed] [Google Scholar]
  11. Köhrer K., Domdey H. Preparation of high molecular weight RNA. Methods Enzymol. 1991;194:398–405. doi: 10.1016/0076-6879(91)94030-g. [DOI] [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Lawson J. E., Behal R. H., Reed L. J. Disruption and mutagenesis of the Saccharomyces cerevisiae PDX1 gene encoding the protein X component of the pyruvate dehydrogenase complex. Biochemistry. 1991 Mar 19;30(11):2834–2839. doi: 10.1021/bi00225a015. [DOI] [PubMed] [Google Scholar]
  14. Lawson J. E., Gawaz M., Klingenberg M., Douglas M. G. Structure-function studies of adenine nucleotide transport in mitochondria. I. Construction and genetic analysis of yeast mutants encoding the ADP/ATP carrier protein of mitochondria. J Biol Chem. 1990 Aug 25;265(24):14195–14201. [PubMed] [Google Scholar]
  15. Lawson J. E., Niu X. D., Reed L. J. Functional analysis of the domains of dihydrolipoamide acetyltransferase from Saccharomyces cerevisiae. Biochemistry. 1991 Nov 26;30(47):11249–11254. doi: 10.1021/bi00111a009. [DOI] [PubMed] [Google Scholar]
  16. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  17. Neagle J., De Marcucci O., Dunbar B., Lindsay J. G. Component X of mammalian pyruvate dehydrogenase complex: structural and functional relationship to the lipoate acetyltransferase (E2) component. FEBS Lett. 1989 Aug 14;253(1-2):11–15. doi: 10.1016/0014-5793(89)80919-6. [DOI] [PubMed] [Google Scholar]
  18. Niu X. D., Browning K. S., Behal R. H., Reed L. J. Cloning and nucleotide sequence of the gene for dihydrolipoamide acetyltransferase from Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7546–7550. doi: 10.1073/pnas.85.20.7546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Perham R. N. Domains, motifs, and linkers in 2-oxo acid dehydrogenase multienzyme complexes: a paradigm in the design of a multifunctional protein. Biochemistry. 1991 Sep 3;30(35):8501–8512. doi: 10.1021/bi00099a001. [DOI] [PubMed] [Google Scholar]
  20. Rahmatullah M., Gopalakrishnan S., Andrews P. C., Chang C. L., Radke G. A., Roche T. E. Subunit associations in the mammalian pyruvate dehydrogenase complex. Structure and role of protein X and the pyruvate dehydrogenase component binding domain of the dihydrolipoyl transacetylase component. J Biol Chem. 1989 Feb 5;264(4):2221–2227. [PubMed] [Google Scholar]
  21. Steensma H. Y., Holterman L., Dekker I., van Sluis C. A., Wenzel T. J. Molecular cloning of the gene for the E1 alpha subunit of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae. Eur J Biochem. 1990 Aug 17;191(3):769–774. doi: 10.1111/j.1432-1033.1990.tb19186.x. [DOI] [PubMed] [Google Scholar]
  22. Wexler I. D., Hemalatha S. G., Patel M. S. Sequence conservation in the alpha and beta subunits of pyruvate dehydrogenase and its similarity to branched-chain alpha-keto acid dehydrogenase. FEBS Lett. 1991 Apr 22;282(1):209–213. doi: 10.1016/0014-5793(91)80479-m. [DOI] [PubMed] [Google Scholar]
  23. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES