Proc. NatI. Acad. Sci. USA Vol. 81, pp. 4475-4479, July 1984 Genetics Differential regulation of the duplicated isocytochrome c genes in yeast (yeast regulation/heme deficiency/glucose repression/cytochrome c regulation) THOMAS M. LAZ, DENNIS F. PIETRAS, AND FRED SHERMAN Departments of Biochemistry and of Radiation Biology and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642 Communicated by Herschel L. Roman, March 26, 1984 ABSTRACT The two unlinked genes CYCI and CYC7 en- acid (ALA) synthetase (10, 12); since ALA is an intermedi- code iso-l-cytochrome c and iso-2-cytochrome c, respectively, ate in the biosynthesis of heme, hemi mutants are deficient in the yeast Saccharomyces cerevisiae. An examination of the in heme and all heme-containing proteins, including the iso- steady-state level of CYCI and CYC7 mRNAs in normal and cytochromes c. In contrast, the CYC2 and CYC3 genes ap- mutant strains grown under different conditions, along with pear to affect only the isocytochromes c. These mutants may previous results of apoprotein levels, demonstrate that CYCI be involved in some aspect of the enzymatic attachment of a and CYC7 have similar and different modes of regulation. heme to the apoisocytochromes c or the transport of the apo- Both CYCI and CYC7 mRNAs are diminished after anaerobic cytochromes c into the mitochondria (9). Whereas hemi and growth. In contrast, CYCI mRNA but not CYC7 mRNA is de- cyc3 mutations can completely block the isocytochrome c creased by heme deficiency in hem) mutants. Although both production, the cyc2 mutation can block at most only 90% of CYCI and CYC7 mRNAs are substantially lowered after the isocytochromes c. Immunological studies of apocyto- growth in glucose medium, there is a difference in the kinetics chromes c demonstrate that hemi, cyc2, and cyc3 mutants of glucose derepression. CYCI mRNA levels rise in the early lack apoiso-1-cytochrome c but contain substantial amounts logarithmic phase of growth before complete exhaustion of of apoiso-2-cytochrome c (9). The absence of apoiso-1-cyto- glucose, whereas CYC7 mRNA levels rise in the late logarith- chrome c and the presence of apoiso-2-cytochrome c in these mic phase when the level of CYCI mRNA has plateaued. For a mutants may be explained by differences in the rates of syn- brief period before cessation of growth, the level of CYC7 thesis or degradation of either apoisocytochromes c or their mRNA attains a level corresponding to the high derepressed respective mRNAs. level of CYCJ mRNA. The high amount of CYC7 mRNA is As part of a systematic investigation of the regulation of surprising because iso-2-cytochrome c constitutes only 5% of the isocytochromes c and to explain the differences in the the total cytochrome c complement in derepressed cells. We accumulation of the apoisocytochromes c, we have exam- suggest that iso-2-cytochrome c has the potential to comprise a ined the steady-state mRNA levels of both isocytochromes c major proportion of cytochrome c under certain physiologic under conditions that have been shown to decrease cyto- conditions that have not been experimentally defined. The chrome c levels. These conditions include the cyc3 and hem] cyc3 mutant, which lacks the ability to attach heme groups to mutations, anaerobic growth, and glucose repression. Al- apocytochromes c, contains both CYCI and CYC7 mRNAs in though some aspects of transcriptional regulation of both normal amounts. Yet, cyc3 mutants contain only apoiso-2-cy- genes have been investigated previously (13-20), we have tochrome c and not apoiso-1-cytochrome c. The lack of accu- chosen to systematically compare the regulation ofthe CYCJ mulation of apoiso-1-cytochrome c in cyc3 mutants, which con- and CYC7 genes. tain CYCI mRNA, suggests that apoiso-1-cytochrome c is ex- Our studies demonstrate that both CYCI and CYC7 tensively regulated by a post-transcriptional process. steady-state mRNA levels were diminished in cells grown anaerobically or under glucose repression, whereas only The total complement of cytochrome c in derepressed cells CYCI mRNA is diminished in hemi strains. However, the of the yeast Saccharomyces cerevisiae is typically composed cyc3 mutation has no effect on either CYCI or CYC7 mRNA of 95% iso-1-cytochrome c and 5% iso-2-cytochrome c (1). levels. Thus, the basis of the differential regulation of the Both iso-1-cytochrome c and iso-2-cytochrome c carry out two apoisocytochromes c in cyc3 strains must be at a post- identical or similar functions in mitochondrial electron trans- transcriptional step. Lastly, glucose derepressions of CYCI port (2). Iso-1-cytochrome c and iso-2-cytochrome c are en- and CYC7 mRNAs are different; the level of CYCI mRNA coded by the unlinked nuclear genes CYCJ and CYC7 (3-6), plateaus by the midlogarithmic growth phase, whereas CYC7 respectively, that are -=80% homologous in their DNA se- mRNA accumulation is initiated and peaks in the late loga- quence (7) and that appear to be evolutionarily related (8). rithmic phase. The biosynthesis of the two isocytochromes c, similar to the biosynthesis of other heme proteins, is decreased by growth MATERIALS AND METHODS in glucose media (catabolite repression) and by growth with Yeast Strains. The following mutant strains were obtained anaerobic conditions (reviewed in ref. 1). However, the for- either from our stock collections or constructed by standard mation of the isocytochromes c from glucose-repressed and genetic methods: DP44-5C (MATa CYCIP CYC7+ hem] lysi anaerobically grown cells shows different kinetics (1). In ad- metl4); DP39-2D (MATa cycl-J CYC7+ hemi lys2 trp2 dition, recent work (9) has shown that the expression of the his-); M15-4A (MATa CYCI+ CYC7+ cyc3-10 lys2); and CYCI and CYC7 genes is affected differently by mutations at M15-4D (MA Ta cycl-J CYC7+ lys2 his] trp2). Normal the following three other loci: hem] (ref. 10), cyc2 (ref. 11), strains included D311-3A (MATa CYCI+ CYC7+ lys2 his] and cyc3 (ref. 11). The hemi locus encodes 8-aminolevulinic trp2), M15-4B (MATa CYCI+ CYC7+ lys2), and D273-10B (MATa CYCJ+ CYC7+). The cycl-J mutation is a deletion the locus and The publication costs of this article were defrayed in part by page charge encompassing entire CYCI adjacent regions payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: ALA, 8-aminolevulinic acid. 4475 Downloaded by guest on October 1, 2021 4476 Genetics: Laz et aL Proc. NatL Acad. Sci. USA 81 (1984) Cells for the glucose derepression study were obtained by overnight growth in 1 liter of YPD medium at 30'C and were harvested at appropriate intervals during the growth cycle. RNA Analysis. Total RNA was isolated, electrophoresed on 1.5% agarose denaturing gels, and transferred to nitrocel- Nick translation lulose paper essentially as described (22). Single-stranded Heat denaturation M13 mp9 phage DNAs with CYC) and CYC7 inserts were used for hybridization probes as schematically illustrated in Fig. 1. The CYCJ probe, denoted mCYC1, contains a 345- * Immobilized specific mRNA base-pair EcoRI to Taq I insert in the M13 mp9, whereas the fO" +( probe + (CYC1 mRNA) CYC7 probe, mCYC7, contains a 349-base-pair Sau3A to (mCYCl) t \- 5'-AM 3' Hpa II insert in the M13 mp9. With the A of the initiating N ATG codon corresponding to nucleotide position 1, the II mCYC1 insert encompasses nucleotide positions 7 through Hybridization 352 and the mCYC7 insert encompasses nucleotide positions 72 through 277. In all cases, RNA preparations were checked quantitatively by ethidium bromide fluorescence af- ter agarose gel electrophoresis and absorbance at 260 nm. Prehybridization and hybridization were carried out as de- scribed (22), with the following modifications. Typically, the probes for the RNA analysis consisted of 0.2-0.3 ,g of unla- 5' AAMAA 3' beled phage mCYC1 or mCYC7 DNA and -0.3 ,tg of dena- tured M13 mp9 replicative form that had been nick-translat- FIG. 1. Schematic representation of the hybridization of a single- stranded M13 with an insert, a nick-translated replicative form (RF) ed to a specific activity of 4 x 107 cpm/,ug with [32P]idCTP. of M13 without an insert, and a mRNA transcribed from a region After overnight hybridization, the filters were washed with encompassing the insert. gentle shaking; four washes were carried out for 5 min each at 55°C in the first wash solution (0.3 M sodium chloride/0.03 (21). The hem) and cyc3-10 mutations used in this study M sodium citrate, pH 7/0.1% NaDodSO4) and six washes were obtained from the mutants GT38-7A (10) and B-614 (8, were carried out for 1 hr each at 55°C in the second wash 9), respectively. solution (15 mM sodium chloride/1.5 mM sodium citrate, pH Culture Media. YPD medium contains 1% yeast extract 7/0.1% NaDodSO4). Under these conditions, there was no (Difco), 2% peptone (Difco), and 2% glucose and is used for significant cross-hybridization between the mCYC1 probe growth of normal strains. YPD + ET medium is YPD medi- and CYC7 mRNA and between the mCYC7 probe and CYC1 um containing ergosterol and Tween 80 (Sigma) as described mRNA. Because the mCYC1 and mCYC7 probes contain (9). YPD + ALA medium is YPD mediurt supplemented inserts of almost identical length, the relative amounts of with ALA (Sigma) as described (9). YP3%D medium is a CYC1 and CYC7 mRNAs can be compared when the same high-glucose medium containing 1% yeast extract, 2% pep- preparation of labeled M13 mp9 is used for hybridization. tone, and 3% glucose.