Apr 5, 2015 - either Schleicher & Schuell (nitrocellulose), MSI (Nytran), or Du ... standard conditions (21), and then probed by incubation in the same buffer containing ...... ask whether excision is possible from other sites, suggesting that the ...
THEJOURNALOF BIOLOGICAL CHEMISTRY
Vol. 268, No. 10, Issue of April 5, pp. 7372-7381,1%3 Printed in U.S.A.
0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.
Peptide Splicing in theVacuolar ATPase Subunit A from Candida tropicalis” (Received for publication, October 12, 1992)
Howard H. GUS&Jin Xu, Michael Gallagher, and GaryE. Dean7 From the 4Department of Physiology and Biophysics and Departmentof Molecular Genetics, Biochemistry, and Microbiology University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524
Subunit A of the vacuolar proton pump appears tobe in eukaryotic cells, including secretory vesicles, coated vesiresponsible for theATP hydrolysis which is coupled to cles, lysosomes, the tram-Golgi network, and the vacuoles of the pumping of protons into a variety of intracellular fungi and plants (for reviews see Refs. 1-7). These proton acid compartments, including the fungal vacuole. We pumps generate an electrochemical gradient for protons report here thecloning and sequence determination of across these intracellularmembranes which provides the drivthe gene encoding subunit A from Candida tropicalis. ing force for active transport of a variety of substances into Southern blot hybridizationanalysisindicates that the organellar lumen. there is a single gene which encodes this protein. The Allof the H’-ATPases purified to date are multisubunit gene contains a single intron at the extreme 5’-end of proteins, with subunits of approximate molecular masses of the coding region. The gene is predicted to encode a 16, 31, 39, 42, 60, 70 kDa, and one which has been variously polypeptide of 1088 residues with acalculated molecular mass of 119,019 daltons, yet the maturepolypep- estimated as 100-116 kDa (8-10). It has been estimated that tide appears to be approximately 67 kDa, indicating there are equimolar amounts of the 70- (subunit A) and 60that this protein probably undergoes the same sort of kDa (subunit B) subunits andthat each of these is in roughly processing that isevidenced in thehomologous protein 3:l ratio with the other cytosolic subunits (the VI domain). from Saccharomyces cerevisiae in which an approxi- Because these two subunits andthe membrane-associated 16mately 50-kDa polypeptide (the spacer) isspliced out kDa proteolipid subunit have been shown to have some sequence similarity with the corresponding FIFOATP synthase of the mature protein. The Candida gene, with and without this middle portion, has been expressed in S. subunits (in the range of 20-25% identity), because of the cerevisiae and found to restore a Saccharomyces sub- apparent stoichiometry mentioned above, and because of the unit A deletion mutant (tfpl-At3) to apparently wild- apparent ball-and-stalk arrangement of the structure seen in type growth at pH 7.6, and normal vacuolar acidifi- the electron microscope (ll),it has been postulated that the cation. The peptide sequence of the two predictedma- overall vacuolar ATPase structure is similar to that of the ture ends is very similar to the sequences of the anal- FIFO ATPase(12). ogous proteins from Daucus carota, S. cerevisiae, and Subunit A is thought to carry the ATP hydrolytic site of Neurospora crassa (60.5, 87.4, and 72.9% identity, the enzyme (8, 13-15). Recently, Kane et al. (16) have demrespectively), but the middle portion bears only very onstrated that the protein encoded by the subunit A gene limited homology with the Saccharomyces protein se- (TFP-1(17)) fromSaccharomycescereuisiae, an organism quence. Processing of the gene product occurs in S. distantly related to Candida tropicalis, is synthesized as a cerevisiae, Escherichia coli,and in rabbit reticulocytemediated in vitro translation, indicating that the ex- precursor of 119 kDa in mass which is subsequently processed of69 kDa as judged by SDScision is probably autocatalytic. The limited sequence to yield amatureprotein identity seen between the Saccharomyces and Candida polyacrylamide gel electrophoresis. This Saccharomyces 69spacer domains may considerably narrow the function-kDa protein is immunologically identical to the subunit A allyimportant regions responsible forthe excision protein from the vacuolar ATPase. Astonishingly, a 50-kDa segment (termed the spacer domain) is excised from the event. middle of the 119-kDa precursor and appears to be a stable protein. Even more surprisingly, this same 50-kDa protein (also termed the VDE, for ymal-_derived element) appearsto Vacuolar proton-pumping adenosine triphosphatases (H+- have endonuclease activity which may mediate its insertion ATPases) serve to acidify certain intracellular compartments into aunique site in aSaccharomyces TFP-1 gene lacking the VDE (18).We report here the cloning of the subunit A gene * This work was supported by American Heart Association Grant from C. tropicalis, a pathogenic organism for which relatively AHA 87-1025, National Institutes of Health (NIH) Grant R01little molecular genetic information is known. Theintact GM39555, and NIH Grant Pol-AI28392 to University the of Cincin- Candida gene functions in S. cereuisiae, complementing a nati Fungal Center. The costs of publication of this article were TFP-1 deletion mutant (tfpl-A8). The protein that is predefrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 dicted to be encoded by this gene is 119 kDa, but the mature protein appears to be approximately 67 kDa. These are the U.S.C. Section 1734 solelyto indicate this fact. The nucleotide sequencefs) reportedin thispaper hus been submitted only two organisms (and genes) currently known to have an accession numberfs) insert of this nature.We present evidence that theC. tropicalis to the GenBankTM/EMBLDataBankwith M64984. subunit A protein is processed post-translationally in a man5 Current address: Dept. of Pharmacology, Yale University, P. 0. ner similar to thatof the Saccharomyces TFP-1 gene product, Box 3333, New Haven, CT 06510. but comparison of the internal peptide sequences from Sac1[ To whom correspondence should be addressed Tel.: 513-558charomyces and Candida reveals relatively little primary 0065: Fax: 513-558-8474.
7372
C. tropicalis Vacuolar ATPase Subunit A
7373
structure similarity between the two excised spacer domains.(22). Two liters of midlog yeast cells were collected by centrifugation 4500 X g for 3min a t room temperature, washed twice with 100 ml The similarities existing between these two excised domains aoft distilled water and resuspended in 100 ml of Buffer I (1 M sorbital, may provide insight into this unusual processing event. 10 mM Mes-Tris, pH 6.9,5mM MgC12,l mM dithiothreitol), incubated at 30 "C for 90 min after 10 mg of Zymolyase (ICN) and 400 p1 of 0.4% glusulase were added. The spheroplasts were collected by spinEXPERIMENTALPROCEDURES ning a t 1000 x g for 3 min, washed twice with Buffer I, resuspended Materials-DNA modification and restriction enzymes were from in ice-cold Buffer I1 (10 mM Mes-Tris, pH 6.9, 0.1 mM MgCL, 12% New England Biolabs, GIBCO-Bethesda Research Laboratories, or Ficoll-400), and homogenized in a loose-fitting homogenizer. The Boehringer-Mannheim. Sequenase sequencing kits were purchased unbroken cells and cell debris were pelleted by spinning at 4500 X g from United States Biochemical Corp. Blotting media were from for 5 min at 4 "C. 15 ml of supernatant was layered on top of 15 ml either Schleicher & Schuell (nitrocellulose), MSI (Nytran), or Du of Buffer I1 in a centrifuge tube for a swinging bucket rotor (SW 281, Pont (Genescreen Plus).All radiochemicals were from Du Pont-New and centrifuged at 51,900 X g (20,500 rpm, SW28) for 30 min at 4 "C. England Nuclear. Oligonucleotidesynthesis reagents were from ABN. The white layer floating on top of the tube containing vacuoles was All other reagents were reagent grade or better. collected, suspended in 15 ml of Buffer 11, transferred to a new Strains and VectorsUsed-Bacterial strain: XL1-Blue: F'recAl centrifuge tube, and, on top of it, 15 mlof Buffer I11 (10 mM MesendAl gyrA96 thi hsdR17 supE44 relAl lac- (F' = proAB Tris,pH 6.9,0.5 mM MgC12, 8% Ficoll-400) was layered. After lacIqZA(M15)TnlO tetR);BL21 (DE3),which has the T7 polymerase centrifugation as before, the vacuoles contained in the white layer on gene integrated into its genome with an inducible Lac Z promoter, top were converted to vacuolar vesicles by diluting them first in one was used to express genes with a T7 promoter. S. cereuisiae strains: volume of 2 X Buffer IV (20 mM Mes-Tris, pH 6.9, 10 mM MgC12,50 SF838-5Aa (63), MATa ade6 gal2 leu2-3,112 ura3-52 (T. Stevens); mM KC1) and then in two volumes of Buffer IV (10 mM Mes-Tris, SF83&5Aa/tfpl-A8 (2542) MATa ade6 gal2 leu2-3,112 ura3-52 pH 6.9, 5 mM MgCl,, 25 mM KCl). The vacuolar vesicles werepelleted tfp1::LEUZ (P. Kane); 1-7A, MATa adel00 leu2-112 ura3-52 his4- (37,000 X g, 20 min, 4 "C), resuspended in three volumes of Buffer 119 (J. C. Loper). C. tropicalis ATCC750 (from the American Type IV, frozen quickly in liquid nitrogen, and stored at -80 "C. Culture Collection) was used for generating genomic libraries and for Quinacrine FluorescenceAssay-The fluorescent dye quinacrine DNA and RNA isolation. was used to label the acidic vacuoles. The procedure used was similar Gene Cloning,Sequencing,and SequenceAnalysis-Two degenerate to thatdescribed (23). Cells were grown to fresh saturation in YEPD, anti-sense-oriented oligonucleotides were designed to hybridize with pelleted by spinning at 3000 rpm for 2 min, resuspended in YEPD the carrot gene sequence (19) between nucleotide positions 1156 and buffered at pH 7.6 containing 50 p~ quinacrine, and incubated at 1103 (GD1081: TCTAGATACCIGTRTAAATGGARGCCTCAC"C for 10 min. Cells were then pelleted and washed with and GAGCRGCGACAGGCATGTTAGARGTGTT) and between nucle- 30 resuspended in cold YEPD, pH 7.6, kept on ice and examined under otides 1500 and 1445(GD1079: CCGCGGRAARTGTTTTCTTT- a microscope within 1 h. A Zeiss PhotoMicroscope I1was used to GIGCCAICTTYTTGTCIAAACCCCARAAGACCTGRACGAT). A, examine the cells and take phase contrast and fluorescent photos. C, G, and T have their usual meanings; R = A or G, Y = T or C and For fluorescent micrographs, a set of filters was used with excitation I is deoxyinosine triphosphate. These and various other deoxyoligo- bandpass of 450-490 nm and emission bandpass of515-545 nm. nucleotides used for DNA sequencing were synthesized using a Phar- Kodak p3200 BW film was used. Phase contrast photos were taken macia Gene Assembler. The first oligonucleotide was used to screen with automatic exposure and fluorescent photos taken with a 10-s a C. tropicalis genomic library (with an average insert size of 10 kb)' exposure time. of Sau3A partially digested genomic DNA cloned into the unique Proton TransportAssay-Proton transport of vacuolar vesicles was BamHI site of pABlOS), which was kindly provided by Dr. Jack Loper measured by the quenching of acridine orange fluorescence similar to (20). Replica filters were prehybridized for 16 h at 57 "C in 6 X SSC, that described (24). The vacuolar vesicles prepared as described above 1 X Denhardt's solution, 0.1% SDS, and 100 pg/ml denatured salmon were thawed on ice, diluted (500 pg/ml final) in assay buffer (20 RIM sperm DNA. The filters were hybridized in 70 ml of the same solution Tris-HC1, pH 8.0, 25 mM KC1, 5 mMMgC12, 1 p M acridine orange), for 16 h at 42 'C with 0.3 pg of the GD1081 labeled to 2 X lo9 dpm/ and reactions were started by adding 250 p~ Na2+/Mg2"ATP,pH pg using polynucleotide kinase and [Y-~*P]ATP.Filters were then 7.0. Nigericin (10 p ~ was ) used to assess the degree of acidification washed for 10 min a t room temperature in 6 X SSC, 0.1% SDS and at the end of reaction. The fluorescence measurements were perthen two times for 15 min each at 42 'C in the same buffer and formed on a SPEX DM 300CN, with excitation wavelength of 493 permitted to autoradiograph for 24 h (21). The filters were stripped nm and emission wavelength of530 nm. Inhibitor concentrations: of radiolabel by incubating them in 1 X SSPE, 50% formamide for 1 sodium azide, 500 p M ;vanadate, 50 pM; bafilomycin, 50 nM. h a t 64 'C, and then reprobed with GD1079 as for GD1081. HybridZn Vitro Transcription and Translation-Plasmids having genes of izing clones were picked, replated, and screened again, using the same interest cloned downstream of the T7 phage promoter in Bluescript methods. Successful clones were used to generate restriction maps. SKII+ were first linearized with a restriction enzyme cut at the 3'Restriction fragments of the cloned genes were subcloned into Blueend of the gene. 5 pg of the linear DNA was then transcribed in vitro script plasmids and the DNA sequences of a part of the insert DNA was determined. All DNA sequencing was performed by the dideoxy with 50 units of T7 polymerase (GIBCO-Bethesda Research Laboratories) in the presence of 40 units of RNasin for 60 min at 37 "C method as described (21) on double-stranded plasmid DNA. Deletions and cleaned up by phenol/chloroform extraction and ethanolprecipwere obtained with the Stratagene ExoIII/mung bean deletion kit; itation. A small portion (4 pl) of the reaction mix was transcribed in both strands were sequenced in their entirety. Computer analysis was parallel using 10 pCi of [a-32P]ATP.After reaction, the labeled RNA performed using DNANALYZE (Gregory Wernke, University of Cincinnati), and Clone and Align software from Scientific and Educa- was analyzed by formaldehyde-denaturing electrophoresis and autoradiography to assess the quality of the RNA transcribed. Half of the tional Software. Northern Blot Analysis-Total RNA was isolated from C. tropicalis newly transcribed RNA was then translated with 50 pl of rabbit reticulocyte lysate (Promega) in the presence of 36S-labeledmethioas described (21). RNAwas denatured in 50.7% formamide and from Du Pont-New England Nuclear Research separated by electrophoresis through 0.75% agarose gels in 0.22 M nine (Trar~s~~S-label Products; 10 mCi/ml, 1200 Ci/mmol). The products were finally formaldehyde. Capillary transfer toGenescreen Plus membrane was performed for 12 h in a buffer containing 0.025 M NaP04, pH 6.5. analyzed by denaturing SDS-polyacrylamide gel electrophoresis as The membrane was baked at 80 "C for 2 h, prehybridized under well as, or following, immunoprecipitation. Bacterial Expression-Genes of interest were inserted into Bluestandard conditions (21), and thenprobed by incubation in the same buffer containing double-stranded DNA probe at approximately lo6 script vectors downstream of the T7phage promoter and transformed cpm/ml, 10' cpmlpg. The probe was labeled with [32P]dATP by into bacterial strain BL21 (DE3) which has an inducible T7 polymerase gene integrated intothe genome as described above. When cells polymerase chain reaction amplification (21). Isolation of Vacuolar Vesicle-Isolation of vacuolar vesicles from had grown to a density of Am = 0.3, isopropyl-1-thio-@-D-galactopywild type and mutantS. cereuisiue cells were similar to thatdescribed ranoside was added to a final concentration of 0.3 mM to induce the T7 polymerase. At various times, cells were harvested by centrifugation in a microcentrifuge for 10 s, resuspended in SDS-urea buffer The abbreviations used are: kb, kilobase(s); bp, base pair(s); TBS, (1% SDS, 9 M urea, 5% 2-mercaptoethanol, 1 X stacking gel buffer), Tris-buffered saline; PBS, phosphate-buffered saline; Mes, 4-mor- boiled for 10 min and cleared by centrifugation in an Eppendorf pholineethanesulfonic acid Tris, 2-amino-2-hydroxymethylpropane- microcentrifuge for 15 min. The supernatants were then loaded on 1,3-&01. SDS gels for analysis.
7374
C. tropicalis Vacuolar ATPase Subunit A
Western Blot Analysis-Zn Vitro translation products and whole cell lysatesof bacteria expressing genesof interest were separated by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose filters.The filters were blocked with10% milk, 0.1% Tween 20 in either TBS (Tris-buffered saline solution:50 mM Tris-HC1, pH 7.4, 137 mM NaCl) or PBS (phosphate-buffered saline solution),washed once in TBS (or PBS) plus 0.1% Tween 20 and once in TBS, incubated with primary antibodies TBS in or PBS (anti-c-myccell culture medium from Dr. Linda Parysek, 10 X dilution; or anti-Flag antibody purchased from Immunex,50 X dilution) for 4 h at room temperature or overnight at 4 "C. The filters were then washed four times with 0.1% Tween 20 in TBS and incubated with horseradish peroxidase-labeled goat-anti-mouse antibody (Sigma, 1000 X dilution) for 2 h at 4 "C. Finally, the filters were washed once with 0.1% Tween 20, twice with 0.3% Tween 20, and again twice with 0.1% Tween 20, all in TBS. The signals were detected with the ECL Western blotting detection reagents (Amersham International) by soaking the filters in equal volumesof solutions 1 and 2 from the kit for 60 s; film was immediately exposed for between 1 s and 5 min. Immunoprecipitation-Theprimaryantibodiesanti-c-mycand anti-Flag, and the secondary antibodies goat-anti-mouseand rabbitanti-mouse antibodies (Sigma)were bound to protein A attached to agarose beads (Schleicher & Schuell) and covalently linked to the beadsaccording to the manufacturer'smanual of the AFFINICA antibody orientation kit (from Schleicher& Schuell). 10 p1 of the in vitro translation products were diluted in 200 pl of PBS and rocked overnight with 50 pl of antibodies linked to agarose beads, and 2 pl of anti-c-myc or anti-Flag were added with the secondary antibodylinked beads. After incubation, the beads were pelleted by spinning 1 min in an Eppendorf microcentrifuge, and the supernatant was discarded. The beads were then washed twice with 1 mlof PBS and soaked in Laemmli SDS sample buffer (2% SDS, 10% glycerol, 5% 2-mercaptoethanol,1 X stacking gel buffer) for 5 min. The supernatants were boiled for 2 min and loaded onto an SDSgel for analysis. Southern blotting, yeast expression, and other DNA and RNA preparations and manipulations were carried out essentially as described (21).
t o display diminished growth at pH values higher than 5.5 (25, 26). Thismutant grows extremely slowly at pH 7.6 compared with thewild-type parent (Fig. 3, A and B ) . When pHG661Y was transferred into these mutants, the cells grew a t p H 7.6 essentially as well as the parent (Fig. 3). Five out of five transformed colonies showed identical growth curves a t p H7.6 which were identical to the wild-type parent (data not shown), indicating that the Candida gene functions as well as the native gene, presumably by permitting the expressed protein to replace the deleted native protein in the pump structure. To show that it was indeed the predicted gene that was restoring function, the gene was disrupted by cutting at the unique BstXI site (position 3069, Fig. 1)in the gene, blunt-ending with T4 DNA polymerase in the presence of excess deoxynucleoside triphosphates (and re-ligating) the plasmid. In three out of three colonies, the disrupted gene failed to restore wild-type viability at pH 7.6 (Fig. 3). Two methodswere used to assess whether expression of the Candida gene conferred vacuolar acidification upon the deletion strain used. The first was an in vivo technique, using quinacrine fluorescence to estimate the degree to which the vacuoles of these cellscould be acidified by the vacuolar proton pump. Quinacrine isa weak base that accumulates in acidic compartments; strong vacuolarfluorescence of cells incubated with 50 FM quinacrine indicates that the compartment is ata low pH. It was observed that the tfpl-A8 mutant complemented with pHG661Y displayed a fluorescence comparable to thatof the wild-type parent cell, while the tfpl-A8 mutant and tfpl-A8 carrying the disrupted Candida gene failed to show fluorescence (datanotshown).Fromthese experiments, it appeared as though the Candida gene subunit A gene could indeed replace the wild-type Saccharomyces TFP-1 gene. T o further demonstrate the acidification properties of the vacuoles from each of the above strains, the rate RESULTS of acridine orange uptake into isolated vacuoles was deterCloning of the C. tropicalis 70-kDa Gene-Inspection of the mined.Acridine orangeisanother fluorescent weak base known subunit A peptidesequences revealsa number of which accumulates in acidic compartments with a concomiregions that are shared among them. Degenerate oligonucletant decrease in total fluorescence within the cuvette. Measotides were designed that were predicted to recognize the urement of ATP-dependentacridineorange fluorescence DNA sequences encoding several of these regions; two oligoquenching is commonly used to examine ATPase-mediated nucleotides, GD1079 and GD1081, that hybridized well to avesicularlumen.Vacuolar vesicles of S. cerevisiae and C. tropicalis protonpumpinginto single discrete bands in digests were isolated froma randomly chosen example of each of the genomic DNA(datanotshown) were used t o probe a C. above strains andassayed for vacuolar proton pump activity. tropicalis genomic DNA library. Approximately 15,000 coloThis acidification was not inhibitedby vanadate, an inhibitor nies of the C. tropicalis library described above were screened of the P-type of ATP-dependent ion pumps, norby oligomysequentially with theabove two probes. Twelve colonies were cin, azide, or efrapeptin, agents known to inhibit the mitodetectedthat hybridized withboth.Restrictionmapping analysis indicated that they were identical copies of the same chondrial ATP synthase; it was, however, inhibited by baficonstruct (named pHG601 (Fig. 1)) and contained the DNA lomycin AI (27), a specific inhibitor of all known vacuolar fragment diagramed in the restriction map Fig. in 1; this map ATPases. Nigericin, whichpermits theexchange of potassium if one of also illustrates the DNA sequencing strategyfollowed in de- or sodium for protons, abolishes the proton gradient the alkali cations is present, permitting an estimation of the terminingits complete DNAsequence (discussedbelow). degree of acidification obtained in thevacuole. The wild-type Southernblotting (Fig. 2) of electrophoreticallyseparated Saccharomyces strain and tfpl-A8 carrying pHG661Y were genomic C. tropicalis DNA restricted with BamHI, HindIII, a DNA fragment correspond- indistinguishable in these experiments, indicating that proton &ID, or EcoRI and probed with pumping was comparable in both, while the strains inwhich ing to nucleotides 1335-2719 resulted in single bands in all digests, indicating that the geneencoding the C. tropicalis either oneor both nonfunctionalgenes were present displayed no fluorescence quenching,indicatingthattheyare comsubunit A is a single-copy gene. Expression of the Gene in Saccharomyces-To prove that pletely incapable of acidification (data not shown). DNA and RNA Analysis-DNA sequence analysis indicated the cloned gene encoded the vacuolar ATPasesubunit A beganwith protein, a 5.7-kb SalI-BglII fragment (from pHG601, Fig. 1) that alarge openreadingframeexistedthat codon containing the entire gene was subcloned into thesingle-copy nucleotide position 464 and ended with the termination shuttle vector YCp50 t o yield pHG661Y (Fig. 1)and used to beginning at position 3725 (Fig. 4); this open reading frame transform a S. cerevisiae mutant deleted for the entire TFP- was predictedto encodea protein which was reasonably 1 gene (SF838-5Aa/tfpl-A8 (2542) (16)). Growthof mutants homologous to other known subunit A peptide sequences at carboxyl termini, butlacked homology t o most deleted for at least several genes encoding theSaccharomyces the amino and vacuolar ATPase subunitshave been previously demonstrated of these in the middle region (discussed below). The open
7375
C. tropicalis Vacuolar ATPase Subunit A Subunit A
A pHG6OI
1
S
1
B
I
H
X
P
" s8- " " " " "
c-
" a
-"
" t "
t
-
__t
"
B pHG66IY
I
S
I
B
I
H
X
I
I
w
P
as
H
H
pHG603 I,
,/
I
S
B
H
I
X
am
P
dr
H
pHG672
pHG672FM
pHG672MF
pHG672FN
pHG672FC
pHG672MN
pHG672MC
pHG674
pHG674FJM
pHG674MJF
FIG. 1. Map of the subunit A gene and derived constructs. A , the DNA fragment shared by all of the clones recovered from the initial cloning is indicated. Also included is an abbreviated restriction map and sequencing strategy used to determine the DNA sequence from the HindIII site at position 1 to the HindIII site at position 5352. Restriction sites indicated S, SalI; B, BamHI; H, HindIII; X, XbaI; P, PstI; N , NcoI; P u , PuuII; R, EcoRI; Bs,BstXI; Sp, SpeI; Xm, XmnI; Bg, BglII; RV, EcoRV; S2, SacII; BII, BstEII; M , MluI; S I , Sad; C , ChI; Bg/B, fused BglIIIBamHI sites. The putative coding regions are indicated with bold arrows, the spacer domain is shown by bars, and the single intron is presented as anopen box. An unidentified reading frame (URF)is indicated B, selected constructs were derived from the subunit Agene, as described in the text. Plasmid pHG661Y: A 5.7-kDa fragment between the SalI site and the BglII site in pHG601 containing the entire gene was subcloned into the Sal1 and BamHI sites within the tetracycline resistance gene ofYCp50 and used to express the Candida subunit A gene in S. cereuisiae. Plasmid pHG129 is a construct carrying additional silent restriction sites. pHG603 is a construct lacking the spacer domain. Plasmid pHG603 is identical to pHG661Y except that the spacer domain has been deleted. C , epitope-tagged constructs with their parents. Six constructs were epitope-tagged with one or both of the Flag and c-myc epitopic domains, each derived from plasmid pHG672. pHG672 contains the entire Candida subunit A gene fused with the vector SKII+ phage promoter T7 sequence, carrying a synthetic ribosome binding site and lacking the intron. Two other epitope-tagged constructs were derived from plasmid pHG674, a plasmid identical to pHG672 except that the spacer region was deleted as for pHG603. The plasmid, when linearized with XmnI and transcribed in uitro with T7 polymerase, gave rise to a single RNA species of 2.0 kb which was then translated into a dominant band of 67 kDa. Each construct is labeled with the name of the plasmid that carries it. The Flag domain is indicated by a boxed F , and thec-myc epitope is indicated by a boxed M.
reading frame lacked a start codon, however, and we therefore searched for potential upstream ribosome binding sites. A good candidate for a start codon was found at position 378, with perfect intron consensus sequences. polymerase chain reaction amplification of whole cell RNA between nucleotides 361 and 543 of the gene sequence yielded a single product band of about 80 nucleotide which, when subcloned and sequenced, demonstrated that the region between bases 381
and 463 was missing. This indicates that an intron separates the start codon ATG from the rest of the coding region, as predicted based on theintron consensus sequences. The mRNA would therefore result in the addition of a single methionine residue to theextreme 5'-end of the open reading frame described above, yielding a protein with 1,088 amino acids and a predicted molecular mass of 119,019 daltons. This molecular mass would be in striking contrast with the
C.tropicalis Vacuolar A TPaw Subunit A
7376
amino acid number 2 to 283 (nucleotide numbers 463-1309), was about 75% identical to the N. crmsa N-terminal half of theprotein, andthat the Candidacarboxyldomain from amino acid number 745 to the end(nucleotide numbers 2723 to 3724) showed the equivalent identity, but that the central domain was completely lacking in the N. crassa sequence. One explanation of this additional region is that the RNA - 23.lkb might be spliced, despite the absence of obvious splice junctions. To test this possibility, Northern blots of total C. - 9.4kB tropicalis RNA probed with each of the regions of the cDNA - 6.6kb (N-terminal domain (nucleotides 433to 1313)), centralspacer domain (nucleotides 1328 to 2722)) and C-terminal domain - 4.4kb (nucleotides 2961 to 3714)) were carried out to determine whether different sized mRNA’s were detectable, indicating the occurrence of mRNA splicing. For all three probes, only a single RNA species of approximately 3.5 kb in size was detected; there was no evidence of hybridization with smaller - 2.3kb RNA species (Fig. 51, suggesting that a single mRNA is used - 2.0kb to produce the entire 119-kDa protein. A second explanation for this additionalregion is that, like several viral mRNAs, ribosomes involved in translating the subunit A mRNA might skip over the intervening protein sequence and join the amino and carboxyl termini during the process of translation. If this were the case,a frameshift introduced into the spacer region should have no effect upon the resulting protein. To permit testing of this hypothesis (and additional manipulations), silent restriction sites (BstII at position 913, MluI at position 1573, SacI at position 2530, - 0.56kb and ClaI at position 3805) were first placed into the gene cloned into Bluescript SKII(+),asindicatedin Fig. 1 (pHG129). Additionally, a BamHI sitewas inserted atposition FIG. 2. Southern blots of restricted C. tropicalis genomic 3148 in such a way that the existing PvuII site at position DNA. Southern analysis of C. tropicalis genomic DNA restricted with 3129 was destroyed,making theremainingPvuIIsiteat the following enzymes:lane A , BamHI; lane B, HindIII; lane C, PstI; lane D,EcoRI; and probed with a DNA probe extending from nucleo- position 2184 unique. The XbaI-SpeI fragment from pHG129 tide 1335 to 2719 (Fig. 6). Washing stringency was 62 “C in 0.1 X was used to replace the gene downstream of the XbaI site in SSC. Fragments of X DNA cut with HindIII DNA serve as size pHG661Y and the resulting plasmid used to transform the markers on the right. tfpl-A8 deletion strain; the altered gene complemented the deletion strain for both viability at high pH and vacuolar acidification as well as the unaltered form (data not shown). Frameshifts were then introduced into this gene in the samemannerastheBstXI-generated deletion described above, but using the newly generated SacI and MluI restriction sites in the spacer domain. The SacI disruptionproduced a 4-nucleotide deletion beginning at nucleotide position 2529 (amino acid number 660)) resulting in translational termination 31residues downstreamat nucleotide number 2623, while the MluI disruption resulted in a 4-nucleotide insertion at nucleotide number 1572 (amino acid number 371) and termination 2 residues further at nucleotide number 1580. When transformed into the t f p l - A 8 mutant, the resulting cells behaved exactly the same as the BstXI-frameshifted mutant and were incapable of growth at high pH (data not shown), FIG. 3. Growth of parent and transformed tfpl-A8 deletion indicating that theRNA was translated into a single protein needed to be translated mutants. For both Petri dishes: upper right, wild-type (SF838-5A) and suggesting that the entire protein cells carrying YCp50 grow equivalently well at pH 7.6 and 5.5, while in order tobe functional. the tfpl-As strain carrying YCp50 (upper left. tfp-1::LEUZ) shows Protein Expression-Recently, Kane et al. (16) have devery poor growth at pH 7.6. Lower right, t f p l - A 8 cells carrying scribed a novel post-translational modification that appears pHG661Y ( YCp50 + CT70,for C. tropicalis 70-kDa subunit) grow as well as wild-type a t both pH values, while (lower left) tfpl-A8 carrying to occur with theS. cerevisiae subunit A protein. This protein the disrupted Candida subunit A (tfp-l::LEUZ/YCp50 + CT70/ is initially expressed as a precursor protein of about 118 kDa; subsequent modification excises approximately 50 kDa of the BstXI) gene resembles the tfpl-A8mutant. internal portionof the protein (termed the “spacer” domain), mass of every other known vacuolarATPase subunitA, which followed by rejoining of the amino and carboxyl termini of all have molecular masses of between 66 and 70 kDa. Com- the precursor. In the process, the peptide sequence Gly-Cysparison of the predicted amino acid sequence with the N. (50-kDa spacer domain)-Cys-Gly apparently resolves to Glycrussa subunit A protein sequences (28) indicated that the N- Cys-Gly. T o determine whether the same phenomenon might terminalportion of the Candida protein,extendingfrom occur with the Candida subunit A,we designed a series of A
B
C
D
C. Vacuolar tropicalis
ATPase Subunit A
7377
n
G T I I I C A C T A C A G T f f i T ~ I T T A G ~ C ~ A A G A A A A G A A A T T M A C G I C T I I C A I I A G A I G A C A C C A A I G A A I C C C ~ I A I f f i I C A540
A G A L E N A R K E I K R L S L D D T N E S Q Y G Q (27) ~ T C T A T T C T G T T T C C ~ T C C ~ I I G I I A I T G C C G ~ C A T G A I I ~ ~ ~ A T G I ~ C A T G T A C G M I I ~ ~ ~ T I A A A G I I ~630 ~~ICAIGAIMIIT I Y S V S G P V V I A E N M I G C A M Y E L V K V G H D N L (57) AGIIGGGG~GTTATTAG~IIMT~~~TGAI~GCMCCAIICAAGTITATGMGAAACIGCAGGGGTCACTGII~~~IGAICCAGITII 720 V
G
E
V
I
R
I
N
G
D
K
A
T
I
Q
V
Y
E
E
T
A
G
V
I
V
G
D
P
V
L
(87)
M G A A C T ~ T A A A C C A T T A T C I G I I G ~ T I A ~ ~ ~ I C C I ~ ~ ~ I I T ~ I ~ ~ ~ ~ C T A I I I A T G A I G G I A I I C A A A G A C C I T I A A A A G C C A810 IIAA R T G K P L S V E L G P G L M E T I Y D G I Q R P L K A I K ( l l 7 ) A G A T G A A T C C C ~ T C I A T T T A T A T C C C M G A ~ I A I T G A I G T I C C I G C I T I A T C M G M C T G I T C ~ I A T G A ~ I I C A C I C C A ~900 ~~IC~II D E S Q S I Y I P R G I D V P A L S R T V Q Y D F I P G Q L ( l 4 7 ) G M A G T T ~ ~ ~ T G A T C A T A T C A C I ~ I ~ A C A I I I T I ~ I I C I A T I I A I G ~ C I C I I I A I T G G A I G A C C A I A A G A I T I I G I I A C C I C C990
K V G D H I T G G D I F G S I Y E N S L L D D H K I L L P P ~ l 7 7 MGAGCAAGAGG1ACTATTACTTCIAITGCIGMGCCffiIICIIAIAAIGIIGAAGAACCAGTITIffiAAGTIGMIITGAIffiTMGM 1080
)
R A R G T I T S I A E A G S Y N V E E P V L E V E F D G K K ( 2 0 7 ) A C A T ~ T A C T C T A T G A T G C A I A C A T G G C C A G I T A G A G T I C C ~ G A C C A G I I G C I G ~ T I G A C I G C T G A I C A I C C A I I G I T G A C C1170 ~~~ H K Y S M M H T W P V R V P R P V A E K L T A D H P L L T G ( 2 3 7 ) T C A A A G A G T C T T ~ A T T C I T I A I T C C C A T G T G I T C M ~ ~ ~ I ~ ~ ~ I A C I A C I I G I A T C C C A ~ I I I T ~ ~ ~ T I G I ~ I ~ C I G I1260 IATTTC Q R V L D S L F P C V Q G G T T C I P G A F G C G K I V I S ( 2 6 7 ) TCAATCTTTGTCCAAATTCTCCAACICTGAIGIIAITATCIAIGITffiTIGTIICACTAAAffiTACICAAGTCAIGAI~TGAIffiI~1350 Q S L S K F S N S D V I I Y V G C F T K G I O V M M A D G A ( 2 9 7 ) CGACMATCTAITGAATCTATIGMGITffiTGACAAAGICATGIAAAGATGGIAIGCCMGAGAAGTIGIT~ITACCAAGAffiIIA1440 D K S I E S I E V G D K V M G K D G M P R E V V G L P R G Y ( 3 2 7 ) T G A T G A T A T G T A C M ~ ~ ~ T T C G I C ~ C I T I C I A G T A C T A G A C G I ~ I G C T ~ I C C G M ~ I I G A I ~ ~ ~ A I I I C A C I G I I T C I G C1530 IGAICA D D M Y K V R O L S S I R R N A K S E G L M D F I V S A D H ( 3 5 7 ) I M A C T T A T C T T G A A A A C T ~ C ~ G A T G I C A A G A T I G C I A C A C G T A A A A T I f f i I ~ A A C A C C I A I A C I G G I G T I A C I T T C I A I G I T I I1620 K L I L K T K Q D V K I A T R K I G G N T Y T G V T F Y V L ( 3 8 7 ) GGiUAAGACTAAGACTffiTATTGAAIIAGIIAAAGCCMGACTAAAGIIITCffiTCAICAIATCCAIffiICiUAAI~GCTGAAGiUAA 1710 E K T K T G I E L V K A K T K V F G H H I H G Q N G A E E K ( 4 1 7 )
AGCTGCTACTTITGCTGCTGATTGACICIAAAGAAIACAIIGATIGGAICAITGAAGCIAGAGATIAIGIACMGITGATGAAAIIGT
1800
A A T F A A G I D S K E Y I D W I I E A R D Y V Q V D E I V ( 4 4 7 ) CAAGACCAGCACCACTCAAATGAICAACCCAGTTCATITIGAAICIffiIAAACICffiIAACIffiIIACACGAACACAAGC~CAAAIC 1890 K T S T T O M I N P V H F E S G K L G N W L H E H K Q N K S ( 4 7 7 )
ACTTGCTCCACAATTGTTACTIGIIGIACIT~IffiIAIIffiAAAIGTI~ICIICIGCTTICACCAIGAACTCCAAAGATGA1980 L A P O L G Y L L G T W A G I G N V K S S A F T M N S K D D ( 5 0 7 ) IGTTAAATTAGCTACAAGAATIAIGAACIACICIICAAAATI~AIGACITGITCITCIACIGMICCffiTG~CICAAIGICGCTGA 2070 V K L A T R I M N Y S S K L G M T C S S I E S G E L N V A E ( 5 3 7 )
FIG. 4. Geneandpredictedprotein sequences for the C. tropicalis vacuolar ATPase subunit A. Predicted protein molecular weight = 119,019 with 1,088 amino acid residues. The spacer region is underlined. A single intron exists at theN terminus separating only the start codon ATG from the rest of the coding region.
AAACGAAGAAGAATTTTTCAAIAACCTIffiIGCIGAAAAffiATGAAGCIffiIGAIIICACTITIGAIGMTIIACCGATGCTAIffiAIGA 2160 N E E E F F N N L G A E K D E A G D F T F D E F T D A M D E ( 5 6 7 ) ATTGACTATCAATGTICAT~TGCAGCIGCAAGAAGMG~CAAIIIGIIGIGGMTGCIIIGAAATCTCIIGGTTICAGAGCCAAGIC 2250 L
I
I
N
V
H
G
A
A
A
S
K
K
N
N
L
L
H
N
A
L
K
S
L
G
F
R
A
K
S
(
5
9
7
)
TACTGATATTGTCAAGAGTATICCICAACAIAITGCIGIIGAIGATATIGTIGICAGAG~TCIIIGAITGCCffiTIIAGTTGAIGCIGC2340 T D I V K S I P Q H I A V D D I V V R E S L I A G L V D A A ( 6 2 7 ) T~TAATGTTGAAACCAAAICCAAIffiIICTAIIGAA~IGIIGTIAGMCTICTIICAGACATGICGCTAGAffiTCTTGICAAGAIIGC 2430 G N V E I K S N G S I E A V V R I S F R H V A R G L V K I A ( 6 5 7 )
TCATTCTTIGGGTATTGAAICATCTAIIAATAIIAAAGAIACICACAIIGAIGCIGCIffiTGTIAGACAAGAAIITGCIIGIATTGICAA 2520 H
S
L
G
I
E
S
S
I
N
I
K
D
I
H
I
D
A
A
G
V
R
O
E
F
A
C
I
V
N
(
6
8
7
)
TTTGACT~TGCTCCACTT~IGGTGIICIIICIMAIGTGCACIIGCAAGAAACCAAACICCAGIIGTC~TIIACCAGAGACCCAGI 2610 L
T
G
A
P
L
A
G
V
L
S
K
C
A
L
A
R
N
Q
T
P
V
V
K
F
I
R
D
P
V
(
7
0
7
)
TTTGTTCAACTTTGATITGAICAAAICIGCAAAAGAAAACIATTATffiIAIIACITTGIGMGAAACIGAICAICAAIICCITIIAIC 2700 L F N F D L I K S A K E N Y Y G I T L A E E I D H Q F L L S ( 7 3 7 ) C M C A T G C T T ~ T G C A C ~ C I G I f f i I G A A C G I f f i T A A T G A G A I G I G A A G I I T I G A I G G A A T ~ C C C A G M T I G I T I A C I G M A I I I C2790 N M A L V H N C G E R G N E M A E V L M E F P E L F I E I S ( 7 6 7 )
TGGIAGAAAAGAACCAATTAIGAAACGTACCACITIffiITGCCAATACTTCIMIAIGCCAGICGCIGCCAGAG~TAITIAIAC
2880
G R K E P I M K R T T L V A N I S N M P V A A R E A S I Y T ( 7 9 7 ) T f f i T A T T A C A T T G G C T G A A T A I I T C A G A G A I C M f f i T M G M I G I T I C T A I G A T T G C T G A I I C I I C I I C A C G I T ~ I G ~ G A G 2970 G I T L A E Y F R D Q G K N V S M I A D S S S R W A E A L R ( 8 2 7 ) A G A A A I T T C T f f i T A G A T I G T G A A A I G C C T G C T G A T C M f f i I I I C C C A G C T I A I I I ~ T G C I A A A I I G T I C T I I C I A I G A G C G T G C3060 E I S G R L G E M P A D Q G F P A Y L G A K L A S F Y E R A ( 8 5 7 )
CGGTAAAGCCACTGCTTTGIICACCAGAIAGAGITffiITCAGIIICIAIIGTTGCIGCTGTTICTCCAGCTffiTffiIGATITCICIGA 3150 G K A I A L G S P D R V G S V S I V A A V S P A G G D F S D ( 8 8 7 ) T C C A G T I A C I A C I I C T A C T T I G I A T T A C T C ~ G I I I I C T ~ I I f f i A I A A G A A A I I G C C A A A G A A A A C A I I I C C C A I C I A T I A A 3240 P V T T S T L G I T Q V F W G L D K K L A Q R K H F P S I N ( 9 1 7 ) CACCAGTGIIICTIAITCT~TACACCMIGIIIIGMCMAIACTAIGAIICCAACIATCCAGAAIICCCACAAITGAGAGACAAAAI 3330 T S V S Y S K Y T N V L N K Y Y D S N Y P E F P Q L R D K I ( 9 4 7 )
T A G A G M A I T T I A I C I A A T ~ I G ~ G ~ I I ~ ~ ~ ~ C ~ G T I G I I C ~ I I A G I T ~ ~ ~ I M A I C T ~ A I I G I C T G A I I3 C 4 2T0G A T ~ G A ~ I A C I ~ ~
~~
~~~~~~~~
~~~
~
~
~
R E I L S N A E E - L E Q V V ~ Q L V G K S A L S D S D K I I L ( 9 7 7 ) A G A T G T T G C T A C C T T G A T T A G A A G A I T I C I T G C M C ~ I f f i I I A I I C I I C A I A I G A I G C A I I C I G T C C M I I I f f i A A G A C I T I I G A3510 D V A I L I K E D F L Q Q N G Y S S Y D A F C P I W K I F D ( l O O 7 ) IATGATGAGAGCATTTATTTCAIAIIAIGAIGMGCAC~GCAAIIGCCAAIffiIGCICAAIffiICIAAAIIAGCIGAAAGIACIAG 3600 M M R A F I S Y Y D E A Q K A I A N G A Q W S K L A E S T S ( l O 3 7 ) T G A T G T T A A A C A T G C T G T T T C T I C A G C I A A A I I C I I I G M C C A T C A A G A f f i T C ~ G M f f i I G ~ G M I T I f f i A G A I I I A I I M C 3690 D V K H A V S S A K F F E P S R G Q K E G E K E F G D L L T ( l O 6 7 ) C A C T A T C I C C G A A A G A T T T G T G ~ A G ~ T M T C G I I A G A I C C A I G T I T C A C C T I G T I G I I G I I C A G T T C T I A G A A I T I I I I C C I 3780 (1078) 3870
3960 4050
4140 4230 4320
4410 4500 4590 4680 4770 4860
4950 5040 5130 5220 5310 5400
5433
experiments to examine its processing. As a first stepin analyzing whether the same sort of novel processing occurs with the Candida protein, a gene (pHG603, Fig. 1) was constructed which lacked the middle spacer domain and which contained the presumptive resolved sequence Gly-Cys-Gly (Fig. 6). This gene, subcloned into the singlecopy vector YCp50, was subsequently used to transform the tfpl-A8 deletion strain and was found to restore V-ATPase function just as well as the intactCandida gene, judged both by growth at pH 7.6 and vacuolar acidification (datanot shown), suggesting that the Candida subunit A undergoes peptide splicing in yeast. I n Vitro Expression-Both the naturalgene and thespacer-
less gene were subcloned into Bluescript vectors and used to express the corresponding proteins in vitro. For these purposes, the single intron at the 5’-end was deleted anda ribosome binding site sequence C C A C C D G (where the underlined ATGis the start codon), was inserted and the resulting construct placed downstream of the Bluescript T7 promoter. The resulting plasmid pHG672 (Fig. 1) was linearized, and the gene was transcribed i n vitro with T7 polymerase. The products showed only the full size RNA, yet when translated in vitro, gave rise to a major band of about 51kDa (size of the spacer protein), aweaker 67-kDa band (size of the mature subunitA), and faint bands of higher molecular weight (Fig. 7A, lane 1 ). When the spacer region was deleted, however
C. Vacuolar tropicalis
7378
ATPase S u b u n i t A
(plasmid pHG674, Fig. l), themajor 51-kDa band disappeared I n Vitro Translation and Immunoprecipitation of Tagged and the 67-kDa band became dominant (Fig. 7A, lane Z ) , Subunit A-The tagged genes were transcribed in uitro with (only full length indicating that the 51-kDapolypeptide is encodedby the T 7 polymerase, andtheresultingRNAs messages were observed) were translated with rabbit reticuspacer domain andthat peptide splicing occurs inuitro. Tagging the Proteins-Although therelative molecular locyte lysate. Fig. 7B shows the "S-labeled, translated prodweights of the protein productssuggested that the precursor ucts separated on a 10% reducing SDS-acrylamide gel. Full protein was being modified in this unusualway, we wished to length constructs gave rise to a dominant band of about 51 kDa, a 67-kDa band, and bands of higher molecular mass, demonstrate that the two ends were infactbeingjoined together anddecided to use epitope tagging to accomplish this while the spacer-deleted constructslacked the major 51-kDa goal. Monoclonal antibodies against the Flag epitope (peptideband and the bands above 67 kDa, but showed a dominant 67-kDa band. This result indicates thattagging both ends of sequence: DYKDDDDK),andthe c-myc epitope(peptide sequence: EQKLISEEDL) are widely available. We reasoned the protein with the two short peptides does not affect the that the peptide-splicing process might not involve the ex- peptide-splicing process in vitro. Moreover, when the in uitrotreme N and C termini, and that placing the above peptide translated productswere immunoprecipitated with antibodies was sequences a t these termini might similarly be unaffected by recognizing oneortheothertags,the67-kDaband the process. Eight constructs (Fig. 1) were made which put precipitated by both the anti-FLAG antibodyrecognizing the the tagged intact subunit A gene (pHG672 derivatives) or the Flag epitope tagged at the N-terminal and the anti-c-myc spacerless gene (pHG674 derivatives) downstream of the T7 antibody recognizing the c-myc peptide attached at the Cterminal, while the 51-kDa bandcould not be precipitated by promoterinSKII+(plasmidspHG672FM,pHG672MF, either antibody(Fig. 7C). The result indicates that the mature pHG672FC, pHG672MC, pHG672FN, pHG672MN, pHG674FJM, and pHG674MJF). The PstI-Sac1 insert from protein represented by the 67-kDa band has both the N and pHG672MC (carrying the c-myc epitope at the C terminus) C termini, whereas the 51-kDa band derived from a portion wasused to replace the coding region downstream of the of the full length precursor protein in the middle. That the unique PstI site in pHG661Y and used to transform the tfpl- 51-kDa band is only present in the productof the full length A8 deletion strain; the resultingcell grew at pH7.6 as well as gene, and the 67-kDa bands derived from the full length and size the wild-type (data not shown), indicating thatmodification spacerless version of the gene have exactly the same was derived from the of a t least the 3'-end of the gene has little or no effect upon strongly indicates that the 51-kDa band domain the subunit's function. The 5'-end modifications were not spacer region of the gene. Antisera raised to the spacer were never of sufficiently high titer tobe of any use. tested for vacuolar ATPase function. Bacterial Expression of the Genes-We expressed the tagged genes in bacteria and demonstrated that peptide splicing also occurs in prokaryotic cells. The plasmids carrying the above constructs were transformed into E. coli strain BLZl(DE3) which has the T7polymerase gene integrated into the bacterial chromosome under Lac Z control. Upon addition of isopropyl-1-thio-P-D-galactopyranoside, T 7 polymerase is induced and initiates the synthesis of the gene product under T 7 control carried on the plasmid present in the bacteria. We 3.5KB expressed the subunitA geneand itsspacerless versiontagged with Flag peptideat the N-terminal and c-myc peptide at the C-terminal (plasmid pHG672FM and pHG672FJM)in these bacteria, separated the whole cell lysates on a 10% reducing SDS-acrylamide gel, transferred the gel protein pattern to a nitrocellulose filter, and blotted with the anti-Flag and antic-myc antibodies. As shown in Fig. 70, theinduced whole cell lysate of the full length construct (pHG672FM)were probed with anti-FLAG and anti-c-myc antibodies (lanes 1 and Z ) , and both antibodies recognized a band of more than 100 kDa full length FIG.5. Northern blots of C. tropicalis RNA. 10 pg of total and a band of 67 kDa, representing the presumptive RNA from C. tropicalis was electrophoretically separated on a 0.75% precursor protein and the processed mature protein, respecagarosegel and transferred to a Genescreen Plus replica, and the tively; the spacerless construct encoding the 67-kDa protein replica was probed with polymerase chain reaction-derived radiola- alone, however, gave rise to a single band of 67 kDa which beled portions of the C. tropicalis subunit A gene, corresponding to: N , the N-terminal portion (nucleotides 433-1313); S, spacer domain was recognized by the anti-c-myc antibody (lane 3 ) ; no band (nucleotides 1328-2722); and C, the C-terminal portion (nucleotides at 51 kDa was recognized by either antibody in any of the constructs. The additional band of 85 kDa in lane2 recognized 2961-3714).
N
C
S
-
1290
1300
1 3 1 1330 L 1320
2720 2710
-
2750 2740 2730 GAACGTdoTAATWTQQCTQ
CTQATdTATTATCTATdT~ ( I A A C O T W T A A T ~ A A T E I A O A T ~ Q ~ T O
FIG.6. Spacerless gene sequence. The DNA sequence of pHG603 in the region of the predicted 51-kDa spacer domain is indicated. Nucleotide numbers correspondto those presented in Fig. 6.
C. tropicalis Vacuolar ATPase Subunit A
7379
TABLE I Repeats found in the C. tropicalis subunit A gene sequence The minimumrepeatlengthreportedis 12 bases. Palindromic sequences are indicated by the symbol P.
4
-
m
N
0
0
-
Fragment from base
N
4
- 97.4kD - 69kD
Direct repeats 522 1558 2331 2783
2478 2972 - 30iD
64
- 46kD 2624 3909
Inverted repeats 64 1163 1469 2624 3711 3909
Repeated from base
813 2419
1163 1469 3711
Size of repeat
Repeat sequence, 5‘ + 3’
12 13 14 15
ATGAATCCCAAT TGTCAAGATTGCT TTGATGCTGCTGGT GAAATTTCTGGTAGA
14 (P) 12 (P) 12 (P) 14 (P) 12(P) 12 (P)
AAATGTATACATTT TTGACCGGTCAA TCTAGTACTAGA GATTTGATCAAATC CTGAAGCTTCAG ATATATATATAT
It encodes a protein which equivalently replaces the endogenous Saccharomyces subunit A gene, as judged by its ability to restore the tfpl-A8 deletion strain to wild-type growth at --c\ici high pH and to apparently normal vacuolar acidification. The -200kD 97.4kD3.4-kb gene is interrupted by an 83-bp single intron which 69kD separates the methionine initiation codon from the restof the -97.4kD 45kD coding sequence. The intronlexon boundary sequences con-69kD form to the consensus sequences derived by Mount (29) and a sequence TACTAAC, present within this intron, has been -45kD 30kD shown tobe necessary in S. cereuisiae for the efficient removal of introns by RNA splicing (30, 31). A second unidentified reading frame (URF) is located in FIG. 7. A, i n vitro translation products of full size and the spacer- the DNA sequence indicated in Fig. 1 and reported in Fig. 4, less subunit A gene. RNAs transcribed in vitro from the Bluescript T7 promoter were translated in rabbitreticulocytelysates in the beginning with nucleotide 5127 and extending to nucleotide A gene. No presence of 35S-labeled methionine, separated on a 7.5% reducing 3922 in the opposite direction to the subunit SDS-Crylamide gel, and fluorographed. Lysates were from: lane 1, homology to anypreviously identified gene or cDNA has been t f p l - A 8 carrying pHG672; lane 2, t f p l - A 8 carrying pHG674. Molec- made. If this open reading frame actually constitutes a tranular weight markers are on the right. (The bands of high molecular scribed unit, there exists only a 195-nucleotideregion between weight on lane I are spillovers from a positive control onthe adjacent the C termini of subunit A and theopposing second gene. lane.) Note the presence of the 51-kDa spacer protein in lane 1. B, i n There area number of unusual repeatedsequences throughvitro translation of the tagged subunit A. Selected gene constructs illustrated in Fig. 1were transcribed in vitrowith T7 polymerase, and out the subunit A gene, both inverted and direct, shown in the resulting RNAs were translated with rabbit reticulocyte lysate in Table I. Upstream of the presumptive translation initiation the presence of 35S-labeled methionine andseparatedon a10% site, there are also three sequences which exhibited strong reducing SDS-acrylamide gel. Lane 1, pHG672FM; lane 2, homology to a CCAAT-like transcriptional element,TCAAT, pHG674FJM; lane 3, pHG672MF; lane 4 , pHG674MJF lane 5, 368 of the sequence presented. This pHG672FN; lane 6, pHG672FC; lane 7, pHG672MN; and lane 8, a t positions 165,315, and pHG672MC. C, immunoprecipitations of the subunitA gene products. “ C A T motif has been shown to bind various DNA-binding In the autoradiograph at the left, the in vitro translated products of proteins including human transcription factors CPl and CP2 pHG672FM (lanes I and 2 ) and pHG674FJM (lanes 3 and 4 ) were as well as the comparable HAP2IHAP3 yeast transcription immunoprecipitated with anti-Flag (lanes I and 3 ) and anti-c-myc factors (32). (lanes 2 and 4 ) antibodies. The autoradiograph at the right displays The mature mRNA hasbeen used to predict a polypeptide the nonimmunoprecipitated translation products for pHG672FM 1,088amino acids with a molecularmass of 119,019 daltons. of (lane I ) and pHG674FJM ( l a n e 2) for reference. D, Western blot of the tagged subunit A expressed in bacteria. E. coli strain BL21(DE3) The data supporting use of the suggested start site are: 1) (lanes I and 2 ) and there isa termination codon upstream of this pointa t nucleowas transformedwithplasmidspHG672FM pHG672FJM (lane 3 ) and grown to A m -0.3, induced with isopropyl- tide number 327,2) there is no other startcodon between this 1-thio-P-D-galactopyranoside, collected, and lysed in SDS-urea buffer. termination and our predicted start site, and 3) any other These lysates were then separatedon a10% reducing SDS-acrylamide gel and transferred to nitrocellulose, which were probed with anti- translationproduct would not begin until nucleotide 378. Flag and anti-c-myc antibodies. Anti-Flag (lane I ) and anti-c-myc Comparison of the Candida subunit A predicted protein seantibodies (lanes 2 and 3) were then used to probe the nitrocellulose quence with that of the predicted subunit A sequences from replicas. Neurospora (28),carrot(19),and cow (33) indicated that there is a n approximately 51-kDa peptide sequence inserted by the c-myc antibody may represent the spacer-containing into the middle of the protein in a similar manner to that C-terminal fragment, an intermediateof the peptide-splicing seen in the Saccharomyces TFP-1 gene (16). In the Sacchaprocess, suggesting that the process might start with a cleav- romyces gene, a n approximately 50-kDa spaceris cleaved from age at theN terminus of the spacer domain. the middle of the subunit A protein and the two ends are spliced back together. Several lines of evidence suggest that DISCUSSION subunit A of C. tropicalis V-ATPase also undergoes a peptideWe have described the isolation of the single gene which splicing process similar to that seen in the subunit A (TFPencodes the C. tropicalis vacuolar ATPase subunit A protein. 1)gene from S. cereuisiae. These are as follows: 1)Comparison
7380
C. tropicalis VacuolarATPase Subunit A A 10
140
130
120
F
~
20
T
~
40
30
~
K
S
I
E
50
S
I
70
60
~
D
CT SC
Y T ~ F W L E K I K ~ I - - - - E L ~ G ~ I K ~ N G A E ~ T F ~ I D S ~ Y I W I I ~ ~ E I V K .FE.ITFPMK.APDGRIV....EVS.SYPISEGPERAN.LVESYRK.--SN.A.FE.T.....LSLLOSE.RKA.Y.~A.ILY.-----.DHFFDYH3
CT SC
K S L A H ) - - - - - - - L G n L G I I G ~ ~ ~ ~ D ~ ~ ~ S S ~ ~ S S T E ~ ~ A E N E ~ ~ - - - - - - ..I(FMT1EGPKV.A....L.I.D.LSDRAT.SM .-R.TS.HE.VTE.AE..IILCAEYKDRK.Pp..K ~ Y S K V V R O R O I R
CT
F T D ~ E L T I H V B G A M S K L ~ ~ K S L G ~ ~ I ~ I ~ E I A M D I ~ S L I ~ L M ~ ~ ~ I W V V R T S ~ ~ V .P..D.IVG...---LK.G..N..SFLST.N.GT..TPL...I.SD.Y.--1DEBO.K.TIK.IBIS.RD...SL.R...
100
~
~
L
S
210
280
""""_
400 380
290
300
390
220
310
230
320
420 440
240
330
430
170 190
160
150
200
CT SC
~
S
~
..A...N.L....SIEC..N....N.......R....IK....RET..S.V.-K.PB.AB..DS~.~..CI(.T.E.VVR.PaS.RRLS.T.K.~
110
410
L
so ~
C
sc
~
80
CT SC
250
340
470 450
-
-
-
180
270
260
350
~
360
370
460
I E S S I N I W T A I D A A O V R P E F A C - I ~ T G A P ~ S ~ ~ Q T ~ - - F ~ ~ ~ L I ~ G I ~ E T D E Q P L L S L W . V .AEPAKV.EM.TICHKISYA.YEtSG.DV.LN......OSKKPR.APAM.A.ECRO.Y.E.Q~DD......SDDS......A.PW....
FIG. 8. Comparison of subunit A B peptide sequences. A , comparison of C.1330 tropthe internal spacer regions from 2750 2740 2730 2720 2710 1320 1310 1300 1290 CMATQTTATTAKTATQTT Tuul/""/moczT TmnUQTwJ icalis and S. cereuisine, with the Candida ....c.cc...........c ....u.T/""/..Tro. sequences above the Saccharomyces. Conventions are the same as in Fig. 9. There is a 35.9% identity between the two sequences. Note that the N and C AATWT"TTTaW termini are both shown as Cys residues. .A......T.....C.CC......... .....A . . . . . A . . . . . C . . . . . It is not possible, in the absence of pepC tide sequence data, to know if one or both of these aminoacids may be absent CT M A G A - - L E N A R M I K R L S L - - D D T ~ S Q Y ~ I Y S V S G ~ I ~ ~ I ~ L ~ ~ V G ~ I( R8 6I) N W ~ T I ~ T SC ...................................................................................... ( 8 8 ) in the mature protein. B, comparison of NC ..PQ-QNG ~ G 1 A T . ......... K V..D...V...........Q...........Q............M....... ( 78) the nucleotide sequences flanking the ET .MDFSK.PKI.D.D.----------..TF.YYAG......T.CD.A.A......R...SE....I..LE..U.........S..S....... ( 82) DC - " "_ P S W G D . .TTFE.SEK..E..YVRK......V.DG.G.A......R......I..I..LE..S...........LH.N..... ( 84) spacer region in Saccharomyces and Candida. C, the mature protein sequences for CT T G K P L S V E L G H j l l 3 E T I Y D G I m ( P L K A I K D E S Q S I Y I ~ G I D V P ~ ~ ~ ~ F ~ ~ - L ~ D E I ~ I F O S I ~ S( 1L7L9D) D ~ I L L P ~ the vacuolar ATPase subunit A proteins SC ..................... Q.......E............T...D..I104....K-PP.....S....Y..VP....ISS........S (170) NC ............. LNN.....Q...EK.AW.N........AT...D.IMKWE...-T-U......A...VW.N....FISV......... (168) from C. tropicalis (CT),S. cereuisine BT ............ 1.GA.F ...Q...SD.SSQT........VN.S....D.MJ....CKN.R..S.......Y.IVN...-.IK...H....N (173) D€ .A..........ILGN.F...Q....T.AKR.GDV.....VS....DI(DTLWE.Q.KK-IGE..LL....LYAM...-.~.BVA...D. (174) ( S C ) ,N. crassa ( N C ) ,Bos taurus (BT), and D.carota (DC)were compared. Dots CT R G A I T S I A E A G S Y N V E E P n E F ~ S ~ ~ ~ ~ T ~ E ~ L T ~ ~ D S( 2L7 1 )F ~ T ~ I ~ indicate identity in the sequences, while SC ..T..W..P..E.TLD.KI.........SDFTLY...........T...S..Y..........A............................ (271) the residues which are different are inNC ..T..R...K.E.T...KI.........TE.RI.Q.........A...ES.NQ.F.V......A...S.....VA...............V. (260) ET .. n . Y . . PP.N.DTSDV ... L..E.I.E.F.HVQV....QV...T...P.N...........A..........A................. (265) dicated; hyphens indicate absence of the DC M.K ..W . P . .Q.SLWT ...L..Q.V.KQFTMLQ.....T.....S..A..T..........A...S.L...CA..............A.. (266) residue. The presumed site of the excised spacer in the C. tropicalis sequence is CT K F S N S D V I I W ~ E R G N P F P E L F - T E I S ~ P I ~ T T L V ~ T S ~ ~ I Y T G I (362) ~ ~ I ~ S S ~ W A SC (362) underlined and marked with an asterisk. NC ........ (351) ,
BT DC
.Y..................S...RD....T-M.M.KV.S.....A.......................S.....M.YB...H...T.... .Y....FW................D..Q.RI.LPD..E.SV...........................I......M.Y....H...T....
CT SC NC ET DC
E A W 1 E I S G R L G P I P M ) ~ F P A ~ G ~ F ~ ~ T ~ G S ~ R V G S V S I V M V S P ~ F( 4S5 4D) ~ T S ~ I ~ ~ ..................................... V.......T............D.........A....................... (454) .......................................... P.E ......G....P.........SA....V.................. (443) ..........A.....S.Y......R.........RVKC..N.E.E......G....P.........SA....V.................. (448) ..........A.....S.Y....A.R..........VKC..G.E.N...T..G....P.........SA..S.V.................. (450)
CT SC NC ET DC
1 N T S V S Y S K Y T H V L H K W D S N Y P E F H ) ~ K I R E I L S N A E E F D (546) ................F.........V...RMK..........,,......,................,..........T............ (546) ,,,....,..LT1.D.W . ~ D..R..,R..QL..DS...D.............P......U...............D..Q.......~ . . (535) V.WLI.....HRA.DE...KAFT..VP..T.AK...QEE.D.AEI......AS.AET.....E..K...D........TP..R...FY..VG (540) V.WLI.....STA.ESF.FKFDSD.IDI.T.A..V.~DD.NEI......D..AET.....ET.K.LR..Y.A..AFTP..K...FY.SVW (542)
CT SC NC ET DC
-ISYYDULO~IANGAO------WSKLAESTSDVIMAVS-------SAKFFEPSR--LEIYLTTISERFAWLSE (618) (617) ........A......V....N------.....D..G.......-------.S........E.-E.EGE--F.~.S.~.....STD (607) ,.KII*IGFH........Q.QN------.N.VR.A.Q.LQAPLK-------.L..~..E..EKIC..Y"W.~DI(FASVID. (618) .LSNM.AF..M.RR.VETT..SDHKIT..IIR.~EIL~.-------.M..~.VM)(jU.I.AD--YMIL.E~NA.RSLED ... N N . ~ . ~ . N Q . ~ . . G - - - - - - ~ ~ K I . Y I L I . . ~ G D - V . Q . . E D . ~ . E D ~ . G . ~ . ~ D . . ~(623) ~~E~
of the N and C termini of the predicted subunit A sequences from Candida and Saccharomyces with subunit A sequences from other species show high similarity, while the spacer domains show identity with no other known protein (andonly limited identity between the Saccharomyces and Candida domains, discussed below). 2) There are no RNA splicing consensus sequences found in the middle of the coding region, and Northern analysis revealed only the full size mRNAwith probes hybridizing to N- and C-terminal and spacer regions, implying that mRNA splicing, aside from that seen at the5'end of the gene, was unlikely. 3) Frameshifts in the predicted spacer domain severely compromised growth at high pH, characteristic of cells lacking a functional vacuolar ATPase. This strongly suggests that a ribosome-skipping mechanism is not responsible for subunit A processing. Furthermore, the Candida gene, when transformed into the tfpl-A8 deletion mutant, fully restored vacuolar ATPase function, suggesting
(356)
(358)
that thepredicted 180-kDa Candida protein might need some modification to be assembled in the place of the endogenous 67-kDa protein in the vacuolar ATPase enzyme complex. 4) The spacerless gene functioned as well as the wild type gene encoding the 119-kDa protein. 5) I n uitro translation of the full length Candida gene produced predominantly proteins with M , values of 51 and 67 and trace amounts of proteins with M , values above 100; in contrast, the spacerless gene gave rise to the single dominant 67-kDa protein and none of the 51-kDa species or species above 100 kDa. 6) We epitopetagged the protein with a shortFlag peptide at theN terminus andanother one with c-myc at the C terminus. I n vitro translated products of the full length tagged gene were immunoprecipitated with antibodies against Flag and c-myc epitopes, pulling down the 67-kDa protein and the proteins above 100 kDa, but not the 51-kDa band. This indicates that the Candida subunit A gene is used to produce a 119-kDa
C. tropicalis VacuolarATPase Subunit A protein from which is derived a 67-kDa protein containing both N and C termini and a 51-kDa protein cleaved from the mid-region. 7) When the full length gene was expressed in bacteria and Western blotted, a 110-kDa band and the 67kDa band,butnot the 51-kDa band, were recognized by antibodies against the tags at both ends. Because the c-myc antibody, recognizingthe C-terminal portionof the full-length precursor, appears to also immunoprecipitateaprotein of approximately 85 kDa, we propose that theN-terminal cleavage is made first, resulting in an antigenically cryptic Nterminal fragment, and an85-kDa C-terminal fragment. This is envisioned to be followed by a second C-terminal cleavage which produces the 51-kDa spacer protein and is coupled to the ligation of the N-terminal portion to theC-terminal part to result in a mature 67-kDa protein which functions as the vacuolar ATPase subunit A. The second cleavage is suggested to be coupled to the ligation step because of the absence of any smaller fragments which might otherwise be expected to be seen. As first suggested by Kane et al. (16), it is probable that this is an autocatalytic event, given its occurrence in vitro, in bacteria, and in yeast. The Spacer Domain-The Candida spacer domain is a protein that would have a predicted size of 51,514 kDa, if it is assumed that only 1of the 2 cysteine residues at either end is retained by the mature protein; its precise size cannot, however, be known until its mature N and C termini have been determined. Comparison of the Candida and Saccharomyces protein spacer domains (Fig. 8 A ) indicates that there are comparatively few regions in which any appreciable identity exists between these two polypeptides. Overall identity in this region between the two proteins is approximately 35.9%; the few regions of identity are located in the regions corresponding to what wouldbe the N andCtermini of the resulting mature spacer proteins. It may be that the portions of the protein which are responsible for the excision process will be found to be located in these regions. Obvious tests of this proposal include the replacement of individual residues within these regions and the construction of deletion mutations at various points in the protein. To date, we have not been successful in placing the insert into other proteins to ask whether excision is possible from other sites, suggesting that thenative structureof the precursor may also be important. As indicated below (in Fig. 8C) the protein sequences flanking the spacer domain are nearly invariant between the Candida and Saccharomyces precursor proteins and separation of spacer excision function from vacuolar ATPase function may not be trivial. Uchida et al. (34) firstnoted an amino acid sequence similarity between the HO nuclease and theSaccharomyces spacer domains. Interestingly, Gimble et al. (18)have recently shown that thespacer-derived protein (which they termedthe !MA1 derived element, or VDE) from the Saccharomyces TFP-1 gene functionsasa site-specific nuclease andappears to mediate precise meiotic insertion of the element into engineered strains that have a TFP-1 gene which lacks the element. The sequence that is cleaved by the VDE-derived protein is composed of the two half-sites which flank the VDE in the TFP-1 gene, as shown in Fig. 8B. Whether the Candidaderived spacer protein is also capable of nuclease activity is an open question.
7381
The Subunit A Protein-Comparison of the mature Candida subunit A-predicted protein sequence (ignoring the spacer region) with the predicted subunit A protein sequences from other species indicates that thereis overall identity with the mature form of the S. cerevisiae, Neurospora, bovine, and carrot predicted protein sequences of 87,72,65, and 60%, respectively, as shown in Fig. 8C. It has been previously noted that the known subunit A protein sequences show identity with the protein sequences for the F1 subunit (approximately 25% (13)) and this is true also for the Candida subunit A protein.Inaddition, the lack of functional effect of the modifications that we have made to theprotein’s C terminus suggest that this region is not involved in interactions with other subunits. The residues in the middle third of each of the various proteins seem to be relatively invariant. Acknowledgments-We thank Drs. Tom H. Stevens and Patricia Kane for sharing data prior to publication and for the gift of the tfplA8 deletion mutant and wild-type, Dr. John C. Loper for the use of the C. tropicalis library, Dr. Linda Parysek for the gift of anti-c-myc monoclonal antibody, and Dr. Karlheinz Altendorffor the gift of bafilomycin A,. REFERENCES 1. Mellman, I., Fuchs, R., and Helenius, A. (1986) Annu. Reu. Biochem. 66, 663-700 2. Rudnick, G. (1986) Annu. Reu. Physiol. 48,403-413 3. Anraku, Y., Umemoto, N., Hirata, R., and Wada, Y . (1989) J. Bioenerg. Biomembr. 2 1 , 589-603 4. Nelson, N. (1989) J. Bioenerg. Biomembr. 21,553-571 5. Stone, D. K., Crider, B. P., Sudhof, T. C., and Xie,X. S. (1989) J. Bioenerg. Biomembr. 21,605-620 6. AI-Awqati, Q. (1986) Annu. Reo. Cell Biol. 2,179-199 7. Forgac, M. (1989) Physiol. Reu. 6 9 , 765-796 8. Arai, H., Berne, M., Terres, G., Terres, H., Puopolo, K., and Forgac, M. (1987) Biochemistry 26,6632-6638 9. Arai, H., Terres, G., Pink, S., and Forgac, M. (1988) J. Biol. Chem. 2 6 3 , 8796-8802 10. Xie, X.-S., and Stone, D. K. (1986) J. Biol. Chem. 261,2492-2495 11. Bowman, B. J., Dschida, W. J., Harris, T., and Bowman, E. J. (1989) J. Biol. Chem. 2 6 4 , 15606-15612 12. Adachi, I., Puopolo, K., Marquez-Sterling, N., Arai, H., and Forgac, M. (1990) J. Biol. Chem. 266,967-973 13. Mandala, S., and Taiz, L. (1986) J. Bwl. Chem. 2 6 1 , 12850-12855 14. Moriyama, Y., and Nelson, N. (1987) J. Biol. Chem. 262,14723-14729 15. Percy, J. M., and Apps, D. K. (1986) Biochem. J. 239.77-81 16. Kane, P. M., Yamashiro, C. T., Wolczyk, D. F., Neff, N., Goebl, M., and Stevens, T. H. (1990) Science 260,651-657 17. Shih, C.-K., Wagner, R., Feinstein, S., Kanik-Ennulat, C., and Neff, N. (1988) Mol. Cell Biol. 8, 3094-3103 18. Gimble, F. S., and Thorner, J. (1992) Nature 357,301-306 19. Zimniak, L., Dittrich, P.,Gogarten, J. P., Kibak, H., and Taiz, L. (1988) J. Biol. Chem. 263,9102-9112 20. Chen, C., Turi, T. G., Sanglard, D., and Loper, J. C. (1987) Biochem. Biophys. Res. Commun. 143,1311-1317 21. Ausubel, F. M., Brent, R., Kingston, R. E. Moore, D. D., Seidman, J. D., and Struhl, K. (1991) Current Protocols hMolecular Biology, John Wiley & Sons, New York 22. Uchida, E., Ohsumi, Y., and Anraku, Y. (1985) J. Biol. Chem. 2 6 0 , 10901095 23. Preston, R. A., Murphy, R. F., and Jones, E. W. (1989) Proc. Natl. Acad. Sci. U. S. A. 8 6 , 7027-7031 24. Wieczorek, H., Weerth, S., Schindlbeck, M., and Klein, U. (1989) J. Biol. Chem. 2 6 4 , 11143-11148 25. Nelson, H., and Nelson, N. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 35033.507
26. Yamashiro, C. T., Kane, P. M.,Wolczyk, D. F.,Preston, R. A., and Stevens, T. H. (1990) Mol. Cell Biol. 10,3737-3749 27. Bowman, E. J., Siehers, A., and Altendorf, K. (1988) Proc. Natl. Acad. Sei. U. S. A. 8 5 , 7972-7976 Bowman, B. J. (1988) J. Biol. Chem. 2 6 3 , 28. Bowman, E. J., Tenney, K., and 1 ~ W A- - I Ann1 29. Mount, S. M. (1982) Nucleic Acids Res. 1 0 , 459-472 30. Langford, C. J . , Klinz, F.-J., Donath, C., and Gallwitz, D. (1984) Cell 36, 645-653 31. Myslinski, E., Segault, V., and Branlant, C. (1990) Science 247,1213-1216 32. Chodosh, L. A., Olesen, J., Hahn, S., Baldwin, A. S., Guarente, L., and Sharp, P. A. (1988) Cell 53,25-35 33. Pan. Y . X.. Xu. d., Strasser, J. E., Howell, M., and Dean, G. E. (1991) FEBS”&-&3,89-92 34. Hirata, R., Obsumi, Y., Nakano, A., Kawasaki, H., Suzuki, K., and Anraku, Y. (1990) J . Biol. Chem. 266,6726-6733 - _ ”
~
~
~~
“”_
