Molecular cloning and tissue distribution of alternatively spliced

Aug 25, 1992 - Tony D. Southall , Selim Terhzaz , Pablo Cabrero , Venkateswara R. ..... Ricardo Luis de Mendon?a , Ewald Beck , Franklin David Rumjane...
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Biochemistry 1992, 31, 7600-7608

7600

Molecular Cloning and Tissue Distribution of Alternatively Spliced mRNAs Encoding Possible Mammalian Homologues of the Yeast Secretory Pathway Calcium Pump+,e Ann-Marie Gunteski-Hamblin,* David M. Clarke,$ and Gary E. Shull’J Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Mail Location 524, Cincinnati, Ohio 45267-0524. and Department of Medicine, University of Toronto, Toronto, Ontario M5S lA8. Canada Received February 12, 1992; Revised Manuscript Received May 1, 1992

ABSTRACT: Rat stomach and testis cDNAs corresponding to two alternatively spliced mRNAs encoding variants of a P-type ion-transport ATPase that closely resembles the yeast secretory pathway Ca2+pump have been isolated and characterized. A partial kidney cDNA was identified previously using an oligonucleotide probe corresponding to part of the sarcoplasmic reticulum Ca2+-ATPase [Gunteski-Hamblin, A,, Greeb, J., & Shull, G. E. (1988) J . B i d . Chem. 263, 15032-150401. In the present study, we first isolated and characterized a stomach cDNA that contains the entire coding sequence. The 919 amino acid enzyme has the same apparent transmembrane organization and contains all of the conserved domains present in other P-type ATPases. Northern blot analyses demonstrate that 3.9- and 5-kilobase mRNAs corresponding to the cDNA were present in all tissues examined, suggesting that the protein it encodes performs a housekeeping function. Rat testis also contained a 3.7-kilobase mRNA that hybridized with a probe from the 5‘ end of the stomach cDNA but did not hybridize with a probe from the 3’ end. Cloning and characterization of cDNAs corresponding to the smaller testis mRNA revealed that it is derived from the same gene but encodes a variant of the enzyme in which the C-terminal residue, Val-919, is replaced by the sequence Phe-9 19-Tyr-Pro-Lys-Ile-923. Similarity comparisons show that the two enzymes are more closely related to the known Ca2+ pumps than to other P-type ATPases. They exhibit 23% amino acid identity with the plasma membrane Ca2+-ATPase, 33% identity with the sarcoplasmic reticulum Ca2+-ATPase,and 50% identity with the yeast secretory pathway Ca2+-ATPase. On the basis of these comparisons, it seems likely that they are mammalian homologues of the yeast secretory pathway Ca2+ pump.

ATP-dependent Ca2+pumps of the P-type family of enzymes play an essential role in the control of intracellular Ca2+ concentrations(Pietrobon et al., 1990). Two distinct classes of mammalian P-type Ca2+pumps have been identified and extensively characterized using biochemical and molecular biological techniques. These are the plasma membrane Ca2+ATPases (PMCA class), which extrude Ca2+from the cell, and the sarco(endo)plasmicreticulum Ca2+-ATPases(SERCA class), which sequester Ca2+ within intracellular storage vesicles. Full-length cDNAs encoding four calmodulinsensitiveplasma membrane Ca2+pumps @hull& Greeb, 1988; Verma et al., 1988; Greeb k Shull, 1989; Strehler et al., 1990) and three sarco(endo)plasmicreticulum Ca2+pumps (MacLennan et al., 1985; Brandl et al., 1986; Burk et al., 1989) that are the products of separate genes have been identified, and additional diversity is generated by alternative ~

~~

t This work was supported by National Institutes of Health Grant

HL41558 (to G.E.S.) and by Medical Research Councilof Canada Grant MT 3399 (to David H. MacLennan), which provided support for D.M.C. e The nucleotide sequences in this paper have been submitted to GenBankunder AccessionNumbersM93017 (Figure2) andM93018 (Figure 6).

* Correspondence should be sent to this author.

t University of Cincinnati College of 5 University of Toronto.

Medicine.

Abbreviations: SR, sarcoplasmic reticulum; ER, endoplasmic reticulum;PMCA, plasma membraneCa2+-ATPase;SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase;SDS, sodium dodecyl sulfate; FITC, fluorescein isothiocyanate; FSBA, 5’-[p-(fluorosulfonyl)benzoyl]adenoATP sine; ClRATP, 7-4-[N-(2-chloroethyl)-N-methylamino]benzylamide kb, kilobases; nts, nucleotides; bp, base pairs.

splicing of exons encoding the C-terminal regions (Strehler et al., 1989; Brandl et al., 1987; Lytton & MacLennan, 1988; Gunteski-Hamblinet al., 1988). By pumping Ca2+outof the cytoplasm, both classes of pumps serve to maintain low resting levelsof cytosolicCa2+,and SERCA pumps have the additional function of maintaining intracellular Ca2+ stores that must be available for release into the cytosol during Ca2+ signaling events. There is evidencefor the existenceof additional mammalian Ca2+ pumps besides those that have already been characterized. ATP-dependent Ca2+uptake activity has been shown in coated vesicles (Blitz et al., 1977), lysosomes (Klempner, 1985), nuclei (Nicotera et al., 1989), and Golgi vesicles of lactating mammary glands (Virk et al., 1985; Watters, 1984; Neville et al., 1981). The coated vesicle Ca2+pump reacts with antibodies directed against the SR Ca2+-ATPase(Blitz et al., 1977) and, therefore, could be a member of the SERCA class of pumps, but its exact identity has not been determined. The Ca2+pump identified in nuclei requires calmodulin, like the PMCA pumps, but has a relatively low sensitivity to vanadate (Nicotera et al., 1989). There is insufficientinformation regarding the biochemical properties of the nuclear membrane pump or of the lysosomal pump to determine whether they might be related to the SERCA or PMCA pumps. The Golgi Ca2+ pump is sensitive to vanadate (Virk et al., 1985), a characteristic of the P-type ATPases, but has other characteristics, such as a different apparent Ca2+ affinity and insensitivity to quercetin, that seem to distinguish it from the PMCA and SERCA pumps (Virk et al., 1985).

0006-2960/92/043 1-7600%03.00/0 0 1992 American Chemical Society

Biochemistry, Vol. 31, No. 33, 1992 7601

Rat Homologues of Yeast Secretory Pathway Ca2+ Pump In a previousstudy (Gunteski-Hamblin et al., 1988),directed at the isolation and characterization of cDNAs encoding Ca2+ATPases of intracellular and plasma membranes, we identified a partial rat kidney cDNA, clone RK9- 11, encoding a protein that exhibited significant amino acid similarity to several known Ca2+ pumps; however, on the basis of the degree of similarity it did not appear to be a member of the SERCA or PMCA families of Ca2+-ATPases. This raised the possibility that it represented a third distinct class of mammalian Ca2+pump. In the present study, we have isolated and characterized cDNAs derived from two alternatively spliced mRNAs that encode variants of the protein which differ in their C-terminal sequences. Amino acid similarity comparisons show that the two enzymes are closely related to a yeast P-type ATPase (Rudolph et al., 1989) that seems to function as a secretory pathway Ca2+pump.

EXPERIMENTAL PROCEDURES Rat Brain, Kidney, Stomach, and Testis cDNA Libraries. The rat brain, kidney, and stomach cDNA libraries used in this study, each consisting of 50 000 colonies, were the same libraries used previously in the isolation of various transport. ATPase cDNAs (Gunteski-Hamblin et al., 1988; Shull et al., 1986;Shull & Lingrel, 1986). The rat testis library, obtained from Stratagene, was prepared using cDNA of greater than 2.5 kb in length and the phage vector Uni-ZAP XR. A total of 70 000 plaques were plated on 26 master plates. Replica filters were prepared and processed basically as described by Maniatis et al. (1982). Isolation and Characterization of cDNAs. Replica filters from brain, kidney, and stomach cDNA libraries were prehybridized overnight at 58 OC in 6X SET, 1X Denhardt’s solution, 0.1% SDS, and 100 pg of denatured salmon sperm DNA/mL [see Maniatis et al. (1982) for the composition of SET and Denhardt’s solution) and then hybridized in thesame solution for 36 h at 58 OC with a 519-bp StyI-NsiI fragment (corresponding to nts 2245-2764 shown in Figure 2) of clone RK9- 11 (Gunteski-Hamblin et al., 1988)that had been labeled with [a-32P]dCTP using the random primer oligolabeling kit from Pharmacia LKB Biotechnology,Inc. Filters were washed three times for 30 min each at 58 OC in 6X SET, 0.1% SDS, and analyzed by autoradiography. cDNAs that gave positive hybridization signals colony purified, and plasmid DNA was isolated by the alkaline lysis procedure (Maniatis et al., 1982). Restriction mapping and hybridization analysis indicated that all of the clones belonged to the same class as clone RK9-11 and identified clone RS10-31 as the longest cDNA. DNA sequence analysis of clone RS10-31 was performed by the chemical cleavage procedure (Maxam & Gilbert, 1980). The rat testis library was screened using two PstI restriction fragments of clone RS10-31 (nts 776-1965 and nts 19662518) under essentially the same conditions as described above. Ten cDNAs that gave positive hybridization signals were plaque purified and the pBluescript phagemid portion of the h Uni-ZAP vector, which contained the cDNA insert, was excised and used to infect XL1 Blue cells as described by the supplier (Stratagene). Restriction mapping and hybridization analysis demonstrated that the testis cDNAs belonged to two closely related classes. DNA sequence analysis was performed by thedideoxy chain termination method (Sanger et al., 1977) using the Pharmacia T7 sequencing kit (Pharmacia LKB Biotechnology, Inc.) and the Sequenase Version 2.0 Kit (U.S. Biochemical). Computer analysis of the DNA sequence was performed using the program of Wernke and Thompson (1989) (DNANALYZE, version 5.1).

Northern Blot Hybridization Analysis. Poly(A)+ RNA was isolated from adult CD rats, and Northern blots were prepared exactly as described previously (Burk et al., 1989). Five micrograms of poly(A)+ RNA, which had been denatured with glyoxal, was run in each lane. Labeling of probes with [ C X - ~ ~ P I ~hybridization CTP, at 42 OC in a solution containing 50% formamide, and washing of filters were also performed exactly as described (Burk et al., 1989). Restriction fragments used as hybridization probes were the following: (i) BstBINsiI fragment (nts 2278-2764) from the C-terminal coding regionof clone RS10-3 1; (ii) ClaI-HpaI fragment (nts 29873595) from the 3’ untranslated sequence of clone RS10-31; (iii) BglI-BspHI fragment (nts 3861-4376) from the 3’ untranslated sequence of testis clone RT7-1; and (iv) Bsu36IDraI fragment (nts 2841-3315 plus 52 nts that seem to have been artifactually derived from an unrelated mRNA) from the 3’untranslated sequenceof clone RT5- 1,which was derived from the testis-specific mRNA.

RESULTS Isolation and Sequence Analysis of cDNAs Encoding a New Member of the P-Type Family of Ion Transport ATPases. In one of our earlier studies, we screened rat brain, kidney, and stomach cDNA libraries using an oligonucleotide probe based on a 23 amino acid sequence from the ATP binding site of the SR Ca2+-ATPase (Gunteski-Hamblin et al., 1988). Among the cDNAs identified was a kidney cDNA, clone RK9-11, that encoded part of a P-type ATPase that exhibited similarity to both the sarcoplasmic reticulum and plasma membrane Ca2+pumps. To isolate a full-length cDNA encoding this pump, we rescreened the original brain, kidney, and stomach libraries using a cDNA probe from clone RK911. One brain, three stomach, and two kidney cDNAs (including clone RK9-11) were identified. The cDNAs were colony purified and examined by restriction endonuclease mapping and Southern blot hybridization analysis. On the basis of the hybridization patterns, it was apparent that all of the cDNAs were derived from a single class of mRNA. Fourofthefivenewclonesisolateddidnotcontainthecomplete open reading frame, but the remaining cDNA, stomach clone RS10-31, had an insert of 3.8 kb, and subsequent sequence analysis showed that it contained the entire coding sequence. In a later phase of this study, we identified testis cDNAs corresponding to both an alternatively spliced testis-specific mRNA that contained a different 3’ end and an mRNA that was apparently identical to the form cloned from the stomach, brain, and kidney libraries. One of the testis cDNAs for the latter mRNA, clone RT7-1, encoded the same protein as the brain, kidney, and stomach cDNAs but contained additional 3’ untranslated sequences that were missing from stomach cDNA RS10-31. The restriction maps for clones RS10-31 and RT7-1 are shown in the upper part of Figure 1, and the nucleotide and deduced amino acid sequences are shown in Figure 2. The bottom part of Figure 1 shows the restriction maps for the cDNAs derived from the testis-specific mRNA, which contains alternative C-terminal coding and 3’ untranslated sequences. The Northern blot analysis used to identify the testis-specific mRNA and the cloning of testis cDNAs for both classes of mRNA will be described in detail in a later section. The composite cDNA sequence for clones RS10-31 and RT7-1 is 4645 nts in length and has a 2757-nt open reading frame. The ATG triplet beginning at nucleotide +1 is the probable translation initiation site since it is in an acceptable context for initiation of translation (Kozak, 1987) and is

7602 Biochemistry, Vol. 31, No. 33, 1992

Gunteski-Hamblin et al.

RS 10-31

RT 7-1

RT 13-1

RT 15-1

FIGURE1: Restriction map for rat stomach and testis cDNAs representing alternatively spliced mRNAs encoding variants of a novel P-type ATPase. Stomach cDNA RS10-31 (nts -178 to 3655) and testis cDNA RT7-1 (nts 2139-4467) are derived from an mRNA expressed in

all tissues. Testis cDNAs RT13-1 (nts 407-3174) and RT15-1 (nts 838-3315) correspond to the testis-specific mRNA with alternative C-terminal coding and 3' untranslated sequences. The cDNAs shown here were used to compile the composite sequences shown in Figures 2 and 6. Open areas represent the coding regions, and dark or hatched areas represent untranslated regions. The arrow indicates the point at which the sequence of the testis-specific transcript diverges from that of the transcript expressed in all tissues. The direction and extent of sequencing are indicated by arrows (lower arrows, coding strand; upper arrows, noncoding strand). preceded by an in-frame stop codon which begins at nt -32. The 178-nt 5' untranslated sequence contains two other ATG triplets, which generate small open reading frames of 10 and 12 codons before encountering in-frame stop codons. One of these small open reading frames (nts -7 to +29) overlaps the 919-codon open reading frame. Because the ATG triplet for this 12-codon open reading frame is in a very poor context for initiation of translation, it seems unlikely that it would interfere with initiation from the ATG triplet at nt +1. However, if it did interfere, then initiation would most likely occur at position +49, thereby eliminating the first 16 amino acids of the predicted translation product. The 1690-nt 3' untranslated sequence terminated with a poly(A) tract that began 19 nts following a consensus polyadenylation signal (Proudfoot & Brownlee, 1976). Two additional potential polyadenylation signals, at nts 3173-3 178 and nts 3994-3999, are present in the 3' untranslated sequence. Northern blot analysis, discussed below, suggests that the first site is utilized in the production of a 3.9-kb mRNA whereas the site at nts 44234429 is used for a 5-kb mRNA. Thededuced primary translation product is 9 19 amino acids in length, has an Mr of 100 500, and contains all of the conserved domains commonly found in P-type ion-transport ATPases. The apparent phosphorylation site (Post & Kume, 1973), Asp-350, occurs in the sequence Cys-Ser-Asp-LysThr-Gly-Thr, which has been found in all eukaryotic P-type ATPases that have been characterized so far. The enzyme also contains sequences resembling those that bind FITC in the Na,K-ATPase (Farley et al., 1984), SR Ca-ATPase (Mitchinson et al., 1982), and plasma membrane Ca-ATPase (Filoteo et al., 1987), as well as sequences resembling the binding site for the ATP analogues FSBA (Ohta et al., 1986) and C l R A T P (Ovchinnikov et al., 1987) in the Na,KATPase (see Figure 8). The regions that bind FITC, FSBA, and ClRATP are thought to form part of the ATP binding site. Hydropathy analysis (Figure 3) indicates that the protein contains up to 10 potential membrane-spanning domains and has a transmembrane organization that is identical to that of

other P-type transport ATPases (Shull & Greeb, 1988). The enzyme has four major hydrophobic domains in the N-terminal half of the protein that correspond to known transmembrane domains in the other ATPases. The C-terminal half of the enzyme contains six hydrophobic domains corresponding to regions that have been proposed as possible membranespanning domains in other transport ATPases. Because the RS 10-31 protein has the same apparent transmembrane organization and contains all of the conserved domains characteristic of the mammalian P-type ATPases, it is clear that is represents a new mammalian member of this family of enzymes. Northern Blot Hybridization Analyses. To determine the mRNA tissue distribution, poly(A)+ RNAs from 12rat tissues were analyzed by Northern blot hybridization using probes from the coding sequence and the 3' untranslated sequence of clone RS10-31 (Figure 4). Both probes detected mRNAs of approximately 3.9 kb and 5 kb in all tissues examined except testis. As described below, the two probes gave different patterns in testis, suggesting the existence of an alternatively spliced mRNA. An additional hybridization signal corresponding to an mRNA of 6 kb was also seen in some tissues. The levels of expression of the 3.9- and 5-kb mRNAs were slightly higher in brain, lung, and stomach than in other tissues but, in general, the mRNA concentrations were remarkably uniform in the various tissues, suggesting that the enzyme performs a housekeeping function. It should be noted that the 3' untranslated sequence probe, consisting of nts 2987-3595, spans the potential polyadenylation signal observed at nts 3 173-3 178. Because the relative intensity of the hybridization signal for the 5-kb mRNA, compared with that of the 3.9-kb mRNA, was greater for the 3' untranslated sequence probe than for the coding sequence probe (compare panels B and C of Figure 4), it is likely that this polyadenylation signal is used for the 3.9-kb mRNA. Consistent with this conclusion, we found that a probe consisting of nts 3861-4376 hybridized only with the 5- and 6-kb mRNAs (Figure 5 ) . The Northern blot data gave no indication that the potential polyadenylation signal at nts 3994-

Rat Homologues of Yeast Secretory Pathway Ca2+Pump

Biochemistry, Vol. 31, No. 33, 1992 7603

TCCCTGCCACCATCCTGCTCGTTCCCTC CGGACGCGGCTMiGCTCCGTGTffiCGCCACCGlGCCGC~GCAGCGCCffiTCGCCCGAffiCGCGTGffiGCGCCCCCGCAGCCCCGCGATGTGGCffilGGCTGACACTAAAGACTTTGlffiCCATCAAC~GTGCffilTTC~TG~A

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MetLysValAlaArgPheGlnLysI 1eProAsnvalGluAsnGluThrMetIleProVal .eulhrSerLysArgAlaSe~l~LeuAlaValSe~luValAlaGlyLeuLe~Gl~laAspLe~GlnAsnG1yLeuAsnLysSe~l~ 10 20 30 40 50 G ~ A G T C A ~ A ~ t G A G C C T T C C A l f f i C T G G ~ T G A G T T T ~ T A ~ ~ A G l ~ A G A T ~ C C A T l A T f f i A A G A A G T A C A l ~ T C T C A G T ~ T ~ T C C C C ~ C A l C A T G C T G C ~ l C T G G C l T C f f i C A G T C A T C A G C G l ~ T 300 AAlGCGTCAGTTl

ValSerhisArgArgAlaPneHisGlyTrpAsffildPneAspI leSerGluAspGluPro.euTrpLysLysTyr:leSe~lnPheLysAsnPro~euIle~tLe~LeuLeuAlaSerAlaval IleSerValLeJMetArgGl nPhe 7c 80 90 100 60 G A 7 G A T G C C G T C f f i T A T C A C T G l f f i C A A l A C T G A l T G A T T t T T C C G T ~ C C ~ T T G T T C A G G A A l A T C G l l C A G A A A A A l C T l l A G A A G A A l ~ A A G T ~ C l T G l G C C A C C A G A A T G C C A ~ G T G l T C G T G A A f f i ~ T l G G 450 AGtA A~~A~pAl~ValkrlleTnrV~lAlalle.euIlevalValTnr~~lAl a P h e V ~ l G l n G l u T ~ r A r g S e r G 1 u ~ y s S e - L e ~ ~ l u G l ~ . e ~ S e r ~ y s L e ~ V a l P r o P r ~ l u C y s H i s C y s V a l A r g G l ~ G l sThr yLysLe~Glu~i 11c 12c 13C 140 15c C T T G C C C G f f i A C ~ ~ ~ C C A G G T G A T A C A G l T T G T C T G l C T G T G G f f i W I ~ A G A G l T C C ~ S C T G A C T ' A C ~ C T T A ~ l C ~ A f f i ~ C G l ~ ~ C ~ T l C C A l t G A T ~ A G T C l A G C T T G A C A G ~ G A ~ A C A A C T C C T T G T l ~ ~ A600 AGGlGACCGCl .euAlaArgAspLtuVal P r f f i l y A s D ~ h r V a 1 C y s L e ~ S e r V a l G l y A s D A r g ~ a l P ~ o A l a A s p ~ e u A ~ ~ ~ e ~ ~ h e G ~ ~ u A l ~ vleAspGluSerSe~LeJlnrGlyGlulhrTnrProCysSer~ys~allnrAla alAsp~euSerI 16C 170 180 190 200 C C T C A A C C f f i C T G t T A C C A A l G ~ A T C l l G C A l C A A G A A G T ~ C A ~ G C ~ ~ T C A l f f i f f i A C T C T G G ~ C A G A T G l G G C ~ A G C A A A ~ T A ~ l G l C A l T G G A A C A G G A G A G A A l T C l G A A l T T G G A G A f f i l C l T l A A G A l G A l G 750 CAffiCA ProGlnProAlaAlaThrAsnGlyAsp~e,Al aSerArgSerAsn1 leAl a ~ n e k t G l y T n ~ L e ~ V a 1 A r g C y s G l y ~ y s A l a ~ ~ s G lIleGlyTnrGlyGlbAsnSerGldPneGlyGluValPhe~sMetMetGlnAla yIleva~ 21c 22C 230 240 250 G A A G A A t C A C C A A I I A A C G C C T C T G C A G A A G A G C A T G G A C C l C ~ G G G C A A G C A G C T G l C C l ~ ~ A C l C C ~ l T f f i T A l A A l A G G l A T C A T C A T G ~ l f f i T T G G C l f f i ~ A C T A ~ ~ G A C A T l C l G ~ l G l l C A C l A T T A900 GTGTAAGl Gl~G1~AlaPro~sThrProle~GlnLysSer((etAspLeJLeJGly~ysGln~euSerPnel~rSerPneGly:leIl~lyl l e 1 IeMetLe~ValGlyTrp.eJLeuGly.ysAsplleLe.GluHetPneTnrl 1eSerValSer 2 60 270 280 290 300 ~ f f i C T G T f f i C T C C A A ~ C C T G A A f f i T C T G C C T A T ' G T ~ T C A C A G T G A C A C l A G C C C ~ G G T G T T A ' G A ~ A A ~ G G l G A A G A A A A G f f i C l A ' T G T A A A W I A A ~ G C C l A ~ T G l ~ G A A A C G C l f f i G C T G C T G T A A T G T G A T l T G T T C1050 AGAl

.euAlaValAlaAlaIlePrffiluGl~Le.ProIlevalValTnr~alTnrLe~A~a~e~Gly~a~MetArgMetval~ys~ysArgAlaI~e~al~ysLys~euPrcIleVa~GldThr~euGlyCysCysAsnValIleCysSer~ 310 329 330 340 ~CTU;PJ\CCCTGACGAAGAAlGAGAlGACTGTlACTCACA~CCTCACTT~AGACGGCCTGCAlGCTGAGG~TA~TffiAGTlGGCTACAATCAGTTTGGTGAAGTGATTGTlGATGGT~TGTlGlTCAlffiAlTCTAlAACCCAGCT 1200

.yslhrG'yThrL~Thr.ysAsffiluHetTnrVa~lnrHislleLedT~~SerAs~ly~e~HisA~aGl~ValTnrGlyValGlylyrAsffil~PneGlyGluValIleVa1AspGlyAspvalValHisGlyPhelyrAsnProAla 360 370 38E 390 400 G T T A G C A G A A T T G ~ G A G G C f f i G C T G l G l G T G ~ C G A T G C T G l C A ~ A G ~ A A C ~ C A C T C ' G A T G f f i A A A G C C A A C T G A A G G A G C C ~ A A l C G C l C l C G C G A l G A A G A l ~ l C l T G A T f f i A C T G C A A C A A G A C l A l A l C A ~1350 CT

ValSerArg!leV~1GluA'aGlyCys~alCysAsnAspAlaVal I leArgAsnAsnr~r~eu~tGly~ysProThrG~uGlyAlaLeuIleAla~euAlaktLysMetGly~euAs~~yLe~Glffil~AspTyrl~eArg.ysAla 41C 42C 430 440 450 ~ T A C C C T T T T I G ~ C T ~ G C A G A A A T t G A T G G t T G n U A C C G A A C A C A G C A ~ A C A G A C C A G A G A T T T G T T T l A T t ~ l G C T T A l G A G C ~ T G A ~ T A A G T A T T G T A C T A C G T A C A A C A G C ~ f f i G C A C A1500 C~G GluTyrProPhckrkrGluGln.yrTrpktAlaYalLysCys~alrl~sArgTn.GlnGlnAspArgPrffi1uIleCysPneMetLysG~~yAlalyrGl~Gl nVa1 IleLysTyrCysTnrThrTyrAsnSerLysGlyGlnTnrLe. 460 47C 480 490 500 G C A C T T A C C C A G U G C f f i f f i A G A ~ T G T A C C ~ C A A G A ~ G C C C A G A ~ f f i G C T C f f i C A G G A C T C C G A G ~ ~ T T G C A C T f f i C G T C T ~ ~ C C G G A C C l f f i f f i C A G C T G A C C C T C C l T G G C C l f f i l A f f i A A l C A ~ ~ C C C T C C 1650 ~GAACl

AlaLe~Th.Gl~GlffilnArgAspLeulyffilffilnGluLysAlaGl~etGlySerAlaGlyLe~Argval.eJA1aLeuAlaSerGlyProAsp~euGlyGlnLe~lnrLeuLeuGly~eu~alGlyIlclleAspProPr~ArgThr

510 52C 530 540 550 ~ T G T G P 9 9 I ; A A C C C G T C A C A A C A C l C A T ~ G C C l C A f f i A G T C T C C A T C A A A A T G A l C A C A G G A G A C l C l C A G G A G A C ~ G C A A T l G C ~ T C G C T A G l C G T C l G ~ T T G l A ~ C l ~ A C ~ C A C A G l C T G T G l C T ~ ~ G A A1800 GlAGAl Glyval y s G l ,AhVal ThrThr-euI 1eA1aSerGlyValSer1 le.ysMet 11eTnrGlyAspSerGlqG1 uThrA1 a: leA1 a1 leA1 aSerArg,e.GlyLe.TyrSerysThrSeffilnSerVal SerGlyGluGIuValAsp 560 570 580 590 600 A C A A T G C A A G T C t W ; C A C C ' l l C A ~ A G A l A G T G C C ~ A f f i ~ G C A G T A T ~ ~ A C A G A G C A A G C C ~ A A G A C A C A ~ ~ A T G ~ A T T A ~ C A A G T C l C l A C ~ A G A A C G f f i l C f f i T ~ G l A G C C A T G A C A f f i A G A T G G f f i l1950 ~TGAlGCAGTG T n ~ t G l u v a l G l n H i s L c ~ S e r G l n l l e ~Pro.ysVaIAlaValPne3rArgAlaSerProArg~is-~sMet al .ys1 l e 1 leLysSerLeuGlnLysAsffilySerVa'ValAl~eflhrGlyAs~GlyValAsnAspAlava1 610 620 63C 640 650 G C T C T G A A ~ C T ~ A T A T T G G A G l l G C A A l G G G C C A ~ C l f f i C A C A G A T G ~ G C ~ G A f f i C l G C G G A C A T G A T C ~ f f i T f f i A T G A l G A T ~ C C ~ C l A T A A l G T C T G C A A T A G A A G A ~ l ~ G G C A T ~ A T2100 AAlAACAll~ AlaLeu~ysAlaAlaAspIleGlyValAl~tGlyClnThrGlylnrAspValCysLysGluAlaAlaAs~et Ile.euvalAspAspAspPheGlnThrIl~etSerAlaIlffiluGluGly~ysGlyI1eTyrArnAsnIleLys 660 670 680 690 700 ~ T T T T G T T A G A ~ C A A C T G A G C A C G A G T A T A G C A G C A ~ A A C ~ A A T C T C A ~ f f i C T A C G ~ ~ T G A A C T T T C C l A A C C C l C T C A A T G C A A l G C A G A T T ~ G l f f i A l C A A l A T T A l A A T G G A T ~ C C C C C f f i C l C A G f f i C C2250 TT~A AsnPheVal ArgPheG1nLeuSerThrSerI1 eAlaA1 OLeJThrLeUI leSerLedAl aThrLeuktAsnPheProAsnProLeuAsnAlaWetG1 n I le-euTrpl leAsnI l e 11eMetAspGlyProProAlaGlnSerLeuGly 710 720 7 30 740 750 G T A G A G C C A G T ~ T A A A G A T G T C A T T C G ~ C C C C C l C f f i A A C l ~ A f f i A ~ C A ~ T l G A C ~ C T T G A l C C ~ A ~ T A C T l G T ~ C A T C A A l A A l C A T T G l C l G l ~ A C ~ ~ G T T T G T C T T C T f f i C G A G A2400 GC~CGAGAC ValGluProValAspjsAspVa11 leArg~ysProProArgAsnTrp~sAspSerlleLHIThrLysASn~~Jlle~euLyslleLeJValSerSerlleI leIleValCysGlyTnr.cuPheVa1PneTrpArgGlu~e~ArgAsp 760 770 78C 790 800 A A T G T C A T A A C A U C C G A G A C A C ~ C A A ~ G A C T T ~ C A C ~ G C ~ ~ C G T G T ~ T T T T G A T A ~ G T T C A A T G C A C ~ G A G T T C C A G A ~ C T C A G A C ~ A A G T C T G T G ~ G A G A T ~ G G A C ~ C T G C A G T A A ~ A A G A T G ~ ~ C T G2550 CTA~GCGG~C~~GGA

AsnralIleThrRoArgAspTnrTn~t~nrPne~nrCysPhe~a~P~ePheAspC(ctPneAsnAla~eJSerSerArgSeffilnlnr~ysServalPh~luIleGlyLeJCysSerAsnLysktPneCyslyrAlava~.euGly

810 820 830 840 850 T C C A l C A T G G ~ t A G T T G C ~ G ~ A ~ C T A C ~ C C C G C C T C T G C A G A A G G T T T T T C A G A C C G A ~ G C C T C A G T A ~ C l ~ l C T G T l G T l l C ~ T f f i G l C T C A C T T C A T C A G l G T G C A ~ G l G T C T G A A A ~ T A l ~ G A 2700 Affi~GAAAGG SerI 1eHetGlyGlnLeJLeuVa~I leT~rPheProPro~euGln~s~alPheG'nlnffil~SerLe~SerIleLeuAs~~e~~euPheLeu~euGlyLeulnrSerSe~ValCysIleVa1SerGluIleI 1eLysLysValGluArg 860 870 880 890 900 A G C A U ; G A A A A G A C T C A G A A G A A C A C T A C T l C A A C A C C A T C G T C T ~ C ~ T G A A G l A l G A T G C A T A l l G C A T T ~ l T l l ~ l C A T T l G C A A A C l A f f i A A T l G C A G l C T G G G A A l C A T T T A G A A G G G C A A G T T C A A G A G G A l A A l G A A G A T ~2850

SerArgGluLysThrGlnLysAsnlnrThrSerTnrProSerSerPne.euGl~Va1 910

3000 3150 3300 3450 3600 3750 3900 4050 4200 4350 4467

FIGURE 2: Composite nucleotide sequence of rat stomach cDNA RS10-31 and rat testis cDNA RT7-1 and deduced amino acid sequence of the protein. The nucleotides are numbered to the right of the sequence with nt +1 corresponding to the probable translation initiation site. Amino acids are numbered below the sequence. Two polyadenylation signals that seem to be functional (see text) are underlined. The phosphorylation site (Asp-350) is underlined and labeled. Stomach cDNA RS10-31 begins at nt -178 and terminates at nt 3655 while testis cDNA RT7-1 begins at nt 2138 and terminates with the poly(A) tract.

0

100

200

300

400

500

600

700 800

900

Amino Acids FIGURE 3: Hydropathy profile of the protein encoded by stomach cDNA RS10-31. Hydropathy values were determined by the procedure of Kyte and Doolittle (1982) using a window of 9 amino acids. Possible membrane-spanning regions are darkened.

3999 is utilized. In testis, the 3' untranslated sequence probe detected low levels of the 3.9- and 5-kb mRNAs, but the major transcript

detected with the coding sequence probe was a 3.7-kb mRNA that did not appear to be expressed in other tissues. The 3' untranslated sequence probe from clone RS10-31 did not hybridize with the 3.7-kb mRNA. This raised the possibility that the 3.7-kb mRNA is an alternatively spliced transcript encoding a testis-specific variant of the protein. S1 nuclease protection analysis of rat testis and stomach poly(A)+RNAs (data not shown) indicated that the sequence of the testisspecific mRNA diverged from that of the stomach mRNA just prior to the stop codon. This suggested that the testisspecific mRNA encoded a protein with an alternative C-terminus. Isolation and Characterization of Rat Testis cDNAs Encoding the Putative Housekeeping and Testis-Specific Forms of the Protein. To isolate cDNAs for the alternative transcript in testis, a rat testis library was screening with a cDNA probe from the coding sequence of clone RS10-31. Ten phage plaques were identified and pulled from the master plates. The pBluescript phagemids containing the cDNA

Gunteski-Hamblin et al.

7604 Biochemistry, Vol. 31, No. 33, 1992

2701 1 AGCAGGGAAAAGACTCAGAAGAACACTACTTCAACACCATCGTCT~TCTT~TTTTAC SerArgG1u LysThrG1nLysAsnThrThrSerThrProSerSerPheLeuG1uPheTyr 910 920 CCCAAGATTTAATCATCTTAGCTGTCCAGAACTCCnTTGTTACTT~CTACTAATTGA ProLys I 1e 923

AGATTGACGTGTCCAAAGCCTCAGGTTTGGCTCTGGCAGAGGAGTGGACAGCAGCTGATT GAGATACAAGCCCATCTGGAGACAGGACTGCCACTGACAGAAGACATGGGCTGTGTCTAA ATCCAATCTGGGACCCGGCCGCATGGCTGCCTTCACCTGTTGTAATTCTTCATACAGCAT CTTGGCACCACCTCTCCGTGGATCTTCACTATCTACGTAAAACAACCC~GACAGCGTT

TCCAGACACTGCATCAGTTTGCCCTTCCCCACATTCCATCATC~GCCAC~GACAAT

* *

CAGTGTGAAAACTGGTCACTTTTCTTTTCTGGTTTCATAAATGTCTTCCATACACTGAAG

GTCCCTTTTGACTTTATTCACCCAAAGTTACTGATTTTTCCTTAAAATAAGATCTCTCC

B

-4.4

CATAGTATGCTACCTGACCTTAAACAAGTACATTGTCTAGGATCCGAGTCTATGAAAA~ AJGACACTAAACACC-poly (A) 3315

C

-

4.4

FIGURE 4: Northern blot hybridization analysis using probes from clone RS10-31. Poly(A)+ RNA from the indicated rat tissues was analyzed using probes from the 3'untranslated region (3' probe) and coding sequence (5' probe) of stomach cDNA RS 10-31 as described under Experimental Procedures. The probe and autoradiographic exposure times were (A) 5' probe, 26 h; (B) 5' probe, 66 h; (C) 3' probe, 6.5 day. The positions and sizes (in kilobases) of the RNA markers are shown on the right.

-7.5 -4.4

-

2.4

FIGURE 5: Northern blot hybridization analysis using a probe from the 3' end of testis cDNA RT7- 1. Poly(A)+RNA from the indicated rat tissues was analyzed using a probe from the extreme 3' end of clone RT7-1 as described under Experimental Procedures. The autoradiographic exposure time was 8 days. The positions and sizes (in kilobases) of the RNA markers are shown on the right.

inserts were excised from the X Uni-ZAP vector, colony purified, and analyzed by restriction endonuclease mapping, Southernblot hybridization, and DNA sequence analysis. One cDNA lacked the C-terminal coding sequence and, therefore, could not be classified. Three partial cDNAs, includingclone RT7-1, encoded an enzyme with the same C-terminus as that encoded by stomach cDNA RS 10-31. As mentioned above, Northern blot hybridization analysis using a cDNA probe from the 3' end of RT7- 1 (Figure 5 ) demonstrated that it was derived from the 5-kb mRNA seen in all tissues. DNA sequence analysis of the remaining six clones demonstrated that they were partial cDNAs derived from the alternatively spliced testis mRNA. The restriction maps for two of these clones, RT13-1 and RT15-1, which were used to determine the sequence of the testis-specific transcript, are shown in the lower part of Figure 1. The sequence corre-

tract

FIGURE 6: Nucleotide sequence from the 3' end of the testis-specific cDNA. The nucleotide sequences of the transcript expressed in all tissues and the testis-specific transcript are identical up to nt 2754. The sequenceshown begins at nt 2701 just before the point at which the two transcriptsdiverge (indicated by avertical arrow). The protein encoded by the testis-specific mRNA has an alternative 5 amino acids in place of the last amino acid of the form expressed in all tissues (see Figure2). Amino acids are numbered below the sequence. Asterisks are shown above the nucleotides that were followed by extensive poly(A) tracts in several cDNAs. A single cDNA, clone RTI 5-1, had a poly(A) tract following nt 3315. Sequencesthat may serve as polyadenylation signals are underlined.

sponding to the alternative 3' coding and untranslated sequence was also determined for the four additional clones. The nucleotide and deduced amino acid sequences immediately preceding and following the point of divergence are shown in Figure 6. The point of divergence between the two cDNA classes occurred between codons 9 18 and 9 19 (arrow in Figure 6). The alternatively spliced testis mRNA encodes a protein in which the last residue of the putative housekeeping form of the enzyme, Va1919, is replaced by the short amino acid sequence Phe-Tyr-Pro-Lys-Ile. The coding sequence was followed by a 3' untranslated sequence that differed slightly in length among the six cDNAs examined. Two apparent polyadenylation sites, separated by about 140 nts, were observed. Several cDNAs had extensive poly(A) tracts following nts 3173 or 3176, and one cDNA had a poly(A) tract at nt 3315. An additional cDNA contained sequences that extended well beyond nt 33 15, but Northern blot analysis with a probe derived from the extra sequence failed to identify an mRNA species (data not shown) suggesting that the extra sequence was a cloning artifact. Just upstream of each of the polyadenylation sites are sequences which are similar, but not identical, to the consensuspolyadenylation signal (underlined in Figure 6). To confirm that the cDNAs with the alternative 3' end correspond to the 3.7-kb mRNA in testis, Northern blot hybridization was performed using a probe from the unique 3' untranslated sequence. As shown in Figure 7, the only mRNA identified was the 3.7-kb testis mRNA, which was not detected in any other tissue. Thus, the 3.7-kb mRNA, which encodes a variant of the enzyme that has an alternative C-terminus, appears to be expressed only in testis. The Novel P-Type ATPase Exhibits High Sequence Similarity to the Yeast Secretory Pathway ea2+ Pump and Other Ca*+-ATPases. Similarity comparisons demonstrate that the amino acid sequence of the P-type ATPase identified in this study most closely resembles those of a yeast secretory pathway Ca2+-ATPase(Rudolph et al., 1989)and the SERCA

Biochemistry, Vol. 31, No. 33, 1992 7605

Rat Homologues of Yeast Secretory Pathway Ca2+Pump

In aligning the RS10-31 protein with PRM1 it was not necessary to introduce extended gaps, whereas extensive gapping was necessary in order to maintain the alignment with SERCA2aand PMCA1a. Theoverallsimilarityin amino acid sequence and general organization strongly suggests that the rat and yeast enzymes are functional homologues. 7.5

DISCUSSION

4.4

In an earlier study (Gunteski-Hamblin et al., 1988), we identified a partial cDNA encoding a P-type ATPase that was more closely related to the SERCA and PMCA Ca2+ pumps than to other mammalian P-type ATPases. In order to determine the complete amino acid sequence of the protein, and to obtain information regarding its tissue distribution,we carried out additional cDNA cloning studies and Northern blot hybridization analyses. A stomach cDNA containing the complete coding sequence was isolated and characterized, and Northern blot analyses demonstrated that the corresponding mRNAs are expressed in all tissues, suggesting that the enzyme plays a housekeeping role. Interestingly, a testisspecific mRNA was also identified that seemed to represent an alternatively spliced transcript of the same gene. By isolating and characterizing the corresponding cDNA, we determined that the testis-specific mRNA encodes a variant of the protein that contains an alternative C-terminus. Similarity comparisons reveal that the enzyme encoded by clone RS10-3 1 is more closely related to several known Ca2+ pumps than to other P-type ATPases. It exhibits 23% amino acid identity with the plasma membrane Ca2+-ATPase,33% identity with the SR Ca2+-ATPase, and 50% identity with PMRl, the yeast secretory pathway Ca2+-ATPase(Rudolph et al., 1989). Similarity between the RS10-31 protein and PMRl occurs throughout the two polypeptide chains. Extensive amino acid identity occurs not only in the catalytic domains, which are conserved among all P-type ATPases, but also in the hydrophobic sequences corresponding to the 10 putative transmembrane domains identified in other pumps [discussed in MacLennan et al. (1985), Shull and Greeb (1988), and Clarke et al. (1990)J. These domains are poorly conserved between pumps that differ in their ion specificities. If all ten potential transmembrane domains are considered, the amino acid identity between the two proteins is 63%, considerably greater than the overall identity of 50%. The identity within the predicted transmembrane domains corresponding to those that contain the Ca2+binding residues in the SR Ca2+-ATPaseis greater than 80%. These transmembranedomains are also relatively conserved between the RS 103 1protein and SERCA2a (44%identity in the transmembrane domains vs 33% overall) or PMCAla (38% identity in the transmembrane domains vs 23% overall). On the basis of these comparisons, it seems likely that the protein encoded by clone RSl O-3 1 is a Ca2+-transportingATPase and that it is the mammalian homologue of the yeast secretory pathway Ca2+pump. If this proves to be the case, then it represents a third distinct class of mammalian Ca2+pump and would most likely function in maintaining Ca2+concentrationswithin one or more compartments of the secretory pathway. PMRl was classified as a Ca2+ pump on the basis of its similarity to known Ca2+-ATPasesand the observed effects of Ca2+on the phenotype of pmrl mutants (Rudolph et al., 1989). Null mutants of the PMRl gene grow poorly on solid medium containing 1 pM Ca2+ but grow well on medium containing 20 mM Ca2+. They also exhibit hypersensitivity to EGTA and the anti-calmodulin drug trifluoperizine, and these sensitivitiesare overcomeby addition of Ca2+. Compared

-2.4

FIGURE7: Northern blot hybridization analysis using probes corresponding to the testis-specific mRNA. Poly(A)+ RNA from the indicated rat tissues was analyzed using a cDNA probe from the 3' untranslated region of testis cDNA RTS-I as described under Experimental Procedures. The autoradiographicexposure time was 8 days. The positions and sizes (in kilobases) of the RNA markers are shown on the right. and PMCA classes of mammalian ea2+pumps. Amino acid similarity comparisons between the protein encoded by clone RS10-31, the yeast secretory pathway Ca2+pump (termed PMRl), SERCA2a, and PMCAla are shown in Figure 8. In pairwise comparisons, it exhibits 23% amino acid identity to PMCAla, 33% identity to SERCA2a, and 50% identity to the secretory pathway Ca2+ pump. It should be noted that the amino acid identity between SERCA2a and PMCAla, which are members of two different classes of mammalian Ca2+ pumps, is only 27%. Thus, the degree of similarity observed between the different enzymes is consistent with the possibility that the novel ATPase represents a third distinct class of mammalian Ca2+ pump. The highest degree of amino acid similarity among the four pumps occurs in the major cytoplasmic regions between the second and third transmembrane domains and between the fourth and fifth transmembrane domains. These conserved regions are involved in basic catalytic activities of the P-type ATPases such as ATP binding, phosphorylation, and conformational transitions of the enzyme. Predicted transmembrane domains 4 and 6 also exhibit a relatively high degree of similarity among the four pumps. These putative transmembrane domains contain 4 of the 6 residues (Glu-309, Glu770, Asn-795, Thr-798, Asp-799, and Glu-907 in SERCA2a) that seem to form the high-affinity Ca2+binding sites in the SR Ca2+-ATPases(Clarke et al., 1989). The two additional residues known to be involved in Ca2+binding in the SR Ca2+ pump are in putative transmembranedomains 5 and 8. Three of the six residues, Glu-308, Asn-738, and Asp-742 of the protein encoded by RS 10-31, are conserved in all four pumps. Glu-770 and Thr-798 of SERCA2a correspond to Ala and Met residues, respectively, in the other pumps. Glu-907 of SERCA2a correspondsto Asp residues in the RSl O-3 1protein and PMRl and to Gln in PMCAla. The RS10-31 protein and PMRl exhibit a striking degree of amino acid similarity throughout their lengths. Extended regions of perfect identity between the rat and yeast proteins occur in the catalytic domains and in predicted transmembrane domains 2, 4, 5, 6, and 8. The amino acid identity within these predicted transmembrane domains is 84%, much greater than the overall identity. A significant degree of identity also occurs in the remaining transmembrane domains, in the N-terminal and C-terminal regions, and in the short sequences connecting some of the transmembrane domains.

7606 Biochemistry, Vol. 31, No. 33, 1992

Gunteski-Hamblin et al.

RS10-31

98

PMRl

112

SERCA2a

78

PMCAla

130

RS10-31

194

PMRl

215

SERCAZa

186

PMCAla

249

RS10-31

262

PMRl

283

SERCA2a

253

WAla

369

PMCA1a

.... SVSLAVAAIPEGLPIIVTVTLALGVLRHAKRMIVRRLPSVETLGSVNV .. . ... .. ..... ............ ... ICSDKTGTLTSNHMTVSKLWCL~SMSNK-. . . ........... . . .... .. .. ... .. ...... ........... . ., b E F i E i i i K V I S L i C I A W 1I N IGHFNDPVHGGSW--------IRGAIYVFKIAVALAVAAIPEGLPAVITT~~LGTR~KKNAIVRSLPSVETLGCTSVICSDKTGTLTTN~SVC~FILoKVDGDTC . . . . . ..... .. *. . . . .......... . ... INEKHYKKVP AVQIGKAGLLMSAiTVii L V L Y FVIDTFWVQKRPWLAECTPIY IQYFVKFFIIGVTVLVVAVPEGLPLAVTISLAYSVKKHfKONNL~RHiDAC~~NATAICSDKTGT~NRMTVVQAY

RS10-31

------VTGVGYNQFGEVIVDGDVVHGFY

FWl

_-_--_ LNVLSLDKNKKTKNSNGNLKNY

SERCA2a

SLNEFT1icST;APIiivHKoDKPiKCHQY

WAla

------EPEAIPPNILSYLVTGISVNCAYTSK--------------------------

RS10-31

PMCAla

IDPPRTGVKEAVTTLIASGVSIKMITGDSQETAIAI 580 . . ... . . .... .. .. .. . .... .. . . . . ...... ,. 614 T V Y V k i F E R I LEYSTSY LKSKGKKiEKiiEA~KATINECANSHASEGLRVFGFAKLT~SDS~T~LT----EDLIKD----------LTFTGLIGMNDPPRPNVKFAIEQ~L~VHIIHITGDSENTAVNi . ... . .. .. . *. .... ..... .. .. 6 3 4 K M F V K G A P E G V I D R ~ ~ H I R V G S T K V P M T A G V K ~ K I M S V I R E ~ ~ ~ S ~ ~ D T ~ ~ C - - - - - - - ~ ~ ~ T H D N P L R R E E H H L K DETNLTFVGCVGMLDPPRIE~ASS~KLCRQA~IRVIHITGONKGTAVAI SANFIKY . ... . ILI;K~FKILSAN~EAKVFRPRDRD~IVKTVIEPHASEGLRT-------IC~FRDFPAGEPEPEWD---NENDVV . . ... .. ... .. . . .. . ... .. *. 7 1 8 RIFSKGASEI

RS10-31

ASRLGLYS-------KTSQSVSGEEVDTMEVQHL---SQIVPKVAVFYRASPRHK-----MKI

RS10-31 PMRl SERCAZa

. ... .. .. . ,.. 106-----------------. OKLGKDLSLV~~IVIGMICLVGI

DLLGKQLSFYSFGI I G I IMLVGWLLG------------------KO

I LEHFTISVSLAVAAIPEGLPIVVTVTLALGVMRHVKKRAITLGCCNVICSDKTGTLTKNEHTVTHILTSDGLHAE-..............e. ........a ,

RSWLEHFQI

3

4

395 377

501

Phos Slte

IRME------------------YPFSSEQKWVKCV-HRTQQDRPE . .. .. .. . . .. . LTED~RETLTIGNLCNNASFSQEHAI----FLGNPTDVALLEQ~NFEHPDIRNTVQKVQ~------------------LPFNSKRK~TKIL---NPV~NKC NPAVSRIVEAGCVCNDAVIRNNT------LMGKPTEGALIALAHKHGLOGLWDY

,.. .

374

....

. . . ..

475 496

D G L V E L A T I C A L k iALDYkEAKGVY EKVbEAfiTiiTCiVEbiNVFDTELKGLSiI ~RANACNSVIKQUKKEFTLE~~RDRKSMSVYCTPNKPSRTWS 509

ILPPEKEGG~PRHVGN-KTECALLGFLLDLKRDYQDVRNEIPEEALYKVYTFNSVRKMTVL---KNSM;SF-

596

ICFMKGAY EQVIKYCTTY-NSKG-QTLALTQQQRDLYQQEKA~SAGLRV-------LALASGP-------DLGQ-----------LTLLGLVGI *

PMRl SERCA2a

FITC S i t e

PMCAla

IKSLQKNGSVVAMTGDGVNDAVALKAADIGVPJ4GQTGTDVCKEAAOMILVDDDFQTIHSAI~~GKGIYN 6 9 7 . .. . .. . . . ............... .. .... ... .. . .... .. ........ , ~ K Q I ~ I P V I D - - - - - P K L S V L ~ ~ D K L ~ E M S D D Q ~ - - - A N V I D H ~ N I F A ~ T P E H K - - - - - L N ~ V ~ L R K R G D V V A M T G D G V N D A P A L K L S D I G V S f f i R I G T O V A K E A S D M V L T D D D F S T I L T A I E ~ G K G7I3F3N . . . .. .......... ... . .. ... .. . . . . .. . .. . ... ... 7 5 4 C R i I G I FGQEED---ViAKAFTiRiFoELNPSAQ---RDACLNARCFARVEPSHK-----S~iVEF~iSFDE ITAMTGDGVNDAPALKMEIGIPJ4G-SGTAVAKTASE~LADDNFSTIVAAVEEGRAIYN ... .... .. ... .. , ... AGTDVAKEASDI .... ... . I..L T..D D.N F T S i V ~ ~ R N V ~ O8 5 0 ATKC61 L H P G E D F L C L E G K D F N R R I R N E K G E I E Q ~ R I O K i W ~ ~ L R ~ L A ~ S ~ ~ T DiDSTVSEQRQVVAVTGDGTNDGPALKKADVGFAMG1 ~HTLVKGl

RS10-31

NIKNFVRFOLSTSIAALTLISLATLMNFPNPLNAJ4QILWINI IMDGPPAOSLGVEPVDKDVIRKPPRNWKDSILTKNLILKILVSSIIIVCGTLFVFWREL-------------------------------

e...

PMRl

9

SERCA2a

FSBA/ClRATP S i t e s

798 ..N I ..Q N...... ... . . . .............. ............... . .... . ... . . . . . F L T F ( I L S T S V A A L S L V A L S T A F K L P N P L N A J 4 Q I L W C IIVGTVY IFVKEHA-----------------------------a35 . .. .. .. .. . . ..... . tiMkQF I i Y LI~SNVGEVVCiF~TAALG~~EAiIPVOLLWVNLVTDGLPATALGFNPPDLDIMNKPPRNPKEPLISGW~FFRY~AIGCVVGAA~VG~~FI~DGGPRVSFYQLSHFLQCKEDNPDFEGVD 886 *

PMRl

*

SERCA2a

*

.. . . .. ... *

PMCAla

S ~ S K ~ L O ~ ~ ~ T V N V V ~ V I V A F T G A C ~ T Q D S P L M V ~ L W V N L I M D T L A S L A ~ A l ~ ~ P T E S L L L R K ~ Y G h N K P L I S R T ~ K N ~ ~ G H A F Y O L V V V F T L L F A G ~ K F F D I D S G R N A - - - - - - - - - - - - - - - - - - -9-6 2 5 6 7

RS10-31

RDNVITPRDTTMTFTCFVFFDMFNALSSRSQTKSVF-E IGLCSNKMFCYAVLGSIffiOLLVIY FPPLQKVFQTESLSILOLLFLLGLTSSVCIVSEI IKKVERSREKTQKNTT-STPSSFLEV

PMCAla

919 . ................... ... . ... .... ... .. . . . .. . . . . *. ... *. . . .. E~GKVTARDTTMTFTCFVFFDMFNALAC~HNTKSIF-EI G F F T N K M F N Y A V G L ~ L L ~ ~ ~ CIPFFOSI A I YFKTEKLGISDI LLLLLISSSVFIVDEL-R~L-MTR-K--KNEED~~--YFSN~ 950 . . CAIFE SPY P M ~ ~ L S V L ~ T I E H C N A L N S L S E N Q ~ L L R H P P ~ E N I W L V G S I C ~ S M S L H F ~ I L Y V E - ~ ~ P L I ~ ~ I T P ~ N V T O W ~ ~ K ILE S L P ~ I ~ ~ T L ~ F ~ A ~ N Y L ~ P A9 9I 7 .. ... . P L H A P P S E H Y ~ I V ~ N T ~ ~ L M Q L ~ k E I N A ~ K I H G E R N V F E ~ I F N k A I F C T I V L G TI VF VOV~OGI -I - G ~ P ~ S C S E i i i E Q W ~ W S I F ~ ~ T L L ~ O L i S T I P T ~ ~ L ~ F L ~ E A G H G ~ Q K E E I P E E1092 ELAEDVEE

PMCAla

IOHAERELRRGQILWFRGLNRIQT~DVVNAFQSGGSI~ALRRQPSIASQHHDVTNVSTPTHVVFSSSTASTPVGYPSGECIS

*

PMRl

9 . .

SERCA2a

8

*

9

10

1176

FIGURE8: Amino acid sinklarity comparison. The deduced amino acid sequence for clone RS10-31 is compared with those of the yeast secretory pathway Ca2+ pump, termed PMRl (Rudolph et al., 1989), the cardiac SR Ca2+-ATPase(SERCA2a) (MacLennan et al., 1985), and the plasma membrane Ca2+-ATPase isoform la (PMCAla) (Shull & Greeb, 1988). Gaps (indicated by dashes) were introduced to maintain the alignment. Dots above amino acids of PMR1, SERCA2a, or PMCAl indicate identity to the corresponding residue in the protein encoded by clone RS10-31. Hydrophobic regions which may represent transmembrane domains [identified by the procedure of Eisenberg et al. (1984)l are underlined and numbered. The phosphorylation domain (Phos Site), FITC binding site (Farley et al., 1984; Mitchinson et al., 1982; Filoteo et al., 1987), and the region which binds FSBA (Ohta et al., 1986) and ClRATP (Ovchinnikov et al., 1987) in the Na,KATPase are underlined. Amino acid numbers are shown on the right.

with wild-type cells, pmr 1 mutants have been shown to secrete much higher levels of heterologous proteins such as bovine prochymosin,bovine growth hormone, and a variant of human urinary plasminogen activator (Smith et al., 1985). Outer chain glycosylation is also defective in pmrl mutants; invertase from pmrl strains contains core polysaccharidebut lacks the outer mannose chains (Rudolph et al., 1989), which are added to the core polysaccharide in the Golgi. In their original

study describing the cloning of PMR1, Rudolph et al. (1989) suggested that the aberrant secretion and glycosylation seen in pmrl mutants could result from a failure to sequester Ca2+, with subsequent alterations in cytosolic Ca2+ concentrations or, alternatively, that PMRl might function directly within a compartment of the secretory pathway. Using immunocytochemical and subcellular fractionation techniques, Antebi and Fink have recently shown that PMRl is located in

Rat Homologues of Yeast Secretory Pathway Ca2+ Pump a Golgi-like organelle, confirming that PMRl functions in a compartment of the secretory pathwaye2 A number of investigators have identified a Ca2+pump in Golgi membranes of rat (West, 1981; Virk et al., 1985) and mouse (Watters, 1984;Neville et al., 1981) mammary glands. The enzyme is sensitive to vanadate (Virk et al., 1985), indicating that it is a P-type ATPase. Activity of the pump appears to be at least partly dependent on the existence of a pH gradient across the Golgi membrane (acid interior) (Virk et al., 1985), suggesting that it might transport Ca2+ in exchange for H+ as in the case of the plasma membrane Ca2+ATPase (Niggli et al., 1982; Smallwood et al., 1983). The protein encoded by clone RS10-3 1,which seems to be expressed in all tissues, is a likely candidate for this pump. If the RS 103 1protein and the testis-specificvariant are Golgi Ca2+pumps, it is possible that they are not restricted to this compartment. Fasolato et al. (1991) have identified a large acidic intracellular Ca2+ pool in PC12 cells that is insensitive to thapsigargin, an inhibitor of the SERCA class of Ca2+ pumps (Lyttonet al., 1991). InPC12cells, thispoolconsistsprimarily of secretory granules, but other acidic organelles, including lysosomes, endosomes, and Golgi vesicles might contribute to the pool. Immunocytochemical studies will be needed to determine whether the pumps cloned in our study are located in the Golgi and, if so, whether they might also be present in other organelles of the secretory pathway. The testis-specific isoform of the enzyme has a C-terminus in which the last residue of the RS10-31 protein is replaced by an alternative 5-amino acid sequence. Alternative splicing of exons encoding C-terminal sequences is a common occurrence among Ca2+-ATPasegenes (Strehler et al., 1989;Brandl et al., 1987; Lytton & MacLennan, 1988; Gunteski-Hamblin et al., 1988). It has been suggested that the alternative Ctermini of the SERCA pumps might function as sorting signals (Burk et al., 1989; Lytton & MacLennan, 1988) but, so far, there have been no rigorous experimental tests of this hypothesis. In the case of the PMCA pumps, alternative splicing is known to alter regulatory domains near the Cterminus (Strehler et al., 1989). It will be of interest to determine which cell type(s) in testis express the alternative 923 amino acid isoform. We are currently performing in situ hybridization analysis to address this question. A likely possibility is the Sertoli cell, which is involved in forming the blood testis barrier and serves as the major secretory cell of the seminiferous epithelium (Bardin et al., 1988; Dym, 1977). Using the procedures described by Maruyama and MacLennan (1988), we have made a number of attempts to determine directly whether the RS10-31 protein is a Ca2+ pump, but so far, these experiments have given negative results (data not shown). In our first set of experiments, we transfected COS- 1 cells with constructs containing the wild-type coding sequence but were unable to detect increased Ca2+ uptake in microsomes. Additional experiments were performed using constructs in which the C-terminal coding sequence for the RS10-31 protein had been replaced by that of SERCA3, which resembles an ER retention signal (Burk et al., 1989). We hoped that this approach would allow retention of the protein in the ER and that this would allow an increased expression of Ca2+uptake activity; however, no increase in Ca2+ uptake was detected. The reason for the lack of Ca2+pump activity in these experiments is unclear. Because the appropriate antibodies are not yet available, we have been unable to determine whether the protein is being Gerald R. Fink, personal communication.

Biochemistry, Vol. 31, No. 33, 1992 7607

expressed in our transfection studies. It is conceivable that a second subunit is required for activity, although all of the other Ca2+ pumps that have been identified function as a single polypeptide. A more likely possibility is that the conditions used in this study, which are appropriate for measurement of the activity of the SERCA pumps, are not appropriate for detecting activity of the secretory pathway pumpIn conclusion, we have identified and characterized rat cDNAs encoding possible homologues of the yeast secretory pathway Ca2+pump and have determined their mRNA tissue distribution. Questions concerning the membrane location of these pumps, their biological roles, and whether they do, in fact, function as Ca2+-transportingATPases remain to be determined. To address the latter question, which is central to our understanding of the biological role of these enzymes, it may be necessary to obtain information about the Ca2+ uptake activity of the compartment in which these proteins normally reside, as this should enable the development of a more appropriate assay system. The recent study of Fasolato et al. (1991) may have some relevance in this regard. The acidic Ca2+pool that they identified is quite stable; it does not appear to release CaZ+at a significant rate and the rate of Ca2+uptake into the pool is very low. On the basis of these observations, they suggested that it may represent a dead-end Ca2+pool that does not contribute to short-term fluctuations in cytosolic Ca2+levels that occur during signaling events. If the RS10-31 protein is the pump which serves this pool, then measurement of its activity may require the use of procedures which deplete intravesicular Ca2+prior to conducting Ca2+ uptake assays.

ACKNOWLEDGMENT We thank David MacLennan for useful discussions, Greg Wernke for help with the computer analyses, Jennifer Schroeder for typing the manuscript and preparing the illustrations, and Dr. Randal J. Kaufman, Genetics Institute, Boston, for the gift of the expressionvectors pMT2 and ~91023(B). We also thank Gerald R. Fink and Adam Antebi for sharing the results of their studies on the subcellular localization of PMRl prior to publication.

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