Isolation, Characterization, and in Vitro Expression of a cDNA That ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 266,No , 28, Issue of’ October 5, pp. 18792-18796,1991 Printed in U.S.A.

0 1991 by The American Society for Biochemistry and Molecular Biology, Inc

Isolation, Characterization, andin Vitro Expression of a cDNA That Encodes the Kidney Isoenzymeof the Mitochondrial Glutaminase* (Received for publication, February 14,1991)

Richard A. Shapiro, Lynn Farrell, Maithreyan Srinivasan, and Norman P. CurthoysS From the Department of Biochemistry, Colorado State University, Fort Collins, Colorado 80523

A cDNA that encodes the kidney isoenzyme of the mitochondrial glutaminase (pGA) was generated by recombination of two cDNAs that were isolated from a random-primedrat brain Xgtll library. pGA encodes 674 amino acids which includes an N-terminal sequence of 16 residues that should form an amphipathic helix, typical of a mitochondrial targeting sequence. Residues 73-90 correspondto the N-terminal sequence of the more abundant65-kDa glutaminase peptide. In vitro transcription and translation of pGA yields a 72kDa peptide that is immunoprecipitatedwith glutaminase-specific antibodies. Incubation of the glutaminase precursor with isolated mitochondria yields the 68and 66-kDa peptides that are characteristic of the mature glutaminase. Thus, the two mature glutaminase peptides are synthesized from a single precursor. The complete 3’ nontranslated region of the GA mRNA was characterized by sequencing a GA cDNA (pGA12) that was isolated from an oligo(dT)-primed ratkidney XgtlO library. This segment contains numerous AUrich regions, four potential stem-loop structures, and a 48 base pair repeat of CA dinucleotides. Such domains may contribute to the increased stability of the GAmRNA that occurs in response to metabolic acidosis.

Mitochondrial glutaminase catalyzes the initial reaction in the primary pathway for the renal catabolism of glutamine (1). This activity is a key regulator of the increased renal ammoniagenesis that occurs in response to metabolic acidosis. During acute acidosis, increased renal metabolism of glutamine results primarily from an increased availability of substrate (2, 3) and the rapid removal of the products of the mitochondrial glutaminase and glutamate dehydrogenase reactions (4, 5). However, during chronic acidosis, the acute adaptations are partially compensated (6), and the arterial plasma glutamine concentration is decreased compared to normal (7). In the rat, thecontinued extraction and metabolism of glutamine is accomplished by induction of the mitochondrial glutaminase (8, 9), glutamate dehydrogenase (lo), and the cytosolic phosphoenolpyruvate carboxykinase (11). The increase in glutaminase results from an increased rate of synthesis that correlates with an increased stability of the

* This work was supported in partby National Institutesof Health Grant DK-37124. The costs of publication of this articlewere defrayed in part by the payment of page charges. This article must therefore he hereby marked “aduertisement” inaccordancewith 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in thispaperhas been submitted totheGenBank‘‘“/EMBLDataBankwith accession number(s) M65150. $ T o whom correspondence and reprint requests should be addressed. Tel: 303-491-5566.

GA’ mRNA (12, 13). The kidney type of glutaminase isoenzyme is also expressed in brain and intestine (14). The active form of the mitochondrial glutaminase is a heterotetramer that is composed of 68- and 65-kDa peptides ina 1:3 ratio, respectively (15, 16). Previous studies have established that the two peptides are structurally and immunologically related. Pulse-chase experiments performed using renal proximal tubular epithelial cells (17) and HTC hepatoma cells (18)indicate that thetwo peptides are derived from a 72-kDa precursor. The precursor is initially processed to a 71-kDa intermediate that apparently gives rise to both of the mature subunits. The 65-kDa peptide is generated more rapidly than the 68-kDa peptide, suggesting that the latter may be the product of a covalent modification. Identical results were observed by incubating isolated rat liver mitochondria with the glutaminase precursor that was produced by in uitro translation of acidotic rat renal poly(A+)RNA (17). However, none of these experiments definitively establish that the 68and 65-kDa peptides are derived from the initial translate of a single mRNA. Rat kidney contains two GA mRNAs; a more abundant 6.0kb mRNA and a less abundant 3.4-kbmRNA (19). After separation by size fractionation, either mRNA can be translated in uitro to yield the 72-kDa precursor (19). The levels of both mRNAs are increased in response to metabolic acidosis. An initial glutaminase cDNA was isolated by screening a rat brain Xgtll library with glutaminase-specific antibodies (20). This cDNA contains 1040 bp that encode the C-terminal half of the glutaminase and 62 bp of the 3’ nontranslated region. It was subcloned as two EcoRI fragments to yield the plasmids pGAl and pGA2. The objective of this study was to isolate additional GA cDNA that could be used to deduce the remainder of the glutaminase sequence and toidentify potential sequences that might contribute to its altered stability. A recombinant plasmid that contains the entire coding sequence was used to establish that the two peptides characteristic of the mature glutaminase are derived from a single mRNA. MATERIALS ANDMETHODS

Male Sprague-Dawley rats weighing 200-250 g were obtained from The Charles River Laboratory and maintained on Purina RatChow. [CY-’~P]~CTP (specific activity 3000 Ci/mmol) andnitrocellulose were obtained from Du Pont-New England Nuclear. Restriction enzymes were purchased from Boehringer Mannheim Corp. RNasin was obtained from Promega. T 7 polymerase and Sequenase were products of United States Biochemical Corp. Cell-free protein synthesis was carriedoutusing a rabbit reticulocyte lysatethat was prepared according to the method of Pelham and Jackson (21). An oligo(dT)primed rat kidney X g t l O cDNA library was obtained from Dr. K. Lynch (22). The random-primed rat brain cortexXgtll cDNA library (1.1 X IO6 independent recombinants) was obtained from Clontech Laboratories, Inc. All other biochemicals were obtained from Sigma.

’

The abbreviationsusedare: GA, glutaminase;pBS-SK(-), pBluescriptII-SK(-); kb, kilohase(s); bp, base pair(s).

18792

Renal Mitochondrial GlutaminasecDNA

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EcoRI Pstl Hindlll

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18793

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FIG. 1. Isolation and recombination of various GA cDNAs. The two EcoRI fragmentsof the initialGA cDNA weresubcloned to yield theplasmids pGAl and pGA2(20). pGA12 wasisolated by using pGAl to screen an oligo(dT)-primed rat kidney X g t l O cDNA library. pGA104 and pGA117 were isolated by using the 5’ AluI fragment of pGA2 and pGA104, respectively, t o screena random-primedratbrain Xgtll cDNA library. A cDNA that contains the entire coding sequence of the glutaminase (pGA) was obtained by recombination of pGA117 and pGA104 through a common BglII site.

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cDNA was determined using double-stranded plasmidDNA and the dideoxynucleotide chain termination reaction (26). Sequencing reactions were performed using the Sequenase Version 2.0 kit. Regions with high GC content were resequenced using the dITP labeling mix supplied with the Sequenase kit and by performing the termination reaction for 5 min a t 50 “C. Amino Acid Sequence-Approximately2 pg of glutaminase was immunoprecipitated from a Triton X-100 extract of freshly isolated brain mitochondria (27) and subjected to 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (28).The separated peptideswere electroblotted to a Problott membrane (AppliedBiosystems, Inc.) and stained with Ponceau S. The 65-kDa glutaminase peptide was excised and subjected to 18 cycles of Edman degradation using an Applied Biosystems model 473A gas-phase protein Sequencer (Macromolecular Resources, Colorado State University). I n Vitro Synthesis andProcessing-pGA was transcribed using T 7 polymerase (17,29) and translated in a rabbit reticulocyte lysate (30). I n vitro processing was carried out using isolated rat liver mitochondria as described previously (17). RESULTSANDDISCUSSION

“

“

FIG. 2. Restriction maps and sequencing strategies for pGA and pGA12. The length, position of various restriction sites, and location of initiation and termination codons and polyadenylation signals (PA) within the two cDNAs are drawn to scale. The arrows denote the direction and the length of individual sequencing reactions. Isolation of GA cDNA-The cDNA libraries were screened using nitrocellulcse plaque-lifts(23). Either Escherichia coli C600 or Y1090 cells were infected with 7.5 X lo5 plaque-forming units and plated on five agar dishes (20 X 20 cm). The lifts were hybridized with pGAl (20) or with the restriction fragments described under “Results.” The cDNA probes were purified by low melting temperature agarose gel electrophoresis and labeled (10’ dpmlpg) by random priming (24). Positive plaques were purified by rescreening. The cDNA inserts of the purified XDNA were subcloned into pBS-SK(-) (Stratagene) and characterized by restriction analysis. The DNA isolations, restriction analysis, subcloning, and subsequent recombinationswere performed using standard procedures (25). Nucleotide Sequence Analysis-The nucleotide sequence of pGA

The more abundant GA mRNA, which is 6.0 kb in length, is likely to have a long 3’ -nontranslated region. Initial attempts to screen the oligo(dT)-primed rat kidney cDNA library with pGAl yielded a number of positive clones, the longest of which was termed pGA12 (Fig. 1).However, none of the isolated cDNAs containeda coding sequence that extended 5’ of the initial GA cDNA. Therefore, the 5’-AluI fragment (220 bp) of pGA2wasused to screen a randomprimed rat brain Xgtll cDNA library (Fig. 1).This procedure yielded XGA104 which contains a 2.7-kb insert. Restriction of XGA104 with EcoRI yielded two fragments, a 1.1-kb cDNA that hybridized specifically with pGA2 and a 1.6-kb fragment that hybridized to pGA1. The cDNA insert was isolated by partial digestion with EcoRI, subcloned into pBS-SK(-), and characterized by restriction analysis. The 5’-AluI fragment (510 bp) of pGA104 wasused as aprobe to rescreen the Xgtll library. This screening yielded XGA117 which contains a 1.5kb insert that hybridized to pGA2, but not to pGA1. The insert was subcloned into pBS-SK(-) in the same orientation as pGA104. A XhoI-BgUI fragment of pGA117 was subcloned into pGA104 plasmid that had been restricted with XhoI and

18794

Renal Mitochondrial Glutaminase cDNA

Renal Mitochondrial Glutaminase

cDNA

18795

sequence around the initial Met fits the consensus sequence for an initiation codon (31). In addition, the initial 16 amino acids have the properties characteristic of an N-terminal targeting sequence (32). This sequence is rich in hydrophobic and positively charged amino acidresidues. It contains a R + A 11 singleacidic amino acidresiduewhichis atypical, but is occasionally found inmitochondrial targeting sequences (33). This sequence has a strong propensity to form an a-helix S 3 10 U L (34). When plotted as a helical wheel (Fig. 4), the residues form an amphipathic a-helix that closely resemblesthe helical structure produced by the N-terminal targeting sequence of '+ L other mitochondrial protein precursors (33, 35, 36). FIG.4. a-Helical wheel diagram of the putative N-terminal The N-terminal sequence of the 65-kDa peptide of the brain mitochondrial targeting sequence. The initial 16 amino acid glutaminase was determined by automated Edman degradaresidues of the deduced sequence of the mitochondrial glutaminase tion. Fifteen of the eighteen determined residues are identical are plotted. with positions 73-90 of the deduced amino acid sequence (Fig. 3). The 3 residues in the deduced sequence that differ from the determined sequence are Ser or Thr residues which are recoveredin low yields in the Edman analysis. Thus, the observed identity confirms the validity of the isolated GA cDNAs and establishes the site of proteolytic cleavage that yields the 65-kDa formof the glutaminase (Mr= 66,576). 72The deducedsequence of the kidneyisoenzyme of the 68 mitochondrial glutaminase exhibits no significant homology ,65 to any of the sequences contained in GenBank other than that deduced from a partial cDNA of the liver glutaminase (37). The liver GA cDNA encodes the C-terminal 219 amino acids of a 58-kDa peptide. The initial 150 residues of this sequence exhibit 75% identity with residues 502-651 of the kidney glutaminase. Both of the rat mitochondrial glutaminases lack the highly conserved sequences found the in TrpGtype glutaminase domains (38) and theCys-His-Asp catalytic I triad (39) that are characteristic of various amidotransferases. Thus, the two mitochondrial glutaminase isoenzymes apparently evolved from a common precursor other than the glutaminase subunit of bacterial amidotransferase. Hydropathy analysis (40) of the glutaminase sequence indicates that theprotein lacks a membrane-spanning domain. FIG.5. In vitro processing of the precursor of the mitochon- Thus, glutaminase is not likely to be an integral membrane drial glutaminase synthesized by in vitro transcription and protein. This is consistent with previous submitochondrial translation of pGA. Labeled glutaminase precursor was synthesized fractionation studies which indicate that glutaminase is only by in uitro translation (lane I ) and either immunoprecipitated with loosely associated with the inner surface of the mitochondrial glutaminase antibodies (lane 2 ) or incubated with isolated rat liver mitochondria (lam3 ) .The samples were analyzed by sodium dodecyl inner membrane (41,42). Induction of the rat renal mitochondrial glutaminase in sulfate-polyacrylamide gel electrophoresis and visualized by fluorography. The sizes of the resulting peptides are indicated in kilodaltons. response to acidosis occurs without increasing the rate of transcription of the GA gene (12, 19). The pH-responsive adaptation observedinLLC-PK-F'cells results from an BglII. The resulting plasmid (pGA) contained 3.2 kb ofGA increased stability of the GA mRNA (13). The 3' nontranscDNA. lated region of the GA mRNA contains numerous AU-rich pGA was sequenced according to the strategy outlined in Fig. 2. The region that corresponds to pGAl and pGA2 was sequences (Fig.3). Such sequences have been foundin the 3' sequenced primarily in one direction. The sequence deter- nontranslated regions of the mRNAs of the cytoplasmic phosmined forthis region is identical with that previously reported phoenolpyruvate carboxykinase (43) and of various cytokines with two exceptions. The base located at position 1438 of and proto-oncogenes(44-46). The latter mRNAs exhibit short pGA is a T instead of a C. In eithercase, the resulting codon half-lives and altered stability in response to various stimuli. encodes an Asn residue. The base located at position 1584 of The incorporation of an AU-rich sequencehas been shownto pGA is alsoa T instead of C. This alterationwould introduce increase the turnover of stable mRNA (46). A protein factor a Leu in place of a Pro residue at position 509 of the amino that specifically binds to an AUUUA sequence has been acid sequence. For the remainder of pGA, both strandsof the identified (47). The binding of this protein may target specific cDNA were sequenced.In order to characterize the remainder mRNAs for rapid degradation. The 3' nontranslated region of the 3' nontranslated region, pGA12 was also sequenced of the GA mRNA contains seven AUUUA domains. In addi(Fig. 2). Again, the regions that were identical with pGA were tion, this segment also contains four inverted repeats that sequenced primarily in one direction. However, all regions could form stable stem-loop structures and a stretch of24 repeated CA dinucleotides.Experiments are planned to deterunique to pGA12 were sequenced in both directions. The combined sequenceobtained from pGA and pGA12 is mine if the various AU-rich segments or potential secondary shown in Fig.3. The GA cDNA contains an open reading structures within the GA mRNA impart altered stability and frame that encodes 674 amino acids (Mr = 74,003). The to identify proteins in renal cell extracts that may interact

i

18796

Mitochondrial Glutaminase Renal

with the potential regulatory elements. The 3' nontranslatedregionof the GA mRNA also contains two AAUAAA polyadenylation signals (Fig. 3). The more 5' AAUAAA sequence initiate the processing that in the formation ofthe 3.4-kb GA mRNA. Whenthe 3' EcoRI fragment of pGA12 wasused as a probe forNorthern analysis, it hybridized uniquely to the 6.0-kb GA mRNA (19). Since both the 3.4- and 6 . 0 4 , GA m ~ encode ~ the~ 7 2 -s k ~

cDNA

15. Godfrey, S., Kuhlenschmidt, and T., Curthoys, N. P. (1977) J. Biol. Chem. 2 5 2 , 1927-1931 16. Shapiro, R. A., Haser, W. G., and Curthoys, N. P. (1987) Biochem. J. 2 4 2 , 743-747 17. Perera, S., Chen, T. C., and Curthoys, N. P. (1990) J. Biol. Chem. 265,17764-17770 18. Perera, S. Y., Voith, D. M., and Curthoys, N. P. (1991) Biochem. J. 273,265-270 19. ~ Hwang, J.-J., Perera, s.9 Shapiro, R.A., and Curthoys, N. p. (1991) Biochemistry 3 0 , in press glutaminase precursor(19)9the two mRNAs may be produced 20. Banner, C., Hwang, J.-J., Shapiro, R. A., Wenthold, R. J., Lampel, by use of alternative polyadenylationsites within a common K. A., Nakatami, Y., Thomas, J. W., Huie, D., and Curthoys, N. P. (1988) Mol. Bruin Res. 3 , 247-254 initial transcript. pGAwaslinearized with BamHI, transcribed with T7 21. Pelham, H. R.B., and Jackson, R. J. (1976) Eur. J . Biochem. 6 7 , 247-256 'Ymerase, and in a rabbit lysate*This 22. Burnham, C. E., Hawelu-Johnson,L.,C. Frank, B. M., and procedure yields a 72-kDa peptidethat is immunoprecipitated Lynch, K. R. (1987) Proc. Nutl. Acud. Sci. U. S. A. 8 4 , 5605with glutaminase-specificantibodies (Fig. 5). Isolated rat 5609 renal cortical mitochondria are contaminated with a protease 23. Manniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular that preventstheiruse for in vitro processing studies (17). C1oning:A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, NY Of the 72-kDa precursor with rat liver 24, Feinberg, A. p., and Vogelstein, B. (1984) Ami, Biochem. 137, mitochondria yields the 68- and 65-kDa peptides that are 266-267 characteristic of the mature glutaminase. The apparentratio 25. Davis, L.G., Dibner, M.D., and Battey, J. F. (1986) Basic of the twopeptides is similar to that observedfor the native Methods in Molecular Biology, Elsevier Science Publishing Co., glutaminase (16). This findingfurther establishes that pGA New York 26. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Nutl. encodes the entire sequenceof the mitochondrial glutaminase Acud. Sci. U. S. A. 74,5463-5467 and that the two Peptides are derived froma common Precur- 27. Haser, W. G., Shapiro, R. A., and Curthoys, N. P. (1985) Biochem. sor. This system willbe used to characterize the processing J. 229,399-408 reactions and the functional significanceof the unusual struc- 28. Laemmli, u. K. (1970) Nature 227,680-685 29. Krieg, P. A,, and Melton, D.A. (1987) Methods Enzymol. 1 5 5 , tural properties of the glutaminase. 397-415 30. Jagus, R. (1987) Methods Enzymol. 1 5 2 , 267-276 REFERENCES 31. Kozak, M. (1984) Nucleic Acids Res. 1 2 , 857-872 1. Tannen, R. L., and Sastrasinh, S. (1984) Kidney Znt. 2 5 , 1-10 32. Hartl, F.-U., and Neupert, W. (1990) Science 2 4 7 , 930-938 2. Schrock, H., Chu, C. J., and Goldstein, L. (1980) Biochem. J. 33. Dieckmann, C.L., Koerner, T. J., and Tzagoloff, A. (1984) J. 188,557-560 Biol. Chem. 259,4722-4731 3. Hughey, R. P., Rankin, B. 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