A GTP:AMP Phosphotransferase, Adk2p, in Saccharomyces cerevisiae
2005; Elsevier BV; Volume: 280; Issue: 19 Linguagem: Inglês
10.1074/jbc.m500847200
ISSN1083-351X
AutoresYajuan Gu, Donna M. Gordon, Boominathan Amutha, Debkumar Pain,
Tópico(s)Fungal and yeast genetics research
ResumoAdenylate kinases participate in maintaining the homeostasis of cellular nucleotides. Depending on the yeast strains, the GTP:AMP phosphotransferase is encoded by the nuclear gene ADK2 with or without a single base pair deletion/insertion near the 3′ end of the open reading frame, and the corresponding protein exists as either Adk2p (short) or Adk2p (long) in the mitochondrial matrix. These two forms are identical except that the three C-terminal residues of Adk2p (short) are changed in Adk2p (long), and the latter contains an additional nine amino acids at the C terminus of the protein. The short form of Adk2p has so far been considered to be inactive (Schricker, R., Magdolen, V., Strobel, G., Bogengruber, E., Breitenbach, M., and Bandlow, W. (1995) J. Biol. Chem. 270, 31103–31110). Using purified proteins, we show that at the physiological temperature for yeast growth (30 °C), both short and long forms of Adk2p are enzymatically active. However, in contrast to the short form, Adk2p (long) is quite resistant to thermal inactivation, urea denaturation, and degradation by trypsin. Unfolding of the long form by high concentrations of urea greatly stimulated its import into isolated mitochondria. Using an integration-based gene-swapping approach, we found that regardless of the yeast strains used, the steady state levels of endogenous Adk2p (long) in mitochondria were 5–10-fold lower compared with those of Adk2p (short). Together, these results suggest that the modified C-terminal domain in Adk2p (long) is not essential for enzyme activity, but it contributes to and strengthens protein folding and/or stability and is particularly important for maintaining enzyme activity under stress conditions. Adenylate kinases participate in maintaining the homeostasis of cellular nucleotides. Depending on the yeast strains, the GTP:AMP phosphotransferase is encoded by the nuclear gene ADK2 with or without a single base pair deletion/insertion near the 3′ end of the open reading frame, and the corresponding protein exists as either Adk2p (short) or Adk2p (long) in the mitochondrial matrix. These two forms are identical except that the three C-terminal residues of Adk2p (short) are changed in Adk2p (long), and the latter contains an additional nine amino acids at the C terminus of the protein. The short form of Adk2p has so far been considered to be inactive (Schricker, R., Magdolen, V., Strobel, G., Bogengruber, E., Breitenbach, M., and Bandlow, W. (1995) J. Biol. Chem. 270, 31103–31110). Using purified proteins, we show that at the physiological temperature for yeast growth (30 °C), both short and long forms of Adk2p are enzymatically active. However, in contrast to the short form, Adk2p (long) is quite resistant to thermal inactivation, urea denaturation, and degradation by trypsin. Unfolding of the long form by high concentrations of urea greatly stimulated its import into isolated mitochondria. Using an integration-based gene-swapping approach, we found that regardless of the yeast strains used, the steady state levels of endogenous Adk2p (long) in mitochondria were 5–10-fold lower compared with those of Adk2p (short). Together, these results suggest that the modified C-terminal domain in Adk2p (long) is not essential for enzyme activity, but it contributes to and strengthens protein folding and/or stability and is particularly important for maintaining enzyme activity under stress conditions. Cellular homeostasis of nucleotides is essential for numerous important biological processes. Among several enzymes that participate in maintaining cellular levels of nucleotides are adenylate kinases, which catalyze the reaction ATP (or GTP) + AMP ⇔ ADP (or GDP) + ADP (1Atkinson D.E. Cellular Energy Metabolism and Its Regulation. Academic Press, New York1977: 85-107Crossref Google Scholar, 2Tomasselli A.G. Schirmer R.H. Noda L.H. Eur. J. Biochem. 1979; 93: 257-262Crossref PubMed Scopus (61) Google Scholar, 3Khoo J.C. Russell P.J. Biochim. Biophys. Acta. 1972; 268: 98-101Crossref PubMed Scopus (79) Google Scholar). These kinases provide ADP for oxidative phosphorylation (4Noda L.H. Boyer P.D. The Enzymes. 8. Academic Press, Orlando, FL1973: 279-305Google Scholar) and also control the intracellular AMP level, which serves as a highly sensitive sensor of cellular energy status. Under stress conditions, increased levels of AMP trigger the AMP-activated protein kinase cascade to switch on catabolic pathways that generate ATP while switching off anabolic processes that consume ATP (5Kemp B.E. Mitchelhill K.I. Stapleton D. Michell B.J. Chen Z.-P. Witters L.A. Trends Biochem. Sci. 1999; 24: 22-25Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar, 6Hardie D.G. J. Cell Sci. 2004; 117: 5479-5487Crossref PubMed Scopus (960) Google Scholar). Adenylate kinases therefore occupy a central position in many cellular functions under normal as well as stress conditions. Although ATP-specific adenylate kinases from different organisms have been extensively studied, not much is known about the GTP-specific isoforms. Adenylate kinase in Escherichia coli is indispensable for growth (7Cronan Jr., J.E. Godson G.N. Mol. Gen. Genet. 1972; 116: 199-210Crossref PubMed Scopus (29) Google Scholar). In mammalian cells, at least three (AK1, AK2, and AK3) isoforms of adenylate kinase exist (8Schultz G.E. Cold Spring Harbor Symp. Quant. Biol. 1987; 52: 429-439Crossref PubMed Scopus (62) Google Scholar, 9Yamada M. Sugahara M. Hishitani Y. Nobumoto M. Nakazawa A. J. Mol. Biol. 1998; 280: 551-558Crossref PubMed Scopus (5) Google Scholar, 10Noma T. Fujisawa K. Yamashiro Y. Shinohara M. Nakazawa A. Gondo T. Ishihara T. Yoshinobu K. Biochem. J. 2001; 358: 225-232Crossref PubMed Scopus (64) Google Scholar, 11Tomasselli A.G. Noda L.H. Eur. J. Biochem. 1980; 103: 481-491Crossref PubMed Scopus (66) Google Scholar). AK1 and AK2 use Mg2+ATP as the high energy phosphate donor, whereas AK3 is a GTP:AMP phosphotransferase and uses GTP instead of ATP as a phosphoryl donor. AK1 and AK3 are localized to the cytosol and mitochondrial matrix, respectively. In contrast, AK2 resides in the cytosol and mitochondrial intermembrane space (2Tomasselli A.G. Schirmer R.H. Noda L.H. Eur. J. Biochem. 1979; 93: 257-262Crossref PubMed Scopus (61) Google Scholar, 3Khoo J.C. Russell P.J. Biochim. Biophys. Acta. 1972; 268: 98-101Crossref PubMed Scopus (79) Google Scholar, 4Noda L.H. Boyer P.D. The Enzymes. 8. Academic Press, Orlando, FL1973: 279-305Google Scholar, 12Nobumoto M. Yamada M. Song S. Inouye S. Nakazawa A. J. Biochem. (Tokyo). 1998; 123: 128-135Crossref PubMed Scopus (54) Google Scholar, 13Tanabe T. Yamada M. Noma T. Kajii T. Nakazawa A. J. Biochem. (Tokyo). 1993; 113: 200-207Crossref PubMed Scopus (85) Google Scholar, 14Watanabe K. Kubo S. Eur. J. Biochem. 1982; 123: 587-592Crossref PubMed Scopus (21) Google Scholar). Basic cellular and mitochondrial functions are highly conserved from yeast to human (15Foury F. Kucej M. Curr. Opin. Chem. Biol. 2002; 6: 106-111Crossref PubMed Scopus (39) Google Scholar), and in agreement with this notion, the yeast Saccharomyces cerevisiae also contains three isoforms of adenylate kinase localized to the cytosol and/or mitochondria. As in the mammalian isoforms, all three yeast isozymes are nuclearly encoded. In yeast, the major isoform Adk1p/Aky2p (∼24 kDa) is homologous to mammalian AK2 and displays a dual localization; most (>90%) of the protein is localized to the cytoplasm with a small portion in the mitochondrial intermembrane space (16Magdolen V. Oechsner U. Bandlow W. Curr. Genet. 1987; 12: 405-411Crossref PubMed Scopus (20) Google Scholar, 17Magdolen V. Schricker R. Strobel G. Germaier H. Bandlow W. FEBS Lett. 1992; 299: 267-272Crossref PubMed Scopus (21) Google Scholar, 18Bandlow W. Strobel G. Schricker R. Biochem. J. 1998; 329: 359-367Crossref PubMed Scopus (22) Google Scholar, 19Angermayr M. Strobel G. Zollner A. Korber D. Bandlow W. FEBS Lett. 2001; 508: 427-432Crossref PubMed Scopus (8) Google Scholar, 20Strobel G. Zollner A. Angermayr M. Bandlow W. Mol. Biol. Cell. 2002; 13: 1439-1448Crossref PubMed Scopus (46) Google Scholar). The yeast equivalent of mammalian AK1 is the cytosolic Ura6p (21Schricker R. Magdolen V. Kaniak A. Wolf K. Bandlow W. Gene (Amst.). 1992; 122: 111-118Crossref PubMed Scopus (32) Google Scholar). This yeast protein was identified as a multicopy suppressor of the respiratory-deficient phenotype of adk1 mutants. The gene ADK2/AKY3 (originally called PAK3) was isolated from the S. cerevisiae strain DL1 by a polymerase chain reaction using degenerate oligonucleotide primers corresponding to conserved regions of the nucleoside monophosphate kinases (22Schricker R. Magdolen V. Bandlow W. Mol. Gen. Genet. 1992; 233: 363-371Crossref PubMed Scopus (17) Google Scholar, 23Schricker R. Magdolen V. Strobel G. Bogengruber E. Breitenbach M. Bandlow W. J. Biol. Chem. 1995; 270: 31103-31110Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The corresponding protein Adk2p (225 amino acids) showed 45% identity with both E. coli adenylate kinase and human GTP:AMP phosphotransferase (AK3) and was localized to the matrix side of the mitochondrial inner membrane. A disruption of ADK2 did not produce any detectable phenotype. Moreover, plasmid-borne ADK2 did not restore growth of a temperature-sensitive adk1 E. coli mutant at the non-permissive temperature (39 °C). Based on these results, it was concluded that the DL1 version of ADK2 codes for a non-functional yeast protein (22Schricker R. Magdolen V. Bandlow W. Mol. Gen. Genet. 1992; 233: 363-371Crossref PubMed Scopus (17) Google Scholar, 23Schricker R. Magdolen V. Strobel G. Bogengruber E. Breitenbach M. Bandlow W. J. Biol. Chem. 1995; 270: 31103-31110Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). These studies, however, could not rule out the possibility that the DL1 version of Adk2p was not inherently inactive; rather it was inactivated at 39 °C (which is significantly higher than the usual yeast growth temperature of 30 °C) and thus failed to complement the adk1 E. coli mutant at the non-permissive temperature. Interestingly, random mutagenesis of non-functional ADK2 led to the identification of functional alleles as judged by complementation of the growth phenotype of the adk1 E. coli mutant and GTP:AMP phosphotransferase activity. In each case, the gain of activity was accompanied by a +1 frameshift mutation near the 3′ end of the coding region of ADK2, causing a change in three C-terminal residues and the addition of nine amino acids at the C terminus of Adk2p (Adk2p (long); see Fig. 1A). It was therefore concluded that the lack of activity of the DL1 version of Adk2p (the short form) was due to the absence of the C-terminal domain resulting from a single base pair deletion near the 3′ end of the open reading frame (23Schricker R. Magdolen V. Strobel G. Bogengruber E. Breitenbach M. Bandlow W. J. Biol. Chem. 1995; 270: 31103-31110Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The “so-called” non-functional allele or Adk2p (short) was found to be characteristic of most S. cerevisiae strains (except D273-10B) that are commonly used for laboratory or brewery purposes. In contrast, the alleles carrying the insertion and encoding the active enzyme were found in the more distantly related species Saccharomyces diastaticus, Saccharomyces capensis, and the wine yeasts Malaga and Bordeaux. Since publication of these findings in 1995 (23Schricker R. Magdolen V. Strobel G. Bogengruber E. Breitenbach M. Bandlow W. J. Biol. Chem. 1995; 270: 31103-31110Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar), Adk2p has not received much attention, perhaps because of its presumed non-functionality in most laboratory strains. Here we have investigated the contribution of the modified C-terminal domain in Adk2p (long) to the molecular properties of the enzyme. Contrary to the existing knowledge, we show that it is not essential for enzyme activity. Rather, it contributes to protein folding and/or stability and plays a critical role in maintaining enzyme activity under harsh conditions. Constructs and Yeast Strains—The genes encoding Adk2p (short, accession number AY558457) and Adk2p (long; accession number AY949617) were amplified by PCR from the yeast genomic DNA isolated from BY4741 (Invitrogen) and D273-10B (ATCC 24657), respectively. The PCR products were digested with NdeI and XhoI and cloned into the same sites of pSP64T (24Gordon D.M. Shi Q. Dancis A. Pain D. Hum. Mol. Genet. 1999; 8: 2255-2262Crossref PubMed Scopus (37) Google Scholar) and pET21b (Novagen, Madison, WI). The former was used for cell-free synthesis of proteins. The latter introduced a His6 tag in-frame at the C terminus of the protein and was used for bacterial expression. All constructs were verified by sequencing. Using the PCR-based transplacement cassette approach (25Longtine M.S. McKenzie III, A. Demarini D.J. Shah N.G. Wach A. Brachat A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4160) Google Scholar), the native ADK2 gene in the strains BY4741 and D273-10B/A/H/U (26Paumard P. Vaillier J. Coulary B. Schaeffer J. Soubannier V. Mueller D.M. Brethes D. di Rago J.-P. Velours J. EMBO J. 2002; 21: 221-230Crossref PubMed Scopus (589) Google Scholar) was replaced with the corresponding tagged version of the gene that introduced three hemagglutinin tags in tandem at the C terminus of the short and long forms of Adk2p, respectively. Likewise, modified BY4741 and D273-10B/A/H/U strains were generated that express C-terminally triple hemagglutinin-tagged long and short forms of Adk2p, respectively. The correct integration of the tagged gene was verified by PCR analysis of the genomic DNA. Bacterial Expression, Purification, and Renaturation of Proteins— BL21 (DE3) cells (Stratagene, La Jolla, CA), carrying the plasmid pET21b/Adk2p (short) or pET21b/Adk2p (long), were cultured in M9 medium supplemented with 0.1 mg/ml ampicillin at 37 °C. Cells were switched to 30 °C prior to induction of proteins by the addition of isopropyl-1-thio-β-d-galactopyranoside to 1 mm. For some experiments, radiolabeled proteins were desired; hence, protein induction was carried out in the presence of 25 μCi/ml [35S]Met (1175 Ci/mmol, PerkinElmer Life Sciences). Following incubation at 30 °C for 3 h, cells were fractionated into soluble fractions and inclusion bodies (IB). 1The abbreviations used are: IB, inclusion bodies; Nat, native; Renat, renatured; Ni-NTA, nickel-nitrilotriacetic acid. Both short and long forms of Adk2p were purified essentially as described (27Amutha B. Pain D. Biochem. J. 2003; 370: 805-815Crossref PubMed Google Scholar, 28Gordon D.M. Wang J. Amutha B. Pain D. Biochem. J. 2001; 356: 207-215Crossref PubMed Google Scholar, 29Sepuri N.B.V. Gordon D.M. Pain D. J. Biol. Chem. 1998; 273: 20941-20950Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Briefly, inclusion bodies were solubilized with 50 mm Tris/HCl, pH 8.0, containing 8 m urea, and proteins with the His6 tag were purified on Ni-NTA-agarose (Qiagen, Chatsworth, CA) in the presence of 8 m urea. The elution was done with 50 mm Tris/HCl, pH 8.0, 8 m urea, 0.4 m imidazole. The eluate was dialyzed against buffer A (10 mm Hepes/KOH, pH 7.5, 6 mm MgCl2, 50 mm KCl) containing 6 m urea and stored in aliquots at –80 °C until further use. For renaturation of the urea-denatured short and long forms of Adk2p, proteins were diluted 10-fold with buffer A containing 1% Triton X-100 but no urea. Following incubation at 25 °C for 10 min, samples were centrifuged at 15,000 × g for 10 min at 4 °C. The supernatant fractions containing the renatured (“Renat”) short or the long form of Adk2p will be called SIB/Renat and LIB/Renat, respectively. Adk2p (long) was also purified from the soluble fraction of the bacterial lysate as described above except that no urea was used. The purified protein was stored at –80 °C in the presence of 20% glycerol until further use. Such a preparation will be referred to as “native” Adk2p (long) or LNat. Protein concentrations of the native and”renatured samples were determined using the Micro BCA protein assay kit (Pierce). GTP:AMP Phosphotransferase Activity—Unless otherwise stated, the assay mixture (20 μl) contained Adk2p in 20 mm Hepes/KOH, pH 7.5, 0.6 m sorbitol, 0.1 mg/ml bovine serum albumin, 5 mm KCl, 6 mm MgCl2,1mm dithiothreitol, 0.06 m urea, 0.1% Triton X-100, 1 mm AMP, 1 mm unlabeled GTP plus 1 μCi of [α-32P]GTP (∼3000 Ci/mmol, PerkinElmer Life Sciences). Following incubation at 30 °C for 15 min, the reaction was stopped by the addition of acetic acid to a final concentration of 10 mm. One-tenth of the reaction mixture was spotted onto polyethyleneimine cellulose thin layer chromatography plates (Sigma) and dried with a hair dryer. The plates were then developed with 1 m LiCl2 and 1 m acetic acid (30Griparic L. van der Wel N.N. Orozco I.J. Peters P.J. van der Bliek A.M. J. Biol. Chem. 2004; 279: 18792-18798Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar), air-dried, and exposed to film for 15–60 min. Spots on the autoradiogram were quantitated using the FluorChem 8000 imaging software package (Alpha Innotech Corp., San Leandro, CA). For each sample, the densitometric ratio of GDP/(GDP+GTP) was calculated. The activity of the sample with the highest ratio was considered 100%, and the relative activity of other samples was then normalized. For experiments in Fig. 3, reactions were performed at different temperatures, ranging from 0–65 °C for 15 min. For experiments in Fig. 4A, the reaction mixtures contained different concentrations of urea (0.06–5.6 m), and the assay was performed at 30 °C for 15 min. To determine the ATP:AMP phosphotransferase activity (Fig. 2B), unlabeled and labeled GTP was replaced with unlabeled ATP (1 mm) plus 1 μCi of [γ-32P]ATP (∼3000 Ci/mmol, PerkinElmer Life Sciences).Fig. 4Adk2p (long) is resistant to urea denaturation and degradation by trypsin. A, the activity of SIB/Renat at and LIB/Renat assayed 30 °C in the was presence of increasing urea concentrations. Both proteins were used at a final concentration of 2.5 μg/ml. For details, see legend for Fig. 2A. B, radiolabeled SIB/Renat with and LIB/Renat treated increasing concentrations of trypsin were for 30 min on ice. Samples were analyzed by SDS-PAGE and autoradiography.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 2Both short and long forms of Adk2p are enzymatically active. A, the GTP:AMP phosphotransferase activity of native Adk2p (long) or LNat was compared with the activities of LIB/Renat and SIB/Renat. LNat was purified from the soluble fraction of the bacterial lysate under non-denaturing conditions. The other two samples (LIB/Renat and SIB/Renat) indicate the long and short forms of Adk2p, respectively, that were purified from inclusion bodies in the presence of urea and subsequently renatured in the presence of Triton X-100. All three proteins were used at a final concentration of 0.5 μg/ml in a total volume of 20 μl, and the assays were performed at 30 °C for 15 min. One-tenth of each reaction mixture was analyzed by thin layer chromatography followed by autoradiography. B, the ATP:AMP phosphotransferase activity was examined exactly as in A except that unlabeled and α-32P-labeled GTP was replaced with unlabeled and γ-32P-labeled ATP. The ATP-specific adenylate kinase Adk1p was used as a positive control at a final concentration of 0.5 μg/ml.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Proteolytic Stability—Radiolabeled SIB/Renat or LIB/Renat (4 μg/ml) was incubated with increasing concentrations of trypsin (2–20 μg/ml) in buffer A containing 1% Triton X-100 and 0.48 m urea for 30 min on ice. Trypsin was inactivated with the addition of phenylmethylsulfonyl fluoride (1 mm), and samples were analyzed by SDS-PAGE and autoradiography. Mitochondrial Protein Import—The plasmids pSP64T/Adk2p (short) and pSP64T/Adk2p (long) were linearized with BamHI. Transcription was carried out using the Ribomax-SP6 kit, and 35S-labeled proteins were synthesized in reticulocyte lysate using the manufacturer's protocol (Promega, Madison, WI). For import experiments, the postribosomal supernatant containing the newly synthesized radiolabeled protein was used either directly (native) or after treatment with urea (urea-denatured). The urea treatment was performed as described (24Gordon D.M. Shi Q. Dancis A. Pain D. Hum. Mol. Genet. 1999; 8: 2255-2262Crossref PubMed Scopus (37) Google Scholar). Briefly, an aliquot of postribosomal supernatant (20 μl) was mixed with saturated ammonium sulfate (40 μl) and incubated on ice for 30 min. Samples were centrifuged, and the pellet was solubilized in 20 mm Hepes/KOH, pH 7.5, containing 8 m urea. The procedure for isolation and purification of mitochondria has been described elsewhere (31Murakami H. Pain D. Blobel G. J. Cell Biol. 1988; 107: 2051-2057Crossref PubMed Scopus (279) Google Scholar). Import reactions were performed essentially as described (24Gordon D.M. Shi Q. Dancis A. Pain D. Hum. Mol. Genet. 1999; 8: 2255-2262Crossref PubMed Scopus (37) Google Scholar, 29Sepuri N.B.V. Gordon D.M. Pain D. J. Biol. Chem. 1998; 273: 20941-20950Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 32Gordon D.M. Kogan M. Knight S.A.B. Dancis A. Pain D. Hum. Mol. Genet. 2001; 10: 259-269Crossref PubMed Scopus (32) Google Scholar, 33Schülke N. Sepuri N.B.V. Gordon D.M. Saxena S. Dancis A. Pain D. J. Biol. Chem. 1999; 274: 22847-22854Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Briefly, native or urea-denatured Adk2p (short or long) was incubated with mitochondria (100 μg of proteins) in the presence of ATP (4 mm) and GTP (1 mm) at 30 °C for 30 min. As a control, mitochondria were pretreated with valinomycin (5 μg/ml) to dissipate the membrane potential. For urea-denatured proteins, the final urea concentration in the import reaction was 0.16 m. Following import, reaction mixtures were treated with trypsin (0.2 mg/ml) for 30 min on ice. To inactivate trypsin, samples were diluted with a buffer containing a mixture of protease inhibitors. Mitochondria were reisolated and washed with 10% trichloroacetic acid. Samples were analyzed by SDS-PAGE and autoradiography. We focused on Adk2p corresponding to two representative yeast strains, BY4741 and D273-10B. The former is very popular because mutant strains deleted for individual non-essential genes are commercially available in this background. The latter is widely used for studying various aspects of mitochondrial biogenesis. ADK2 was amplified by PCR from the genomic DNA of these two strains and sequenced. The ADK2 open reading frame in BY4741 codes for a protein of 225 amino acids and will be referred to as Adk2p (short). In D273-10B, a cytosine residue was found to be inserted into the codon 223 of ADK2. This results in changes of three C-terminal residues (RNY to PKL) and the addition of nine amino acids (LNLITSKFG) at the C terminus of the protein; this extended form will be called Adk2p (long) (Fig. 1A). To study the molecular properties of the short and long forms of Adk2p, we took advantage of recombinant proteins. Both forms were individually expressed in bacteria with a C-terminal His6 tag. The C-terminal tag was preferred over an N-terminal tag because most mitochondrial precursor proteins contain the targeting information at their N termini, and a C-terminal His6 tag usually does not interfere with the import and functions of mitochondrial proteins (27Amutha B. Pain D. Biochem. J. 2003; 370: 805-815Crossref PubMed Google Scholar, 28Gordon D.M. Wang J. Amutha B. Pain D. Biochem. J. 2001; 356: 207-215Crossref PubMed Google Scholar). Adk2p (short) was found to be sequestered in the inclusion bodies (Fig. 1B, lanes 3–5). In contrast, Adk2p (long) was distributed equally between the soluble fraction and inclusion bodies (Fig. 1B, lanes 8–10). Short and long forms of Adk2p from inclusion bodies were solubilized with 8 m urea and purified to homogeneity by Ni-NTA-agarose chromatography (Fig. 1B, lanes 6 and 12, respectively). The soluble form of Adk2p (long) was purified exactly the same way (Fig. 1B, lane 11) except that urea was omitted from all buffers; this purified preparation will be referred to as native Adk2p (long) or LNat. Proteins from the inclusion bodies were purified in the presence of high concentrations of urea and hence considered denatured. These proteins must therefore be allowed to renature before they can be used for enzymatic studies. To achieve this goal, urea-denatured proteins were diluted 10-fold with buffer containing a non-ionic detergent (Triton X-100) and centrifuged to determine whether they remained in solution (27Amutha B. Pain D. Biochem. J. 2003; 370: 805-815Crossref PubMed Google Scholar, 28Gordon D.M. Wang J. Amutha B. Pain D. Biochem. J. 2001; 356: 207-215Crossref PubMed Google Scholar). In the absence of detergent, proteins were aggregated and found exclusively in the pellet fractions (data not shown). In 1% Triton X-100, a major portion (>80%) of both short and long forms of Adk2p remained in the supernatant fractions (Fig. 1C). The soluble short and long forms of Adk2p thus obtained (purified from inclusion bodies and subsequently renatured in Triton X-100) will be called SIB/Renat and LIB/Renat, respectively. To measure adenylate kinase activity, most previous studies used a spectrophotometric coupled assay system for the determination of nucleoside diphosphates with pyruvate kinase and lactate dehydrogenase (for example, Ref. 34Ulschmid J.K. Rahlfs S. Schirmer R.H. Mol. Biochem. Parasitol. 2004; 136: 211-220Crossref PubMed Scopus (15) Google Scholar). This method is quite sensitive and reliable at physiological temperatures. However, at higher temperatures, the coupled enzymes may be inactivated, and thus the results may not truly reflect adenylate kinase activity. To examine GTP:AMP phosphotransferase activity of Adk2p, we therefore used an assay that does not depend on other enzymes. We modified a highly sensitive radioactive assay that is normally used to monitor hydrolysis of nucleotides (30Griparic L. van der Wel N.N. Orozco I.J. Peters P.J. van der Bliek A.M. J. Biol. Chem. 2004; 279: 18792-18798Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). Native Adk2p (long) was incubated with [α-32P]GTP in the absence or presence of unlabeled AMP at 30 °C, and samples were analyzed by thin layer chromatography followed by autoradiography and densitometric quantitation of the autoradiogram. The conversion of [α-32P]GTP to [α-32P]GDP was observed only in the presence of the enzyme and AMP (Fig. 2A). As little as 50 ng/ml native Adk2p (long) could be easily assayed (data not shown). Using this method, both LIB/Renat and SIB/Renat were found to be active. The relative activity of LIB/Renat was very similar to that of the native enzyme (LNat) and was ∼1.6 times the value calculated for SIB/Renat. The specific activity of native Adk2p (long) at 30 °C was calculated to be 100 μmol of GTP utilized (or GDP formed) · min–1 · (mg of protein)–1. The activity of all three forms of Adk2p (LNat, LIB/Renat, and SIB/Renat) was specific for GTP, as they failed to efficiently use ATP as the high energy phosphate donor (Fig. 2B). No significant formation of [β-32P]ADP was detected in the presence of Adk2p, [γ-32P]ATP, and unlabeled AMP. Adk1p is an ATP:AMP phosphotransferase, and bacterially expressed and purified Adk1p (data not shown) served as a positive control for these assays. These results suggest that the presence of the modified C-terminal domain of Adk2p (long) may slightly enhance its GTP:AMP phosphotransferase activity at 30 °C, but this domain is not essential for activity or for conferring specificity to GTP as the preferred phosphate donor. For the purpose of comparisons, most experiments in the following sections were performed with LIB/Renat and SIB/Renat because both were purified from inclusion bodies and renatured in an identical manner. Protein function often relies upon a delicate balance between protein stability and flexibility. ATP-specific adenylate kinases from different organisms vary greatly with respect to their optimum temperatures for activity and their stability against thermal denaturation (20Strobel G. Zollner A. Angermayr M. Bandlow W. Mol. Biol. Cell. 2002; 13: 1439-1448Crossref PubMed Scopus (46) Google Scholar, 35Bae E. Phillips Jr., G.N. J. Biol. Chem. 2004; 279: 28202-28208Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 36Vieille C. Krishnamurthy H. Hyun H.-H. Savchenko A. Yan H. Zeikus J.G. Biochem. J. 2003; 372: 577-585Crossref PubMed Google Scholar, 37Haney P.J. Stees M. Konisky J. J. Biol. Chem. 1999; 274: 28453-28458Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 38Okajima T. Kitaguchi D. Fujii K. Matsuoka H. Goto S. Uchiyama S. Kobayashi Y. Tanizawa K. Biosci. Biotechnol. Biochem. 2002; 66: 2112-2124Crossref PubMed Scopus (3) Google Scholar). Such studies for the GTP-specific isozymes are lacking, and we therefore assayed activity of the short and long forms of Adk2p at different temperatures. SIB/Renat exhibited optimum activity at 30 °C in agreement with the optimum growth of most wild type yeast strains at this temperature. It was, however, extremely sensitive to thermal denaturation and was inactiva
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