Proc. Nati. Acad. Sci. USA Vol. 82, pp. 2235-2239, April 1985 Biochemistry Two nonidentical forms of subunit V are functional in yeast cytochrome c oxidase (Saccharomyces cerevisiae/nuclear genes/mitochondria/DNA sequence/gene disruption) MICHAEL G. CUMSKY, CHRISTINE Ko, CYNTHIA E. TRUEBLOOD, AND ROBERT 0. POYTON Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Campus Box 347, Boulder, CO 80309 Communicated by David M. Prescott, November 19, 1984 ABSTRACT In Saccharomyces cerevisiae, the inner mito- ing the gene on a multicopy plasmid, (ii) the ability of the chondrial membrane protein cytochrome c oxidase is com- plasmid to select subunit V-specific mRNA, and (iii) the ap- posed of nine polypeptide subunits. Six of these subunits (IV, pearance of a new cross-reacting subunit V polypeptide in V, VI, VII, VIIa, VIII) are encoded by the nuclear genome, transformant mitochondria (5, 6). and the remaining three (I, II, III) are encoded by mitochon- In this paper, we report that the latter gene, now called drial DNA. We report here the existence of two nonidentical COXSb, encodes a subunit V polypeptide that is similar to, subunit V polypeptides, which are encoded by separate genes but not identical with, the subunit V polypeptide recently within the yeast genome. One gene, COX5a, encodes the poly- sequenced in this laboratory (7). We also report the molecu- peptide Va, normally found in preparations of holocytochrome lar cloning of another gene for subunit V, COX5a. This gene c oxidase. The other gene, COX5b, encodes the polypeptide encodes a polypeptide identical in sequence to the subunit V Vb, which cross-reacts with anti-subunit Va antiserum and re- polypeptide found in the holoenzyme. We show that both stores respiratory competency and cytochrome oxidase activity genes are expressed in yeast and their products can function in transformants of coxSa structural gene mutants. This poly- as bona fide subunits in the holoenzyme. peptide also copurifies with the holoenzyme prepared from these transformants. We have found that COX5b is expressed in vegetatively growing yeast cells, and that the Vb polypeptide MATERIALS AND METHODS can be detected in mitochondria from strain JM28, a cox5a Plasmids, Strains, and Growth Media. The following Sac- mutant. This mutant has 15%-20% residual cytochrome oxi- charomyces cerevisiae strains were used: D273-10B (mata, dase activity, and it respires at 10%-15% the wild-type rate. ATCC 24657); JM28 (mata, leu2-3 leu2-112 his4-580 ura3-52 By disrupting the COX5b gene in this strain, we show that this trpl-289 ade2 coxfa-J) was constructed as described (5). residual activity is directly attributable to the presence of a JM43 (mata leu2-3 leu2-112 his4-580 ura3-52 trpl-289) was chromosomal copy of the COX5b gene. Taken together, these constructed by crossing D273-1OB with AB35-13D (5), then results suggest that Va or Vb can function as cytochrome oxi- backcrossing to D273-10B five times. JM28-511, and JM28- dase subunits in yeast and that Vb may be used under some 552 are transformants of JM28, as described in Results. specific, as yet undefined, physiological conditions. JM28-GD5b2 and JM28-GD5b3 are strains carrying disrup- tions of COXSb also as described in Results. Parental and The biogenesis of a respiratory competent mitochondrion re- transformed yeast strains were grown at 28°C on either YPD, sults from the joint expression of two genomes-mitochon- YPGE, SD, or SCD medium (8), as described in the text. drial and nuclear (1). Over the last decade, studies from a Amino acids and nucleotides were added or deleted as re- number of laboratories have resulted in a fairly thorough un- quired. Solid media contained 2% bacto agar. derstanding of the organization and expression of the mito- Escherichia coli strains used for propagating plasmids chondrial genome (for review, see ref. 2). However, only re- were RR1 (F- pro leu thi lac Y St' r-m- endoI-) and HB101 cently have similar questions begun to be addressed for the (F- pro leu thi lacY Str' r-m- endoI- recA-). A RecA- de- nuclear genome. The hetero-oligomeric membrane protein rivative of strain JM103 (9) was used as a host for all phage cytochrome c oxidase provides a useful model for investigat- cloning. E. coli strains were grown at 37°C in LB or YT me- ing nuclear-mitochondrial interactions in yeast. It is com- dium (10). When grown selectively, transformed strains posed of nine nonidentical polypeptide subunits, the three were grown in either of the above media supplemented with largest of which are mitochondrial in origin (I, II, III), while 100 ,g of ampicillin per ml. the remaining six are nuclear encoded (IV, V, VI, VII, VIIa, The shuttle plasmid YEp13 (11) was used in this study. VIII) (3). The plasmids YEp13-511 and YEp13-552 were isolated from To facilitate studies on the nuclear-encoded subunits of the yeast library constructed by Nasmyth and Tatchell (12). cytochrome oxidase, we have begun to isolate the genes en- Nucleic Acid Hybridizations. High stringency nucleic acid coding these polypeptides. Previously, we reported the mo- hybridizations were done at 680C in 6x NaCl/Cit (lx NaCl/ lecular cloning of two of these genes. The structural gene for Cit is 0.15 M NaCl/0.015 M Na citrate, pH 7.0)/3x Den- subunit VI (COX6) was isolated by using two synthetic oligo- hardt's solution (13)/0.5% NaDodSO4/100 ,ug of sonicated nucleotide probes complementary to the DNA sequence, as salmon testes DNA per ml. Two to three washes were done predicted from its amino acid sequence (4). In addition, we at 680C in 2x NaCI/Cit/0.5% NaDodSO4, followed by an ad- have used a subunit V structural gene mutation to clone a ditional 2-3 washes in lx NaClI/Cit/0.5% NaDodSO4. Re- gene for subunit V, COXS (5). Evidence that this gene was duced stringency hybridizations were at 450C in the same COXS came from (i) the restoration of respiratory competen- hybridization mix; washes were done the same way as de- cy and cytochrome oxidase activity in mutant strains carry- scribed above, except at 450C. All DNA transfers were to nitrocellulose filters, while RNA was transferred to Gene- The publication costs of this article were defrayed in part by page charge Screen (New England Nuclear) after separation on 6% form- payment. This article must therefore be hereby marked "advertisement" aldehyde/1.5% agarose gels (14). in accordance with 18 U.S.C. §1734 solely to indicate this fact. DNA Sequence Analysis. Restriction fragments to be se- 2235 Downloaded by guest on September 25, 2021 2236 Biochemistry: Cumsky et al. Proc. NatL Acad Sci. USA 82 (1985) quenced were subcloned into the M13 phage vectors mp8 00 and mp9 (15) and were sequenced by using the dideoxy N.I method (16). Sequencing reactions were analyzed on 6% /) buffer gradient gels (17) as well as with multiple loadings on I, 6% sequencing gels. I<4R5/ B 1 / t>c, N. Purification of Cytochrome Oxidase. Holocytochrome c oxidase was prepared from various yeast strains, using a scaled-down version of our standard procedure (3), with the following modifications. Mitochondria were prepared from yeast spheroplasts and converted to submitochondrial parti- cles by sonication. Ten milligrams of submitochondrial parti- cles were then extracted with 0.3 vol of 20% Fisher cholic acid, followed by overnight precipitation with 50 mg of am- monium sulfate per ml at 40C. After centrifugation, the su- Vb_ Vb pernatant was subjected to octyl Sepharose chromatography Va- __-- -VVa (0.2-ml bed volume) as described (3), except that the cyto- Vm vm chrome oxidase was eluted in 1 ml of buffer containing 5%, rather than 3%, Triton X-100. The exchange centrifugation and desalting steps were accomplished by ultracentrifuga- tion of the enzyme through the appropriate buffers (3) in a microfuge tube. The tube was supported by an O-ring on the rim of a centrifuge tube that had been filled with the same buffer. A typical preparation yields 10-20 ,ug of cytochrome FIG. 1. Transformants regain cytochrome oxidase subunit V oxidase. polypeptides. Immunoblots of mitochondria (50 ,g) from the indi- Miscellaneous Methods. NaDodSO4/polyacrylamide gel cated strains were carried out as described (5), using anti-subunit V antisera. (A) Strains are D273-1OB and JM43, wild type; JM28, a electrophoresis; immunoblotting; yeast and E. coli transfor- cox5a mutant; and JM28-511, a JM28 transformant carrying the mations; restriction endonuclease analysis; cytochrome oxi- COXSb plasmid YEpl3-511. Positions of subunits Va, Vb, and Vm, dase assays; and the preparation of DNA, RNA, and mito- the mutant form of Va, are indicated. CO, 0.5 ,ug of purified holocy- chondria were as described (5). Poly(A)+ RNA was prepared tochrome c oxidase standard. (B) Same as A. Additional strains are from total yeast RNA by the method of Aviv and Leder (18). JM28-552, the mutant JM28 transformed with the COX5a plasmid YEp13-552; JM43-511, the wild-type strain JM43 transformed with RESULTS YEp13-511. S. cerevisiae Contains Two Nonidentical Genes for Cyto- the vector YEp13 (10), we obtained seven additional trans- chrome Oxidase Subunit V. Previously, we reported the use formants in which the ability to grow on media containing of a cytochrome oxidase subunit V structural gene mutation glycerol and on media lacking leucine cosegregated. To de- to clone the subunit V gene by complementation in yeast (5). termine which subunit V gene they contained, plasmid DNA This mutant, JM28, contains an aberrant form of subunit V, from the seven transformants was screened by Southern designated Vm. In these initial experiments, a hybrid plas- blotting against a COXSb probe.