Isolation of the Yeast Nuclear Gene Encoding the ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257, No. 18, Issue of September 25, pp. 11186-11190, 1982 Printedin U.S.A.

Isolation of the Yeast Nuclear Gene Encoding the Mitochondrial Protein, Cytochrome c Peroxidase* (Received for publication, May 25, 1982) Sherry Goltz#, James Kaput, and Giinter Blobel From The Rockefeller University, Laboratoryof Cell Biology, New York, New York 10021

A good deal of effort has been directed toward studying the yeast mitochondrial genome and its gene products (1). Emphasis has also been placed on characterizing the events involved with the biogenesis and energy-generating functions of mitochondria (2, 3). Although 90% of yeast mitochondrial proteins are coded for by nuclear DNA (4), little is known about the organization, regulation, or chromosomal location of the genes that encode these proteins. Several nuclear DNA mutations have been reported to alter the expression of mitochondrial proteins (5, 6). Only one of these, the OP1 gene, has actually been demonstrated to contain structural gene information (7). The mechanism(s) by which nuclear gene products are incorporated into mitochondria has not been elucidated to any great extent. The identification and the isolation of specific genes from cloned pools of yeast DNA based on their ability to complement well defined mutations in Escherichia coli and yeast proteins has become fairly routine in recent years (8-10). Genes encoding yeast proteins for which mutants have not been selected can be identified using in situ DNA, RNA, or antibody screening procedures that do not depend on functional gene expression (11). Yeast cytochrome c peroxidase is localized to the area between the outer and the inner mitochondrial membranes (12). This nuclearly encoded protein is synthesized both in

vivo and in vitro as a larger molecular weight precursor that is processed to its mature size upon translocation into mitochondria (13). The isolation of defined yeast mutants with alterations in the nuclear gene that codes for this protein has not been reported. We have employed a simple in situ antibody screening procedure to identify the clones in a yeast recombinant DNA library that carry the gene for yeast cytochrome c peroxidase. The radioimmunological assay utilized in this study (14) differs from those employed by other groups (15, 16) to monitor the expression of eukaryotic genes in E. coli in that this assay optimizes the detection of membranebound proteins. The yeast DNA present in the clones isolated using this procedure was characterized with respect to size, restriction endonuclease cleavage sites, and partial DNA sequence. The identification of this DNA as the cytochrome c peroxidase gene is based upon several independent lines of evidence. Antigenically recognizable cytochrome c peroxidase peptides are present in E. coli cells that carry this particular piece of yeast DNA. Recombinant plasmid DNA that contains all or part of this cloned DNA can hybridize to, or select, cytochrome c peroxidase specific mRNA, as determined by hybridization-selected-translation experiments. The nucleotide sequence of a portion of the cloned DNA was determined. This stretch of DNA codes for an 80-aminoacid long peptide whose sequence precisely matches that found in the mature cytochrome c peroxidase (17). The selection scheme used here is not dependent on the functional expression of a given gene product or on the existence of well defined yeast or E. coli mutants. It requires both that one have appropriately prepared antiserum to the protein of interest and that some part of this protein be synthesized in E. coli. As such, this approach may have wider applicability to the study of nuclearly encoded mitochondrial proteins than those used previously. MATERIALS AND METHODS Strains

and Media

The following E. coli strains were used: C600, RR1 and JH103 (18, 19). E. coli CV13 (20) was generously given to us by J. Hicks, Cold Spring Harbor Laboratories. Te yeast strain D273-108 (ATCC25657) was used in this study. Transformed E. oli cells were gro. in the presence of 100 ug/l ampicillin in either LB media (1% Bacto-tryptone. 1% NaC1. 0.5% glyeast extract. 0.1S glucose) or M9 salts (21) supplemented with 1 easamino acids., 1 cose, 20 ug/.l thiamine-HC1 100 ug/ml threonine. 10 ug/ml proline and 10 ug/ml thymine. out in Fortified The growth of single-stranded baoteriophsges in E. oli JM103 as rriod Broth as described (22., 23). Yeast strains were propagated at 30 C in either YPD (1% yeast extract. 25 Bacto-peptone, 3 glucose) or rich medium ith 2% glactose as the carbon source (24). RNAand T DNAPreparations otal yeast RNAwas prepared from D273-10B cells grown to mid-logarithmic phase in rich Plasid DNAwas isolated from bacterial cells medium as described by Naececchini et al (25).

'The "Materials and Methods" are presented in miniprint as

*This investigation was supported by National Institutes of Health Grant GM-27155 and NRSA Grants GM-07873 and GM-07908. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. f To whom reprint requests should be sent.

prepared by the authors. Miniprint is easily read with the aid of a standard magnifying glass. Full size copies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 82C-291, cite the authors, and include a check or money order for $2.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

11186

Downloaded from www.jbc.org by guest, on April 14, 2012

Bacterial clones containing the gene for yeast cytochrome c peroxidase, a nuclearly encoded mitochondrial protein, were isolated from a recombinant DNA library using an in situ radioimmunological assay. Escherichia coli transformants with this cloned DNA, YEp13-CCP, occur in the recombinant pool at a frequency of 10-3-10 - 4 . The YEp13-CCP plasmid contains approximately 3000 base pairs of yeast chromosomal DNA inserted into the Bam H1 site of the vector YEp13. The identification of this cloned DNA is based on 1) its ability to hybridize to, and to select, cytochrome c peroxidase-specific mRNA, 2) its expression in E. coli cells, and 3) DNA sequence analysis of the yeast DNA insert. The selection scheme described here represents a simple, rapid method for the isolation of nuclear genes that encode mitochondrial proteins. This approach is particularly useful for cloning the genes of mitochondrial proteins for which well characterized mutants are not available.

Isolationof the Yeast Cytochrome c PeroxidaseGene grown in the presence l Of

00 ug/ml aipicillin

The method used to isolate and to purify plaid fled froea fuged to

previouly equilibrium

in

9

ediu-

supplemented

as

DNA from trnsformed E.

13mp9 single-stranded DNA as prepared Rplicative fore DNAwa isolated fro

H13mp9 infected edI according to pblished prooedres (22). All NA cas stored in 1io M Tris (pH 81. 1 Me ETA t either 4C or

ater

above.

eas odi-

published proedur-e (21). Eriched plasid DNAfractions were centriin a cesiu chloride-ethidiun bromide density gradient that ontained

.04% n/v sarkonyl NL-90 (Ciba-Geigy). acteriophage from plate lysatno enentially an described (23). was stored in

deSribed

oli cells

11187

-80 C.

Yet

RNA

t -80 C.

Restriction Endonuclease Analyses All restriction enzyes usaed in this etdy ere purehae-d froe Re England Biolabs. Inc. Restriction endonuoleae assys ere perrfor ed according to the aenufactureres iestrwtions. In general, 1-2 ug of DNAwas digested eith 2-4 units of restriction enzyme t 37 C for I h in either 25 or 50 ul reaction ixtree. Reations ere terminated by the addition of 10 1ul of a olution containing 2 sucrose, 1200m ETA and 0.4% e/v brphenl blue per 50 ul1 digestion ixture. Restriction endonuoclea-e digestion products ere separated by eleotrophoresis on either 1 or 2S agarose (SigS, type II) slab gels in 40 M Tris-aoetate (pR 7.8) 5 M sodiu acetate. 1 m EiTA (26). The gels were stained Sith 1 ug of ethidifA bromide per 1 of running buffer following electrophoresis Plasmid Construction The recombinant DNAlibrary screened in this Rtdy was generously given to us by K. na-yth (NRC Laboratories. England). It consists of the vector YEp 13 (20) and the prodct of a partial lau 3A digestion of eat chroosla DNA. The yeast DNA insert ( ean size of 5-20 Ab) were ligated into the ga H cloning site of the vector. ransRforants of E. cli C600 that contain these hybrid planids are resistant to picillin. bt sensitive to tetrcycline. Isolates fro this pol ere maintained either on LB medium With 2 gear and 10

ug/el _picillin or i

15% glycerol at -O°C.

-.

A 1 7 yb ind III digestion prodct of Y2p 13-CCP placid D was iolated frc a 1 agaroe gel by electro-elotion. Thi DNA -as lighted into the Hind III cloning .. eite of E coli 11 was transformed pR322 and 13mp9 doubie-stranded DNAsby standard methods (21). . col1 --.0 li DN (21). -E-..I ueoe - II} I---2-Rn -it -h esi tanc to -. pili -. cells nere transformed ith the K13.p9-ind RII ligation preducte ad plated onto a fresh lawn of 24103 cells in the presence of IPTG and X-gal (23). Plaques formed by phages con tuning the ind III DNA restriction fragment ere identified by color.

Hybridization-Seleted RNATranslation Studies The Selection of specific yeat RNAsby DNA bound to nitrocellulose was accoplished uoing the procedure of Riociardi et al (27). Spercoiled plasmid RDNAs (5-10 g) were converted to their linear forms by restriction endonuolease digestion. elsetrophoreed on a 1% agarose gel as above., and transferrd to nitrocelllose filters. The hybridization. washing and eltion protocols were as described. In general, 50.D. units (approximately 250 ug) of total yeast RNA in 200 u1 of hybridization olution were annealed to 10 ug of filter-bound DNAat S C for6 h. The RNA as fluted fro the filters by incubation in water at 100°C for 5 sin. The luted RNA was precipitated with ethanol 3 times then dissolved in 5 1ulof sneor and frozen i liquid nitrogen. Eluted RN wac translated in 20 ul of a nse-treted rabbit reticulocyte lysate reaction mixture (28) in the presence of 40 uCi S-vethionine (NER.10 nCi/nl). The products of the translation were either prepared directly for electrophoreis onc a 10-15% gradient acrylamlde gel (29) or iunopresipitated with antisern prepared against yeast ytchrome pecrosidase (29). In Viv1 Labeling of Recombinat DNA cotaining E. ccli Cells The aicell labeling technique was prformed using YEpI3, YEpl3-CCP, 191 plasmids in i. coli C600 or RRI1 host trains as desribed (30, 31).

pBR322,

0ith the

and

CCP

Gidelines

RESULTS

Approximately 15,000 clones from a YEpl13-yeast DNA clone bank were screened with an immunoselected cytochrome c peroxidase antiserum using a modified version of the Henning et al. (14) antibody screening procedure. The original protocol was altered to reduce background problems and to enhance sensitivity (see "Materials and Methods"). Putative peroxidase-expressing transformants occur in the pool at a frequency of 2-3 1 - 4 . These clones were purified away from contaminating clones by selection with three successive antibody screenings. Four clones, designated YEp13-CCP 12, 15, 19, and 22, were chosen for further analysis and characterization of their yeast DNA inserts. All four clones contain the same DNA on the basis of restriction endonuclease mapping criteria (data not shown). The clones carry a 3.0-kb2 DNA insert which is not found in the vector, YEp13. It is likely that these recombinants arose by amplification of a single transformant in the original clone bank. 2The abbreviations used are: kb, kilobase; CCP, cytochrome c peroxidase; PBS, phosphate-buffered saline; IPTG, isopropyl-fi-Dthiogalactopyranoside; Xgal, 5-bromo-4-chloro-3-indolyl-,8i-D-galactoside.

TIl Ii~~~~~~~~~~~~' 1-= ,.-j En

LLI [

[

i

t

co

EtI 0

II

-1 i

MM:E I A

2 2°1

FIG. 1. Restriction endonuclease map of YEp13-CCP 19 insert DNA. The location of the YEp13-CCP 19 yeast DNA insert (c) and the restriction sites within this insert are indicated in a. A 1.7-kb HindIII fragment isolated from YEp13-CCP 19 (b) was subcloned into pBr322 and M13mp9 plasmids. This HindIII fragment consists of 346 base pairs of pBr322 DNA ( ) and 1.3 kb of yeast DNA (). The results of additional restriction endonuclease analyses performed on this fragment are also indicated.

A schematic diagram of the restriction map determined for YEp13-CCP 19 insert DNA is presented in Fig. 1. The 3.0-kb insert cannot be cleaved out of the vector DNA using Barn H1 because the ligation of this Sau3A restriction product into the Bamn H1 site of YEp13 failed to regenerate functional Barn H1 cleavage sites. This phenomenon has been observed by others (32). The 1.7-kb HindIII digestion product of YEp13-CCP 19 DNA diagrammed in Fig. lb was ligated into the HindIII cloning site of pBR322 to yield the plasmid pCCP 19A. This restriction fragment contains 346 base pairs of pBR322 DNA in addition to 1.3 kb of the YEpl3-CCP 19 yeast DNA insert. The expression of YEp13-CCP 19 insert DNA in E. coli was examined by probing in vivo labeled bacterial lysates with antibodies to cytochrome c peroxidase. The plasmid-encoded proteins in E. coli cells (C600 or RR1) transformed with either vector (pBR322, YEp13) or recombinant (pCCP 19A, YEpl3-

CCP 19) plasmid DNAs were identified using the maxicell labeling technique (31, 32). The cytochrome c peroxidase peptides in extracts prepared from cells labeled in this manner were immunoprecipitated with total cytochrome c peroxidase antiserum, as indicated under "Materials and Methods." The results obtained upon electrophoresis of these cytochrome c peroxidase-specific proteins are presented in Fig. 2. Maccecchini et al. (13) have previously shown that the molecular weight of in vitro synthesized cytochrome c peroxidase (approximately 39,000) is equivalent to that of the in vivo synthesized precursor form of the protein. This precursor is about 6,000 daltons larger in size than the mature protein. Gasser et al. (33) have observed a third form of the protein which is intermediate in size to these proteins. These authors postulate a two step mechanism for the conversion of the precursor cytochrome c peroxidase protein to its mature form. Two plasmid-dependent proteins in a maxicell lysate from YEp13-CCP 19 transformed cells are recognized by cyto-

Downloaded from www.jbc.org by guest, on April 14, 2012

Screening of Reom.binant DNAClones The radi-oianological reening procedure developed by Henning et al (14) for the detection f eebrane-asciated g. coli protoetn an mdified for use in this study. Rbinant DNA-contining . coil cees nere groan to stationary phase at 37C in LB sedim with 100 g/ml apicillin. The cultures were diluted with L, plated ont LB medium with 10 ug/l ampicillin and 1% w/v agar and allowed to grow overnight at 37 C. Colonies were transferred onto Whatan no. 40 filter paper circles (87 - diameter). Thee filters were treated as described (14), air dried and ither ..reened aith antibody immediately or stored at -20°C. Filters were incubated ith constant agitation at roo temperature for 2-4 h in 20 n/filter of PB (10 nMsdie phosphate. pH 7.2, 0.14 NC1) ith either O.5% Bovine serb albuein (frac. V, Sigma) r 0.5% oval.lrin (grade IlZ. Signa). Atiser in 15 1ml of PBS0.5% ovalbain bffer sae allowed to react ith the filters for 12-15 h at 0C., or 4-6 h at rooa temperature. These filters ere1 shed, then incubated overnight at 0°C in15 1 of PBS-0.5% ovalburih containing 0.4 uCi of I-protein A (EN, 95 uCi/.l, 92 uCi/ng). The filters sere cashed for a total of h ith four changes of PBS-0.5 Triton X-100. The air dried filters re placed on Kodak XAR_-5 film and autoradiographed at -80 C using one intensifying screen (Dupont, lightning-plus). The imunoselected cytochroe c prolidase antiserun used here sas prepared by Ktuyoshi ihara. Since many rabbit antiseru preparations cntain considerable amountsof antibodies against bacterial proteins especiallyy those elicited with Freund's adjuant) it may. i general. be necessary either to use sern bhich has been affinity selected to prified antigen or to remove any bacterial cross-reacting antibodies by adsorption to bacterial lysate-linked sepharose beads (15) prior to preening cells with this radio un.oassay.

Containment Conditions The experiments outlined in in s study were performed in accordance of the National Institutes of Heath for Recabinhant DNA Research.

l O °-

Downloaded from www.jbc.org by guest, on April 14, 2012

Isolation of the Yeast Cytochrome c Peroxidase Gene 190 200 Gly Tyr Glu Gly Pro Trp Gly Ala Ala ASn Asn Val Phe Thr Asn Glu Phe Tyr Leu Asn GGATAC GAAGGG CCA TGG GGAGCC GCT AAC AAC GTC TTT ACCAAT GAG TTT TAC TTG AAC 210 220 Leu Leu Asn Glu Asp Trp Lys Leu Glu Lys Asn Asp Ale Asn A.. Glu Gin Trp Asp Ser TTG TTG AATGAA GAC TGG AAA TTG GAA AAG AAC GAC GCGAAC AAC GAA CAG TGG GAC TCT 230 240 Lys Ser Gly Tyr Met Met Leu Pro Thr Asp Tyr Ser Leu Ile Gin Asp Pro Lys Tyr Leu AAG AGC GGC TACATG ATG CTG CCC ACT GATTAT TCT TTG ATT CAG GAT CCCAAG TAC TTA 260 250 Ser Ile Val Lys Glu Tyr Als Asn Asp Gln Asp Lys Phe Phe Lye Asp Phe Ser Lys Ala AGC ATT GTG AAAGAA TAC GCT AAT GAC CAGGAC AAG TTC TTC AAG GAT TTTTCC AAA GCT

11189

the dipeptide Gly-Pro. Recently, Land et al. (35) have successfully employed this property to identify the arginine vasopressin-neurophysin II gene. Mature cytochrome c peroxidase contains Gly-Pro sequences at amino acid positions 4344, 121-122, and 188-189. The digestion of pCCP 19A insert DNA with Sau 96I yields cleavage products with sizes that are consistent with this placement of Gly-Pro sequences (Fig. 4b). DISCUSSION

" s

1

-1

'C

Ii

_~~

i

H

H s

11 0

I

I

O n 0

z

I

O

M e

1

1

I

I

M

i

.