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• W2054499068 abstract "Cyclic AMP acting on protein kinase A controls sporulation and encystation in social and solitary amoebas. In Dictyostelium discoideum, adenylate cyclase R (ACR), is essential for spore encapsulation. In addition to its cyclase (AC) domain, ACR harbors seven transmembrane helices, a histidine kinase domain, and two receiver domains. We investigated the role of these domains in the regulation of AC activity. Expression of an ACR-YFP fusion protein in acr− cells rescued their sporulation defective phenotype and revealed that ACR is associated with the nuclear envelope and endoplasmic reticulum. Loss of the transmembrane helices (ΔTM) caused a 60% reduction of AC activity, but ΔTM-ACR still rescued the acr− phenotype. The isolated AC domain was properly expressed but inactive. Mutation of three essential ATP-binding residues in the histidine kinase domain did not affect the AC activity or phenotypic rescue. Mutation of the essential phosphoryl-accepting aspartate in receivers 1, 2, or both had only modest effects on AC activity and did not affect phenotypic rescue, indicating that AC activity is not critically regulated by phosphorelay. Remarkably, the dimerizing histidine phosphoacceptor subdomain, which in ACR lacks the canonical histidine for autophosphorylation, was essential for AC activity. Transformation of wild-type cells with an ACR allele (ΔCRA) that is truncated after this domain inhibited AC activity of endogenous ACR and replicated the acr− phenotype. Combined with the observation that the isolated AC domain was inactive, the dominant-negative effect of ΔCRA strongly suggests that the defunct phosphoacceptor domain acquired a novel role in enforcing dimerization of the AC domain. Cyclic AMP acting on protein kinase A controls sporulation and encystation in social and solitary amoebas. In Dictyostelium discoideum, adenylate cyclase R (ACR), is essential for spore encapsulation. In addition to its cyclase (AC) domain, ACR harbors seven transmembrane helices, a histidine kinase domain, and two receiver domains. We investigated the role of these domains in the regulation of AC activity. Expression of an ACR-YFP fusion protein in acr− cells rescued their sporulation defective phenotype and revealed that ACR is associated with the nuclear envelope and endoplasmic reticulum. Loss of the transmembrane helices (ΔTM) caused a 60% reduction of AC activity, but ΔTM-ACR still rescued the acr− phenotype. The isolated AC domain was properly expressed but inactive. Mutation of three essential ATP-binding residues in the histidine kinase domain did not affect the AC activity or phenotypic rescue. Mutation of the essential phosphoryl-accepting aspartate in receivers 1, 2, or both had only modest effects on AC activity and did not affect phenotypic rescue, indicating that AC activity is not critically regulated by phosphorelay. Remarkably, the dimerizing histidine phosphoacceptor subdomain, which in ACR lacks the canonical histidine for autophosphorylation, was essential for AC activity. Transformation of wild-type cells with an ACR allele (ΔCRA) that is truncated after this domain inhibited AC activity of endogenous ACR and replicated the acr− phenotype. Combined with the observation that the isolated AC domain was inactive, the dominant-negative effect of ΔCRA strongly suggests that the defunct phosphoacceptor domain acquired a novel role in enforcing dimerization of the AC domain. The ATP derivative cAMP is the most deeply conserved signaling intermediate in all domains of life. In eukaryotes, cAMP is produced by the conserved class III cyclase domain, whereas prokaryotes use five more unrelated catalysts. The class III domain can be subdivided into four subtypes, a–d, with type IIId, which is found in fungi and Euglenozoa, being derived from type IIIc, which is common to both bacteria and Archaea (1.Linder J.U. Schultz J.E. Cellular Signalling. 2003; 15: 1081-1089Crossref PubMed Scopus (143) Google Scholar, 2.Ritchie A.V. van Es S. Fouquet C. Schaap P. Mol. Biol. Evol. 2008; 25: 2109-2118Crossref PubMed Scopus (35) Google Scholar). Type IIIa is the predominant catalyst in metazoa but is also found in protozoa and prokaryotes. In metazoa, it is present as two asymmetrical copies alternating with two sets of six transmembrane (TM) 2The abbreviations used are: TMtransmembraneACadenylate cyclaseRreceiver/response regulatorYFPyellow fluorescent proteinfforwardrreverse. helices in the G-protein-regulated adenylate cyclases and as a single copy in the guanylyl cyclases. Type IIIb domains are very abundant in prokaryotes, where they are typically combined with many other functional domains but are also found in alveolates, Amoebozoa, and mammals. Some of the alveolate enzymes harbor an additional set of six TM helices, whereas others and the mammalian enzyme are soluble proteins (3.Baker D.A. Kelly J.M. Trends Parasitol. 2004; 20: 227-232Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 4.Chen Y. Cann M.J. Litvin T.N. Iourgenko V. Sinclair M.L. Levin L.R. Buck J. Science. 2000; 289: 625-628Crossref PubMed Scopus (681) Google Scholar). The amoebozoan adenylate cyclase ACR from the social amoeba Dictyostelium discoideum is a multidomain protein, which resembles some of the cyanobacterial type IIIb enzymes with respect to domain architecture (5.Kim H.J. Chang W.T. Meima M. Gross J.D. Schaap P. J. Biol. Chem. 1998; 273: 30859-30862Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 6.Söderbom F. Anjard C. Iranfar N. Fuller D. Loomis W.F. Development. 1999; 126: 5463-5471Crossref PubMed Google Scholar). transmembrane adenylate cyclase receiver/response regulator yellow fluorescent protein forward reverse. Social amoebas use cAMP not only as a second messenger for external stimuli but also as a secreted primary signal. In this role, cAMP coordinates the aggregation of starving amoeba and the construction of fruiting bodies, and it also triggers the expression of aggregation genes and prespore genes. As second messenger, cAMP mediates the effect of a range of stimuli that control initiation of multicellular development, maturation of stalk and spore cells, and germination of the spores (7.Aubry L. Firtel R. Ann. Rev. Cell. Dev. Biol. 1999; 15: 469-517Crossref PubMed Scopus (134) Google Scholar, 8.Saran S. Meima M.E. Alvarez-Curto E. Weening K.E. Rozen D.E. Schaap P. J. Muscle Res. Cell Motil. 2002; 23: 793-802Crossref PubMed Scopus (75) Google Scholar). Comparative analysis of cAMP signaling throughout the social amoebas showed that the roles of cAMP in spore formation and germination are evolutionary derived from a deeply conserved role in the encystation of solitary amoebas (2.Ritchie A.V. van Es S. Fouquet C. Schaap P. Mol. Biol. Evol. 2008; 25: 2109-2118Crossref PubMed Scopus (35) Google Scholar, 9.Kawabe Y. Morio T. James J.L. Prescott A.R. Tanaka Y. Schaap P. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 7089-7094Crossref PubMed Scopus (33) Google Scholar). Encystation is of considerable medical relevance because cysts are resistant to biocides and immune clearance and preclude effective treatment of diseases by protozoan pathogens (10.Turner N.A. Russell A.D. Furr J.R. Lloyd D. J. Antimicrob. Chemother. 2000; 46: 27-34Crossref PubMed Scopus (112) Google Scholar, 11.Marciano-Cabral F. Cabral G. Clin. Microbiol. Rev. 2003; 16: 273-307Crossref PubMed Scopus (989) Google Scholar, 12.Gooi P. Lee-Wing M. Brownstein S. El-Defrawy S. Jackson W.B. Mintsioulis G. Cornea. 2008; 27: 246-248Crossref PubMed Scopus (18) Google Scholar). The mechanisms of encystation are little understood, and an understanding of the role of cAMP in this process will have important therapeutic consequences. The type IIIb enzyme ACR/ACB (5.Kim H.J. Chang W.T. Meima M. Gross J.D. Schaap P. J. Biol. Chem. 1998; 273: 30859-30862Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 6.Söderbom F. Anjard C. Iranfar N. Fuller D. Loomis W.F. Development. 1999; 126: 5463-5471Crossref PubMed Google Scholar) is essential for spore maturation and proper stalk cell formation in D. discoideum, which additionally uses two type IIIa enzymes: adenylate cyclase A and adenylate cyclase G. Adenylate cyclase A mainly produces cAMP for secretion and coordination of cell movement (13.Pitt G.S. Milona N. Borleis J. Lin K.C. Reed R.R. Devreotes P.N. Cell. 1992; 69: 305-315Abstract Full Text PDF PubMed Scopus (264) Google Scholar, 14.Kriebel P.W. Parent C.A. IUBMB Life. 2004; 56: 541-546Crossref PubMed Scopus (38) Google Scholar), whereas adenylate cyclase G is required for prespore differentiation and control of spore dormancy (15.van Es S. Virdy K.J. Pitt G.S. Meima M. Sands T.W. Devreotes P.N. Cotter D.A. Schaap P. J. Biol. Chem. 1996; 271: 23623-23625Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 16.Alvarez-Curto E. Saran S. Meima M. Zobel J. Scott C. Schaap P. Development. 2007; 134: 959-966Crossref PubMed Scopus (33) Google Scholar). ACR is a large multidomain protein in which the cyclase catalytic domain is preceded by two receiver domains, a histine kinase region, consisting of a HisKA (histidine kinase phosphoacceptor domain) and HATPase (histidine kinase-, DNA gyrase B-, and phytochrome-like ATPases) C domain and seven TM helices (6.Söderbom F. Anjard C. Iranfar N. Fuller D. Loomis W.F. Development. 1999; 126: 5463-5471Crossref PubMed Google Scholar). ACR activity is present during growth, decreases during aggregation, and increases again after aggregation to reach maximum levels during fruiting body formation (17.Meima M.E. Schaap P. Dev. Biol. 1999; 212: 182-190Crossref PubMed Scopus (46) Google Scholar). Until now, it was not known whether and how the histidine kinase and receiver domains of ACR regulate its cyclase activity. More information is available on the type IIIb cyclase CyaC from the cyanobacterium Spirulina platensis. Like ACR, CyaC also contains a histidine kinase and receiver domain proximal to the carboxyl-terminal AC domain but additionally contains two, more distal GAF (found in cGMP-phosphodiesterases, adenylyl cyclases and formate hydrogen lyase transcriptional activator) domains and a second N-terminal receiver domain. Here, cyclase activity is activated by phosphorylation of the proximal receiver domain by the intrinsic histidine kinase activity (18.Kasahara M. Ohmori M. J. Biol. Chem. 1999; 274: 15167-15172Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). In another cyanobacterium, Anabaena sp., cyclase activity is up-regulated by phosphorylation of the N-terminal receiver domain in response to far-red light via the phytochrome-like sensor histidine kinase AphC (19.Okamoto S. Kasahara M. Kamiya A. Nakahira Y. Ohmori M. Photochem. Photobiol. 2004; 80: 429-433Crossref PubMed Google Scholar). In theory, ACR could also be regulated by any of the 15-sensor histidine kinases that are present in Dictyostelium (20.Anjard C. Loomis W.F. Kuspa A. Dictyostelium Genomics. 1st Ed. Horizon Bioscience, Norfolk, United Kingdom2005: 59-82Google Scholar). In this work, we investigated the cellular localization of ACR and the role of its multiple functional domains in the control of AC activity. Our data show a novel role for the HisKA domain in dimerization of the cyclase domain and unusual localization of the enzyme at the cell nucleus. D. discoideum NC4A2 (21.Shelden E. Knecht D.A. J. Cell Sci. 1995; 108: 1105-1115Crossref PubMed Google Scholar) and acr− cells (see below) were cultured in HL5 medium that was supplemented with 5 μg/ml blasticidin for null mutants and with 10 to 100 μg/ml G418 for cells harboring expression constructs. D. discoideum NC4 was grown in association with Klebsiella aerogenes on standard medium agar. Multicellular development was induced by incubating cells, freed from bacteria or growth medium, at 22 °C on phosphate buffered agar (1.5% agar in 10 mm sodium/potassium phosphate buffer, pH 6.5) at 1.5 × 106 cells/cm2. To generate an AcrA knock-out construct with recyclable selection marker, nucleotides 350–1382 (KO1) and nucleotides 1638–2685 (KO2) of the AcrA-coding region were amplified by PCR from AX2 genomic DNA with primer pairs KO1f/KO1r and KO2f/KO2r, that harbor a KpnI/SalI and BamHI/NotI restriction site, respectively (supplemental Table S1). The KO1 and KO2 fragments were inserted by T/A cloning into vector pCR4-TOPO (Invitrogen), released by KpnI/SalI or BamHI/NotI digestion, and cloned into KpnI/SalI and BamHI/NotI sites that flank the floxed A15::Bsr selection cassette of vector pLPBLP (22.Faix J. Kreppel L. Shaulsky G. Schleicher M. Kimmel A.R. Nucleic Acids Res. 2004; 32: e143Crossref PubMed Scopus (191) Google Scholar) generating pACR-KO (supplemental Fig. S1A). NC4A2 cells were transformed with pACR-KO KpnI/NotI insert by electroporation, selected for growth at 5 μg/ml blasticidin, and cloned out on Klebsiella aerogenes lawns. Clonal isolates were tested for gene disruption by two PCR reactions using primer pairs KO1f/KOt1 and BsrF/KOt2 (supplemental Table S1 and Fig. S1A) and examination of genomic digests by Southern blot. The complete 6372-nucleotide ACR cDNA was cloned by a stepwise approach in which the sequence was divided into four segments, which were amplified and assembled sequentially. The first segment AB (containing the single intron position of ACR) was amplified with primers A and B (supplemental Table S1) by reverse transcription PCR of mRNA isolated from 12 h-starved AX2 cells. Segments CD, EF, and GH were all amplified from AX2 genomic DNA by PCR using primer sets C/D, E/F, and G/H for the three segments, respectively (supplemental Table S1). The primers contained restriction sites that were either already present in the ACR sequence or were introduced by neutral mutations. The four segments were individually cloned into pCR4-TOPO, sequenced to validate correct amplification, and released by their respective pairs of restriction enzymes. The segments were then inserted into pGEM-7Zf(+) (Promega, Madison, WI) step by step at their corresponding restriction sites, as outlined in supplemental Fig. S1B. Next, the full-length ACR cDNA was excised with BamHI and XhoI and inserted into the BamHI/XhoI-digested expression vector pB17S-EYFP. This places ACR under control of the constitutively active A15 promoter and fuses the gene at the C terminus to an enhanced yellow fluorescent protein (YFP) tag (23.Meima M.E. Biondi R.M. Schaap P. Mol. Biol. Cell. 2002; 13: 3870-3877Crossref PubMed Google Scholar). The truncated forms were created by replacing one or several of the four ACR segments (supplemental Fig. S1B) in pGEM-7Zf(+) with a newly amplified truncated version of the relevant region, using a combination of primers ΔT, ΔTK, ΔTKC, ΔTKCR1, and ΔTKCR1R2 (supplemental Table S1) that incorporate a 5′-BamHI site, with reverse primers D or F (supplemental Table S1) for N-terminal truncations, and primer ΔC, with a XhoI site, combined with forward primer G for the C-terminal truncations. The complete truncated ACR cDNA was released from pGEM-7Zf(+) by BamHI/XhoI digestion and inserted into pB17S-EYFP as described above. For the ΔTΔC and ΔTKCR1ΔC double truncations, the ΔT-D and ΔTKCR1-F amplicons were combined with the ΔC construct. The full-length and truncated ACR constructs were transformed into acr− cells by electroporation. Transformants were selected at 10 μg/ml G418, which was gradually increased to 100 μg/ml. Putative dominant-negative fragments were amplified from ACR cDNA using forward primer A and reverse primers ΔCRA and ΔKCRA (supplemental Table S1). After subcloning in pCR4-TOPO, the fragments were inserted into pB17S-EYFP using their BamHI/XhoI restriction sites as described above. Both constructs and empty pB17S-EYFP vector were transformed into wild-type NC4A2 cells, with G418 selection increased to 300 μg/ml. Single or multiple amino acid mutations were introduced by amplifying the entire pCR4-TOPO vector containing the relevant segment of ACR with a mutagenic forward (f) and reverse (r) primer pair. The mutations H613Q, S617A, and S617E of the HRRLS motif in segment AB were performed with primer pairs H613Qf/H613Qr, S617Af/S617Ar and S617Ef/S617Er (supplemental Table S1), respectively. The mutation of three residues in the HATPase-C domain, N769D, D854A, and G856A, were achieved by two PCR reactions with primer pairs N769Df/N769Dr and D854AG856Af/D854AG856Ar on segment CD. Mutations D1010A in the R1 domain and D1089A in the R2 domains were obtained by amplification of segments CD and EF, respectively, with primer pairs D1010Af/D1010Ar and D1089Af/D1089Ar. All mutated segments were validated by DNA sequencing, excised from pCR4-TOPO by their terminal restriction sites, and recombined with the complementary intact ACR segments in pGEM-7ZF(+) to reconstitute the full-length ACR cDNA, as described above. A double D1010A/D1089A mutant was obtained by combining the mutated CD and EF segments with wild-type AB and GH segments (supplemental Fig. S1B). The mutant ACR cDNAs were then inserted into pB17S-EYFP and transformed into acr− cells. Cells transformed with intact or mutant ACR-YFP constructs were harvested during exponential growth, washed once with PB (10 mm sodium/potassium phosphate buffer, pH 6.5) and resuspended in lysis buffer (2 mm MgCl2 and 250 mm sucrose in 10 mm Tris, pH 8.0) to 108 cells/ml. Cells were lysed by passage through 3-μm pore size nuclepore filters. Aliquots of 10 μl cell lysate were added to 10 μl of 2× assay mix (1 mm ATP, 16 mm MgCl2, 0.4 mm 3-isobutyl-1-methylxanthine, and 20 mm dithiothreitol in lysis buffer). After 5 min of incubation on ice, reactions were started by transferring the samples to a 22 °C water bath. Reactions were terminated after 0, 3, 10, 20, and 30 min by adding 10 μl of 0.4 m EDTA (pH 8.0), followed by boiling for 1 min. cAMP was assayed directly in the boiled lysate by isotope dilution assay (17.Meima M.E. Schaap P. Dev. Biol. 1999; 212: 182-190Crossref PubMed Scopus (46) Google Scholar). AC activities were standardized on the amount of YFP tag in the lysates, determined by quantitative Western blot analysis. Cells were harvested and lysed in SDS-PAGE sample buffer, or filter-lysed cells were mixed with sample buffer. Proteins were size-fractionated by SDS-PAGE, transferred to nitrocellulose, and probed with rabbit polyclonal αGFP antibodies (Abcam, Cambridge, UK) For qualitative analysis, blots were incubated with horseradish peroxidase conjugated goat anti-rabbit secondary antibody (Sigma), and detection was performed with the SuperSignal chemoluminescence kit (Pierce). For quantitative analysis, the secondary antibody was IR dye 800-conjugated donkey anti-rabbit antibody (Invitrogen), and signal intensities were analyzed using the Odyssey infrared imaging system (LI-COR, Lincoln, NE). Cells were washed with phosphate buffer, left to adhere to coverslips, and fixed with 15% picric acid/paraformaldehyde. Cells were post-fixed with 70% ethanol, washed with PBS (24.Müller-Taubenberger A. Lupas A.N. Li H. Ecke M. Simmeth E. Gerisch G. EMBO J. 2001; 20: 6772-6782Crossref PubMed Scopus (189) Google Scholar), and were incubated overnight at 8 °C with a polyclonal rabbit anti-GFP antibody (Abcam), diluted 1:100 in PBSB (1% bovine serum albumin in PBS), and 1:100 diluted monoclonal mouse anti-calnexin antibody (24.Müller-Taubenberger A. Lupas A.N. Li H. Ecke M. Simmeth E. Gerisch G. EMBO J. 2001; 20: 6772-6782Crossref PubMed Scopus (189) Google Scholar) to detect ACR-YFP and calnexin, respectively. After three washes with PBS, the cells were incubated for 2 h at 22 °C with 1:100 diluted FITC-conjugated donkey anti-rabbit IgG (Diagnostics Scotland, Edinburgh, Scotland) and 1:500 diluted Alexa Fluor® 594 conjugated goat anti-mouse IgG (Invitrogen) to detect the α-GFP and α-calnexin antibodies, respectively, and simultaneously counterstained with 0.1 μg/ml of the DNA stain DAPI (Invitrogen). Coverslips were washed three times in PBS and mounted to slide glasses with Hydromount (National Diagnostics). To gain initial insight in the functionality of the putative regulatory domains of ACR, we compared their sequences with those of structurally resolved homologous domains. Histidine kinases consist of an HisKA-dimerizing domain that also carries the histidine for autophosphorylation and an ATP binding HATPase-C (HisC) domain. The HisKA domain contains two α-helices, which form an intramolecular dimer with a second HisKA domain, creating a four-helix bundle. Dimerization enables trans-phosphorylation by the HisC domain of two His residues that point outward from two of the four helices. The phosphoryl group is subsequently transferred to the Asp of a receiver (R) domain. The HisKA domain in conjunction with the HisC domain can also act as an R domain-directed phosphatase (25.Hsing W. Russo F.D. Bernd K.K. Silhavy T.J. J. Bacteriol. 1998; 180: 4538-4546Crossref PubMed Google Scholar, 26.Dutta R. Yoshida T. Inouye M. J. Biol. Chem. 2000; 275: 38645-38653Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The ACR HisKA domain contains two adjacent α-helices but lacks the conserved His and other residues that are essential for both autokinase and phosphatase activity (Fig. 1A). Between helix I and the seventh TM domain, ACR harbors another predicted α-helix that contains an HRRLS motif. Apart from a putative His phosphoryl-acceptor, this motif also contains a consensus protein kinase A phosphorylation site (RRxS) (27.Shabb J.B. Chemical reviews. 2001; 101: 2381-2411Crossref PubMed Scopus (275) Google Scholar). The HisC domain is characterized by five conserved sequence motifs (N, G1, F, G2, and G3) that line the ATP-binding pocket (28.West A.H. Stock A.M. Trends Biochem. Sci. 2001; 26: 369-376Abstract Full Text Full Text PDF PubMed Scopus (754) Google Scholar). Alignment of the HisC domain of ACR with structurally resolved HisC domains shows that the residues that define the five motifs are conserved (Fig. 1B). Mutagenesis of the bacterial enzyme CheA showed that residue Asn409 that marks the N motif, residues Asp449 and Gly451 in the G1 motif, and Gly502 in the G2 motif are essential for its kinase activity (29.Hirschman A. Boukhvalova M. VanBruggen R. Wolfe A.J. Stewart R.C. Biochemistry. 2001; 40: 13876-13887Crossref PubMed Scopus (24) Google Scholar, 30.Stewart R.C. VanBruggen R. Ellefson D.D. Wolfe A.J. Biochemistry. 1998; 37: 12269-12279Crossref PubMed Scopus (62) Google Scholar). For EnvZ, which, unlike CheA, can also act as a receiver-directed phosphatase, mutation of Asn347 (equivalent to Asn409) and Asn343 in the N motif, and Phe390 in the F motif (green highlights) leads to loss of kinase, but not phosphatase activity, whereas mutation of Thr402 in the G2 box (pink highlight) has the opposite effect (25.Hsing W. Russo F.D. Bernd K.K. Silhavy T.J. J. Bacteriol. 1998; 180: 4538-4546Crossref PubMed Google Scholar, 31.Dutta R. Inouye M. J. Biol. Chem. 1996; 271: 1424-1429Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). However, most residues required for phosphatase activity reside in the HisKA domain (Fig. 1A). All essential residues for kinase activity are present in ACR, suggesting that its HisC domain could be functional. The ACR HisC domain is however anomalous in harboring a 51-amino acid insertion of low complexity sequence in the first β-sheet. R domains catalyze transfer of a phosphoryl group from a HisKA domain to one of their own Asp residues. They regulate the activities of associated effectors and catalyze autodephosphorylation. Phosphorylation requires association with a Mg2+ ion that is positioned in the conserved active site and commonly causes movement of a conserved Thr or Ser and Tyr or Phe switch residue pair toward the active site. This conformational change then regulates effector activity (28.West A.H. Stock A.M. Trends Biochem. Sci. 2001; 26: 369-376Abstract Full Text Full Text PDF PubMed Scopus (754) Google Scholar, 32.Gao R. Mack T.R. Stock A.M. Trends Biochem. Sci. 2007; 32: 225-234Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Alignment of the ACR R domains with two well characterized R domains shows that both the secondary structure elements and the active site residues (bold/underlined) are conserved in ACR_R1 (Fig. 1C). ACR_R2 lacks two of four active site residues and additionally harbors a 112-amino acid insertion. Neither R1 nor R2 contain a complete Thr/Ser-Tyr/Phe switch residue pair (maroon highlight). The phosphoryl-accepting Asp (red text) is conserved in both R1 and R2, but their other anomalies may well interfere with domain functionality. Due to its large size, the 6.4-kb required amplification and cloning ACR cDNA in four segments into pGEM-7Zf(+) followed by subcloning into the extrachromosomal expression vector pB17S-EYFP. In this vector, ACR expression is driven by the constitutive A15 promoter, and the gene is fused at the C terminus to an enhanced YFP tag. The empty vector (YFP) and the ACR-YFP construct were expressed in an acr− mutant. As shown previously (6.Söderbom F. Anjard C. Iranfar N. Fuller D. Loomis W.F. Development. 1999; 126: 5463-5471Crossref PubMed Google Scholar), acr− cells develop normally up to the fruiting body stage but are then defective in spore encapsulation, whereas they also form stalks that are thinner than those of wild-type cells. Both aspects of the acr− phenotype were restored by transformation with the ACR-YFP construct but not with the empty vector (Fig. 2A). Inspection of the cells by confocal microscopy showed that YFP-tagged ACR was associated with the nuclear envelope and a vesicular network throughout the cell (Fig. 2B). Incubation of fixed acr−/ACR-YFP cells with antibodies raised against GFP/YFP and the endoplasmic reticulum marker calnexin (24.Müller-Taubenberger A. Lupas A.N. Li H. Ecke M. Simmeth E. Gerisch G. EMBO J. 2001; 20: 6772-6782Crossref PubMed Scopus (189) Google Scholar) showed colocalization of ACR-YFP and calnexin around the nuclei and the vesicular network (Fig. 3). This strongly suggests that ACR localizes to the endoplasmic reticulum and nuclear envelope. Western blots of fractionated acr−/ACR-YFP cell lysates, probed with YFP antibodies, confirmed that ACR-YFP is expressed at the expected size of 270 kDa in the nuclear fraction (Fig. 2C). The ΔT ACR-YFP mutant, which lacks the transmembrane domains (Fig. 4) was present predominantly in the cytoplasmic fraction (Fig. 2C) and showed a similar localization as YFP alone over the entire cytoplasm of the cell (Fig. 2B).FIGURE 3Visualization of ACR-YFP and calnexin by immunofluorescence. Axenically grown acr−/ACR-YFP cells were harvested in exponential phase and triple stained with (i) a polyclonal rabbit-anti-GFP antibody, followed by FITC conjugated donkey anti-rabbit IgG; (ii) a monoclonal mouse-anti-calnexin antibody (24.Müller-Taubenberger A. Lupas A.N. Li H. Ecke M. Simmeth E. Gerisch G. EMBO J. 2001; 20: 6772-6782Crossref PubMed Scopus (189) Google Scholar) followed by Alexa Fluor® 594 conjugated goat anti-mouse IgG; and (iii) DAPI to detect ACR-YFP, calnexin, and DNA, respectively. Cells were photographed through the UV, TRITC, and FITC filter sets of a Leica DMLB2 fluorescence microscope. The merged image was prepared with the Qcapture Pro camera software. Scale bar, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4Functions of the different domains of ACR in adenylate cyclase regulation. A, schematic of truncations. Truncated segments of the ACR cDNA lacking transmembrane (T, vertical bars), HisKA (K), HisC (C), receiver (R1 and R2) domains or the C-terminal low complexity region (L) were prepared by PCR amplification and recombined with unaltered segments in pGEM7ZF+ to reconstitute the entire (but truncated) cDNAs, which were then transferred to vector pB17S-EYFP and transformed into acr− cells. B, AC activity of truncated proteins. Lysates of transformed cell lines were incubated with AC assay mix for 30 min and assayed for cAMP production. Data are standardized on the amount of YFP fusion protein in the lysates as determined by quantitative Western blotting (see “Experimental Procedures”). The results represent means and S.D. of four experiments performed on cells from at least two separate transformations. C, complementation of acr− phenotype. All transformants were developed into fruiting bodies, which were examined as described for Fig. 2A for complementation of the spore encapsulation (Sp) and thin stalk (St) phenotype. D, expression levels of truncated proteins. Lysates of 3 × 105 cells that had been transformed with intact and truncated ACR-YFP fusion constructs were size-fractionated by SDS-PAGE. The YFP-fusion proteins were visualized by qualitative Western blotting using an α-GFP antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT) ACR activity can be measured in cell lysates with Mg2+-ATP as a substrate and 3-isobutyl-1-methylxanthine and DTT present to inhibit the phosphodiesterases RegA and PdsA, respectively. The other D. discoideum adenylate cyclases adenylate cyclase G and adenylate cyclase A are only expressed to low levels in vegetative cells and require Mn2+ and/or Mg2+ plus GTPγS, respectively, to be assayed in cell lysates (17.Meima M.E. Schaap P. Dev. Biol. 1999; 212: 182-190Crossref PubMed Scopus (46) Google Scholar). Consequently, acr−/YFP lysates show very little cAMP accumulation, whereas acr−/ACR-YFP lysates produce cAMP at a steady rate of 1.3 pmol/min mg" @default.
• W2054499068 created "2016-06-24" @default.
• W2054499068 creator A5014587020 @default.
• W2054499068 creator A5027973107 @default.
• W2054499068 creator A5032500984 @default.
• W2054499068 date "2010-12-01" @default.
• W2054499068 modified "2023-09-28" @default.
• W2054499068 title "Functional Dissection of Adenylate Cyclase R, an Inducer of Spore Encapsulation" @default.
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