Abstract
The yeast L(+)-lactate cytochrome c oxidoreductase or cytochrome b2 is a component of the mitochondrial intermembrane space. The protein is encoded by the nuclear genome, synthesized as a larger precursor in the cytoplasmic compartment, and then proteolytically processed to its mature form during its import into the mitochondria. The structural gene for yeast cytochrome b2 has been cloned. The complete nucleotide sequence of the gene with its 5' and 3' flanking regions was determined. The deduced primary structure of the cytochrome b2 precursor reveals an unusually long amino terminal extension of 80 amino acids. A variety of potentially significant sequences were identified in the region flanking the structural portion of the gene. Transcript mapping with both S1 nuclease and primer extension methods reveals that the site of RNA synthesis is 56-66 bp downstream from a putative TATA box. By Northern blot analysis and gene disruption, it is shown that there is only a single copy of the cytochrome b2 gene per haploid yeast nucleus. The cloned cytochrome b2 gene was used to probe specific mRNA levels and demonstrate that cytochrome b2 expression is transcriptionally repressed by glucose and induced by lactate. The inactivation of the chromosomal cytochrome b2 gene by integrative transformation led to a deficiency in L(+)-lactate dehydrogenase activity and consequently to the inability to use L(+)-lactate as a sole source of carbon. This is the first reported mutation affecting the structural gene of cytochrome b2.
Full text
PDF![3265](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f2d1/554653/f35cd8dc84ba/emboj00277-0219.png)
![3266](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f2d1/554653/acad9b24ad27/emboj00277-0220.png)
![3267](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f2d1/554653/75265b0404eb/emboj00277-0221.png)
![3268](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f2d1/554653/88c9e8d79eb5/emboj00277-0222.png)
![3269](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f2d1/554653/7d5253c6477f/emboj00277-0223.png)
![3270](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f2d1/554653/33fff568c6a9/emboj00277-0224.png)
![3271](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f2d1/554653/d6f1eaf86f1b/emboj00277-0225.png)
![3272](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f2d1/554653/c10e1bc1a6ec/emboj00277-0226.png)
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bailey J. M., Davidson N. Methylmercury as a reversible denaturing agent for agarose gel electrophoresis. Anal Biochem. 1976 Jan;70(1):75–85. doi: 10.1016/s0003-2697(76)80049-8. [DOI] [PubMed] [Google Scholar]
- Bajwa W., Meyhack B., Rudolph H., Schweingruber A. M., Hinnen A. Structural analysis of the two tandemly repeated acid phosphatase genes in yeast. Nucleic Acids Res. 1984 Oct 25;12(20):7721–7739. doi: 10.1093/nar/12.20.7721. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
- Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
- Breathnach R., Chambon P. Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem. 1981;50:349–383. doi: 10.1146/annurev.bi.50.070181.002025. [DOI] [PubMed] [Google Scholar]
- Broach J. R., Strathern J. N., Hicks J. B. Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene. 1979 Dec;8(1):121–133. doi: 10.1016/0378-1119(79)90012-x. [DOI] [PubMed] [Google Scholar]
- Clavilier L., Péré-Aubert G., Somlo M., Slonimski P. P. Réseau d'interactions entre des génes non liés : régulation synergique ou antagoniste de la synthèse de l'iso-1-cytochrome c, de l'iso-2-cytochrome c et du cytochrome b2. Biochimie. 1976;58(1-2):155–172. doi: 10.1016/s0300-9084(76)80366-5. [DOI] [PubMed] [Google Scholar]
- Daum G., Gasser S. M., Schatz G. Import of proteins into mitochondria. Energy-dependent, two-step processing of the intermembrane space enzyme cytochrome b2 by isolated yeast mitochondria. J Biol Chem. 1982 Nov 10;257(21):13075–13080. [PubMed] [Google Scholar]
- Davis R. W., Thomas M., Cameron J., St John T. P., Scherer S., Padgett R. A. Rapid DNA isolations for enzymatic and hybridization analysis. Methods Enzymol. 1980;65(1):404–411. doi: 10.1016/s0076-6879(80)65051-4. [DOI] [PubMed] [Google Scholar]
- Douglas M. G., Geller B. L., Emr S. D. Intracellular targeting and import of an F1-ATPase beta-subunit-beta-galactosidase hybrid protein into yeast mitochondria. Proc Natl Acad Sci U S A. 1984 Jul;81(13):3983–3987. doi: 10.1073/pnas.81.13.3983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Federoff H. J., Eccleshall T. R., Marmur J. Carbon catabolite repression of maltase synthesis in Saccharomyces carlsbergensis. J Bacteriol. 1983 Oct;156(1):301–307. doi: 10.1128/jb.156.1.301-307.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Frischauf A. M., Lehrach H., Poustka A., Murray N. Lambda replacement vectors carrying polylinker sequences. J Mol Biol. 1983 Nov 15;170(4):827–842. doi: 10.1016/s0022-2836(83)80190-9. [DOI] [PubMed] [Google Scholar]
- GALZY P., SLONIMSKI P. P. Evolution de la constitution enzymatique de la levure cultivée sur acide lactique ou sur glucose comme seule source de carbone. C R Hebd Seances Acad Sci. 1957 Dec 23;245(26):2556–2558. [PubMed] [Google Scholar]
- Gasser S. M., Ohashi A., Daum G., Böhni P. C., Gibson J., Reid G. A., Yonetani T., Schatz G. Imported mitochondrial proteins cytochrome b2 and cytochrome c1 are processed in two steps. Proc Natl Acad Sci U S A. 1982 Jan;79(2):267–271. doi: 10.1073/pnas.79.2.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ghrir R., Becam A. M., Lederer F. Primary structure of flavocytochrome b2 from baker's yeast. Purification by reverse-phase high-pressure liquid chromatography and sequencing of fragment alpha cyanogen bromide peptides. Eur J Biochem. 1984 Feb 15;139(1):59–74. doi: 10.1111/j.1432-1033.1984.tb07976.x. [DOI] [PubMed] [Google Scholar]
- Guarente L., Hoar E. Upstream activation sites of the CYC1 gene of Saccharomyces cerevisiae are active when inverted but not when placed downstream of the "TATA box". Proc Natl Acad Sci U S A. 1984 Dec;81(24):7860–7864. doi: 10.1073/pnas.81.24.7860. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Guarente L., Lalonde B., Gifford P., Alani E. Distinctly regulated tandem upstream activation sites mediate catabolite repression of the CYC1 gene of S. cerevisiae. Cell. 1984 Feb;36(2):503–511. doi: 10.1016/0092-8674(84)90243-5. [DOI] [PubMed] [Google Scholar]
- Guiard B., Buhler J. M. Yeast cytochrome b2 gene: isolation with antibody probes. Biochimie. 1984 Feb;66(2):151–158. doi: 10.1016/0300-9084(84)90204-9. [DOI] [PubMed] [Google Scholar]
- Guiard B., Groudinsky O., Lederer F. Homology between bakers' yeast cytochrome b2 and liver microsomal cytochrome b5. Proc Natl Acad Sci U S A. 1974 Jun;71(6):2539–2543. doi: 10.1073/pnas.71.6.2539. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Guiard B., Lederer F., Jacq C. More similarity between bakers'yeast L-(+)-lactate dehydrogenase and liver microsomal cytochrome b5. Nature. 1975 May 29;255(5507):422–423. doi: 10.1038/255422a0. [DOI] [PubMed] [Google Scholar]
- Hurt E. C., Pesold-Hurt B., Schatz G. The amino-terminal region of an imported mitochondrial precursor polypeptide can direct cytoplasmic dihydrofolate reductase into the mitochondrial matrix. EMBO J. 1984 Dec 20;3(13):3149–3156. doi: 10.1002/j.1460-2075.1984.tb02272.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jacq C., Lederer F. Cytochrome b2 from bakers' yeast (L-lactate dehydrogenase). A double-headed enzyme. Eur J Biochem. 1974 Jan 16;41(2):311–320. doi: 10.1111/j.1432-1033.1974.tb03271.x. [DOI] [PubMed] [Google Scholar]
- Kaput J., Goltz S., Blobel G. Nucleotide sequence of the yeast nuclear gene for cytochrome c peroxidase precursor. Functional implications of the pre sequence for protein transport into mitochondria. J Biol Chem. 1982 Dec 25;257(24):15054–15058. [PubMed] [Google Scholar]
- Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
- LABEYRIE F., SLONIMSKI P. P. MODE D'ACTION DES LACTICODESHYDROG'ENASES LI'EES AUX SYST'EMES FLAVINIQUE ET CYTOCHROMIQUE. Bull Soc Chim Biol (Paris) 1964;46:1793–1828. [PubMed] [Google Scholar]
- 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]
- Lustig A., Levens D., Rabinowitz M. The biogenesis and regulation of yeast mitochondria RNA polymerase. J Biol Chem. 1982 May 25;257(10):5800–5808. [PubMed] [Google Scholar]
- Maccecchini M. L., Rudin Y., Blobel G., Schatz G. Import of proteins into mitochondria: precursor forms of the extramitochondrially made F1-ATPase subunits in yeast. Proc Natl Acad Sci U S A. 1979 Jan;76(1):343–347. doi: 10.1073/pnas.76.1.343. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
- Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
- Ohashi A., Gibson J., Gregor I., Schatz G. Import of proteins into mitochondria. The precursor of cytochrome c1 is processed in two steps, one of them heme-dependent. J Biol Chem. 1982 Nov 10;257(21):13042–13047. [PubMed] [Google Scholar]
- Osinga K. A., De Vries E., Van der Horst G. T., Tabak H. F. Initiation of transcription in yeast mitochondria: analysis of origins of replication and of genes coding for a messenger RNA and a transfer RNA. Nucleic Acids Res. 1984 Feb 24;12(4):1889–1900. doi: 10.1093/nar/12.4.1889. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Reid G. A., Yonetani T., Schatz G. Import of proteins into mitochondria. Import and maturation of the mitochondrial intermembrane space enzymes cytochrome b2 and cytochrome c peroxidase in intact yeast cells. J Biol Chem. 1982 Nov 10;257(21):13068–13074. [PubMed] [Google Scholar]
- Riezman H., Hay R., Witte C., Nelson N., Schatz G. Yeast mitochondrial outer membrane specifically binds cytoplasmically-synthesized precursors of mitochondrial proteins. EMBO J. 1983;2(7):1113–1118. doi: 10.1002/j.1460-2075.1983.tb01554.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
- Risser R. Abelson antigen is expressed on hematopoietic spleen colony-forming cells from mice carrying the Av-2S virus sensitivity gene. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5350–5354. doi: 10.1073/pnas.76.10.5350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
- SOMLO M. INDUCTION DES LACTICO-CYTOCHROME C REDUCTASES (D- ET L-) DE LA LEVURE AEROBIE PAR LES LACTATES (D- ET L-) Biochim Biophys Acta. 1965 Feb 15;97:183–201. [PubMed] [Google Scholar]
- Sadler I., Suda K., Schatz G., Kaudewitz F., Haid A. Sequencing of the nuclear gene for the yeast cytochrome c1 precursor reveals an unusually complex amino-terminal presequence. EMBO J. 1984 Sep;3(9):2137–2143. doi: 10.1002/j.1460-2075.1984.tb02103.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smith M. M., Andrésson O. S. DNA sequences of yeast H3 and H4 histone genes from two non-allelic gene sets encode identical H3 and H4 proteins. J Mol Biol. 1983 Sep 25;169(3):663–690. doi: 10.1016/s0022-2836(83)80164-8. [DOI] [PubMed] [Google Scholar]
- Soberon X., Covarrubias L., Bolivar F. Construction and characterization of new cloning vehicles. IV. Deletion derivatives of pBR322 and pBR325. Gene. 1980 May;9(3-4):287–305. doi: 10.1016/0378-1119(90)90328-o. [DOI] [PubMed] [Google Scholar]
- Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
- St John T. P., Davis R. W. The organization and transcription of the galactose gene cluster of Saccharomyces. J Mol Biol. 1981 Oct 25;152(2):285–315. doi: 10.1016/0022-2836(81)90244-8. [DOI] [PubMed] [Google Scholar]
- Szekely E., Montgomery D. L. Glucose represses transcription of Saccharomyces cerevisiae nuclear genes that encode mitochondrial components. Mol Cell Biol. 1984 May;4(5):939–946. doi: 10.1128/mcb.4.5.939. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weaver R. F., Weissmann C. Mapping of RNA by a modification of the Berk-Sharp procedure: the 5' termini of 15 S beta-globin mRNA precursor and mature 10 s beta-globin mRNA have identical map coordinates. Nucleic Acids Res. 1979 Nov 10;7(5):1175–1193. doi: 10.1093/nar/7.5.1175. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yamamoto K. R., Alberts B. M., Benzinger R., Lawhorne L., Treiber G. Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology. 1970 Mar;40(3):734–744. doi: 10.1016/0042-6822(70)90218-7. [DOI] [PubMed] [Google Scholar]
- Zitomer R. S., Montgomery D. L., Nichols D. L., Hall B. D. Transcriptional regulation of the yeast cytochrome c gene. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3627–3631. doi: 10.1073/pnas.76.8.3627. [DOI] [PMC free article] [PubMed] [Google Scholar]