Guanylyl cyclases with the topology of mammalian adenylyl cyclases and an N-terminal P-type ATPase-like domain in Paramecium, Tetrahymena and Plasmodium
1999; Springer Nature; Volume: 18; Issue: 15 Linguagem: Inglês
10.1093/emboj/18.15.4222
ISSN1460-2075
Autores Tópico(s)Protist diversity and phylogeny
ResumoArticle2 August 1999free access Guanylyl cyclases with the topology of mammalian adenylyl cyclases and an N-terminal P-type ATPase-like domain in Paramecium, Tetrahymena and Plasmodium Jürgen U. Linder Jürgen U. Linder Fakultät für Chemie und Pharmazie, Universität Tübingen, Auf der Morgenstelle 8, D-72076 Tübingen, Germany Search for more papers by this author Peter Engel Peter Engel Fakultät für Chemie und Pharmazie, Universität Tübingen, Auf der Morgenstelle 8, D-72076 Tübingen, Germany Search for more papers by this author Andreas Reimer Andreas Reimer Fakultät für Chemie und Pharmazie, Universität Tübingen, Auf der Morgenstelle 8, D-72076 Tübingen, Germany Search for more papers by this author Thomas Krüger Thomas Krüger Fakultät für Chemie und Pharmazie, Universität Tübingen, Auf der Morgenstelle 8, D-72076 Tübingen, Germany Search for more papers by this author Helmut Plattner Helmut Plattner Fakultät für Biologie, Universität Konstanz, D-78434 Konstanz, Germany Search for more papers by this author Anita Schultz Anita Schultz Fakultät für Chemie und Pharmazie, Universität Tübingen, Auf der Morgenstelle 8, D-72076 Tübingen, Germany Search for more papers by this author Joachim E. Schultz Corresponding Author Joachim E. Schultz Fakultät für Chemie und Pharmazie, Universität Tübingen, Auf der Morgenstelle 8, D-72076 Tübingen, Germany Search for more papers by this author Jürgen U. Linder Jürgen U. Linder Fakultät für Chemie und Pharmazie, Universität Tübingen, Auf der Morgenstelle 8, D-72076 Tübingen, Germany Search for more papers by this author Peter Engel Peter Engel Fakultät für Chemie und Pharmazie, Universität Tübingen, Auf der Morgenstelle 8, D-72076 Tübingen, Germany Search for more papers by this author Andreas Reimer Andreas Reimer Fakultät für Chemie und Pharmazie, Universität Tübingen, Auf der Morgenstelle 8, D-72076 Tübingen, Germany Search for more papers by this author Thomas Krüger Thomas Krüger Fakultät für Chemie und Pharmazie, Universität Tübingen, Auf der Morgenstelle 8, D-72076 Tübingen, Germany Search for more papers by this author Helmut Plattner Helmut Plattner Fakultät für Biologie, Universität Konstanz, D-78434 Konstanz, Germany Search for more papers by this author Anita Schultz Anita Schultz Fakultät für Chemie und Pharmazie, Universität Tübingen, Auf der Morgenstelle 8, D-72076 Tübingen, Germany Search for more papers by this author Joachim E. Schultz Corresponding Author Joachim E. Schultz Fakultät für Chemie und Pharmazie, Universität Tübingen, Auf der Morgenstelle 8, D-72076 Tübingen, Germany Search for more papers by this author Author Information Jürgen U. Linder1, Peter Engel1, Andreas Reimer1, Thomas Krüger1, Helmut Plattner2, Anita Schultz1 and Joachim E. Schultz 1 1Fakultät für Chemie und Pharmazie, Universität Tübingen, Auf der Morgenstelle 8, D-72076 Tübingen, Germany 2Fakultät für Biologie, Universität Konstanz, D-78434 Konstanz, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:4222-4232https://doi.org/10.1093/emboj/18.15.4222 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We cloned a guanylyl cyclase of 280 kDa from the ciliate Paramecium which has an N-terminus similar to that of a P-type ATPase and a C-terminus with a topology identical to mammalian adenylyl cyclases. Respective signature sequence motifs are conserved in both domains. The cytosolic catalytic C1a and C2a segments of the cyclase are inverted. Genes coding for topologically identical proteins with substantial sequence similarities have been cloned from Tetrahymena and were detected in sequences from Plasmodium deposited by the Malaria Genome Project. After 99 point mutations to convert the Paramecium TAA/TAG-Gln triplets to CAA/CAG, together with partial gene synthesis, the gene from Paramecium was heterologously expressed. In Sf9 cells, the holoenzyme is proteolytically processed into the two domains. Immunocytochemistry demonstrates expression of the protein in Paramecium and localizes it to cell surface membranes. The data provide a novel structural link between class III adenylyl and guanylyl cyclases and imply that the protozoan guanylyl cyclases evolved from an ancestral adenylyl cyclase independently of the mammalian guanylyl cyclase isoforms. Further, signal transmission in Ciliophora (Paramecium, Tetrahymena) and in the most important endoparasitic phylum Apicomplexa (Plasmodium) is, quite unexpectedly, closely related. Introduction cAMP is used universally as a second messenger. Therefore, the existence of at least three phylogenetically separate classes of adenylyl cyclases (ACs) is surprising. The membrane-attached ACs from Gram-negative bacteria (class I ACs) possess no sequence or topological similarity to the class II AC toxins from Bordetella pertussis or Bacillus anthracis. Both class I and II ACs are equally distant from the class III isozymes, which are found in mammals as well as in slime molds and many prokaryotes. The class III ACs share sequence similarity in their catalytic domains and, to a lesser extent, with the catalytic regions of mammalian soluble and membrane-bound guanylyl cyclases (GCs) (for a review see Barzu and Danchin, 1994). The currently accepted topology of mammalian ACs comprises two hydrophobic membrane cassettes of six α-helical transmembrane spans (TM), designated M1 and M2, and a catalytic center formed by two conserved cytoplasmic domains of ∼250 amino acids, C1a and C2a, which are localized downstream of M1 and M2, respectively (see Figure 1B for a schematic representation). C1a and C2a precede poorly conserved cytosolic segments of variable length, designated C1b and C2b. Initially it was speculated that the prominent membrane anchors of the ACs might entertain an independent transport activity because their topology is reminiscent of the multidrug resistance gene, the cystic fibrosis gene, various membrane transporters and voltage-gated channels (Krupinski et al., 1989). Yet, all attempts to demonstrate such a secondary function of class III ACs failed. Mammalian ACs have a low basal activity and their hormonal stimulation is mediated predominantly by G-proteins in conjunction with other effectors (for a review see Sunahara et al., 1996). Figure 1.(A) Sequence of the Paramecium guanylyl cyclase. Transmembrane regions are shaded; the four consensus blocks in the P-type ATPase-like domain and the two catalytic domains of the guanylyl cyclase region are in bold. (B) Suggested topology of the guanylyl cyclase. The 22 putative transmembrane spans are depicted as barrels, and cytosolic portions of the protein are shown as a duck head-like shape for P-type ATPases (Toyoshima et al., 1993; Zhang et al., 1998) and as a doughnut shape for the C1a/C2a heterodimer of the cyclase (Tesmer et al., 1997; Zhang et al., 1997). As in several mammalian adenylyl cyclases, a distinct C2b region is absent in the cloned guanylyl cyclase. For the C-terminal cyclase, the short-hand nomenclature used for mammalian adenylyl cyclases is adopted. M1/2 designate two membrane cassettes of six transmembrane spans, C1a, C1b and C2a domains coalesce as a cytosolic catalytic center. Download figure Download PowerPoint We reported that in the protozoans Paramecium and Tetrahymena, cAMP and cGMP formation are biochemical correlates of electrophysiological events. Thus, the control of cAMP levels in Paramecium is linked to a K+ outward current because K+ channel blockers such as tetraethylammonium, Cs+ and quinine inhibit hyperpolarization-activated cAMP formation, and because a mutant, restless, with a defect in the K+ resting conductance is unable to control cAMP formation (Schultz et al., 1992). This raised the possibility that within the AC protein from Paramecium the function of an ion pore which is intimately linked to the regulation of enzyme activity may have been preserved. Indeed, the purified AC of 97 kDa, i.e. comparable in size with the prototypical mammalian versions, displayed both an AC activity and cation conductance (Schultz et al., 1992). cGMP levels in Paramecium and Tetrahymena are regulated by a Ca2+ inward current (Schultz et al., 1986). Paramecium mutants with defects in Ca2+ channel regulation such as pawn or pantophobiac show deficits in cGMP regulation. To understand the molecular properties of Paramecium ACs and GCs, we wished to clone and express candidate cDNAs. As a start, we used a 328 bp genomic DNA sequence from Paramecium which presumably coded for a partial sequence of a mammalian-type AC (Hinrichsen et al., 1995), and expected to clone an AC in which the bulky membrane anchors had retained an ancestral ion conductance. To our surprise, we discovered a GC which is disguised in the topology of mammalian ACs and has an extended N-terminal domain with topological and amino acid sequence similarity to P-type ATPase ion pumps. After 99 point mutations for conversion from the ciliate to the universal DNA code and partial resynthesis of the cDNA, the gene was heterologously expressed. Similar genes were identified by cloning in Tetrahymena and in Plasmodium by searching the data released by the Malaria Genome Project. Our data provide a novel view on the evolution of mammalian ACs and GCs and suggest the existence of a new type of signal transduction in these protozoans which employs P-type ATPase-like structures as receptor. Results Cloning of a Paramecium cyclase with a mammalian AC topology The sequence of a 328 bp PCR product from genomic DNA of Paramecium which displayed 29% amino acid identity to the catalytic C2a region of the rat type III AC was kindly provided by C.Russel and R.D.Hinrichsen (Hinrichsen et al., 1995). Using specific primers, we amplified a 219 bp piece which was used as a probe to screen a Paramecium cDNA library prepared with poly(A)+ mRNA. We obtained two cDNA clones of 4.5 and 7.2 kb, both with a 3′ poly(A) tail. The open reading frame (ORF) was identical in both clones and ended at a TGA stop codon 47 bp upstream of the poly(A) tail. The 7.2 kb clone contained an ORF of 2408 amino acids; an ATG start codon was not identifiable. The 5′ ends of Paramecium genes are hardly ever detected in cDNA libraries because their 5′-untranslated regions (UTRs) usually are very short. A genomic DNA (gDNA) library prepared from EcoRI-digested DNA and a new probe located at the 5′ end of the clone were used to obtain the ATG start. The genomic sequence unequivocally identified an ATG as start just 12 bp upstream of the 5′ end of the 7.2 kb cDNA clone because of an in-frame TGA stop only a further 24 bp upstream. The ORF coded for a protein of 2412 amino acids with a calculated molecular mass of 282.6 kDa (Figure 1). Hydrophobicity analysis indicated five hydrophobic regions with 22 putative TMs. Similarity searches demonstrated that the protein consisted of two large units: an N-terminal half of 1319 amino acids (155 kDa) with similarity to P-type ATPases and a C-terminal half of 982 amino acids (115 kDa) with a topology identical to that of the prototypical mammalian AC (Figure 1A). Both units were linked by a polypeptide of 111 amino acids. To exclude the possibility that we cloned a cDNA which was joined accidentally during library preparation, we cloned a 2.5 kb EcoRI fragment from gDNA which was intronless, and we fully covered the intradomain region. Thus we unequivocally established the presence of this gene at the gDNA level. The Paramecium cyclase unit contained hydrophobic M1 and M2 regions with six TMs each which were followed by C1a- and C2a-positioned domains with sequence similarity to the catalytic loops of metazoan ACs (Figure 1B). The C1a-positioned domain was followed by a hydrophilic stretch of 221 amino acids reminiscent of the C1b region in mammalian ACs; a distinct C2b region was absent. All mammalian ACs have GDCY as a signature sequence in the C1a domain. In the cloned protozoan cyclase, the GDCY motif is present in the C2a-positioned domain (2276–2279), i.e. close to the C-terminus. Similarly, the motif TYMA is invariant in all C2a regions of mammalian ACs whereas in the ciliate cyclase it was located in the C1a-positioned domain (1686–1689), i.e. toward the 5′ end (Figure 2). Further, mammalian ACs contain a VKGKG motif in their C2a catalytic region. The first lysine presumably binds the γ-phosphate of ATP (Tesmer et al., 1997) and is essential for catalysis. In the Paramecium gene, a similar motif, 1793AKGKG1797, was found in the C1a position. Therefore, we conclude that the C1a-positioned and C2a-positioned regions of the Paramecium cyclase were inverted compared with the mammalian AC congeners resulting in a (mammalian nomenclature) M1C2aC1bM2C1a architecture. Recently, the amino acids in the heterodimeric catalytic pocket of metazoan ACs and GCs which are important for substrate specificity, K, D and Q in ACs, E, C and R in GCs, have been determined by X-ray crystallography and site-directed mutagenesis (Tesmer et al., 1997; Zhang et al., 1997; Sunahara et al., 1998; Tucker et al., 1998). In the Paramecium cyclase, these crucial amino acids are GC-like, with a substitution of C by S1748 (Figure 2). On the other hand, several amino acids which form a second, non-catalytic pocket in mammalian ACs were conserved in the protozoan cyclase, i.e. were like those in ACs (Figure 2). The results imply that the cloned gene represents a novel GC which is very closely related to metazoan ACs by topology and primary structure. Below, the name Paramecium GC is adopted for this gene/enzyme. Figure 2.Local alignment of the catalytic C1a- and C2a-positioned regions of the Paramecium GC (PARA) with corresponding mammalian sequences [rat soluble guanylyl cyclase α1- and β1-subunits: sol GCα/β (Nakane et al., 1990); rat type III AC (Bakalyar and Reed, 1990); bovine type VII AC and rat type VIII AC (Cali et al., 1994; Völkel et al., 1996)]. The signature sequences GDCY and TYMA are in bold. Those residues that determine substrate specifity are printed inverted. The boxed amino acids are located in a non-catalytic pocket of the mammalian AC heterodimer and their positions correspond to those which determine the substrate specificity in the catalytic groove (Tesmer et al., 1997; Zhang et al., 1997). The switch of the catalytic domains in the Paramecium GC is apparent. Download figure Download PowerPoint P-type ion transport ATPases were the only proteins with significant similarity to the N-terminal half of the Paramecium GC. It had a predicted membrane topology identical to that of P-type ATPases, i.e. two cassettes of two putative TMs in the N-terminal portion (amino acids 63–110 and 349–407) and one set of six TMs near the C-terminus (amino acids 1121–1319; Figure 1A; Stokes et al., 1994; Zhang et al., 1998). In the cytosolic domains of the P-type ATPase family which has >150 members, several sequence blocks are conserved (Henikoff and Henikoff, 1992). Four of these align to corresponding cytosolic regions of the Paramecium GC. DKTGT(L/I)T, which is located within the large cytoplasmic loop, is an invariant signature sequence in all P-type ATPases. For ion transport to occur, the aspartate must be phosphorylated (Allen and Green, 1976). In the Paramecium GC, DKTGTLT was conserved at the equivalent position (amino acids 461–467). A conserved GDGXND motif present in the hinge region of P-type ATPases was 1025GDSFSD1030 in the Paramecium gene, and the (TSND)GE (SNT) block in the transduction domain was retained with a signifcant E to N change at 240SGNT243 virtually excluding ATPase function (Clarke et al., 1990a; Fagan and Saier, 1994). Finally, the ATP-binding domain in nearly all eukaryotic P-type ATPases involves an indispensable aspartate in a block of moderately conserved amino acids (Clarke et al., 1990b). The corresponding amino acid in the Paramecium P-type ATPase-like domain was Glu848. These decisive deviations of the Paramecium sequence from the ATPase consensus imply that this GC domain has adopted another function. Expression of Paramecium GC Transfection of Paramecium is not yet established. However, heterologous expression of Paramecium genes is impossible because it uses the universal stop codons TAA and TAG for glutamine (Preer et al., 1991). Therefore, we painstakingly changed all 99 TAA and TAG codons to CAA/CAG by site-directed mutagenesis (Deng and Nickoloff, 1992). The 'corrected' ORF was inserted into the bicistronic expression vector pIRES1neo (Rees et al., 1996). However, upon transfection, no HEK293 cells survived the G418 selection. Using a green fluorescent protein (GFP)-based reporter assay (Trouet et al., 1997) with the cyclase domain, we realized that HEK293 cells did not transcribe a functional mRNA compared with cells transfected with a vector containing a bovine type VII AC cDNA (data not shown). Assuming that the problem may be caused by the high AT content of the Paramecium GC gene (66%) and in order to localize the problem, we used the bovine AC as a scaffold to test for proper transcription of individual protozoan cDNA segments. We separately inserted six cDNA sequences of the cyclase domain (N-terminal amino acids, 1380–1420; M1, 1421–1589; C1a-positioned, 1590–1806; C1b-positioned, 1807–1996; M2, 1997–2178; and C2a-positioned, 2179–2412) at the corresponding positions of the bovine type VII AC cDNA using appropriately generated restriction sites. No expression of the GFP reporter gene was observed when M1 or M2 of the protozoan cyclase replaced the respective bovine membrane cassettes. Therefore, we resynthesized the ciliate cDNA coding for M1 and M2 using the standard mammalian codon usage. This introduced a 58% G/C content into ∼34% of the Paramecium GC domain. The partially synthetic gene in pIRESneo was again transfected into HEK293 cells and yielded a G418-selectable cell population. Using an antibody directed against the C1b-positioned domain (K1811–2007), a set of bands at ∼95 kDa was stained specifically in Western blots, indicating protein expression of the M1380–Q2412 GC domain (data not shown). Cell homogenates had membrane-bound GC activities of up to 150 pmol/mg/min (Figure 3A). The Km for MgGTP was 50 μM. The GC activity was unaffected by 50 μM Ca2+, 1 mM EGTA or by Paramecium Ca2+/calmodulin. AC activity was not observed with MgATP as a substrate, yet it was detectable using Mn2+ as a metal cofactor (Figure 3A). Forskolin did not enhance enzyme activity. These results proved that the cyclase domain of the cloned gene constituted a GC in the disguise of what hitherto had been considered to be a prototypical mammalian AC topology. We were unable to express the holoenzyme cDNA in HEK293 cells. In the hope of obtaining higher expression levels of the GC domain and an expression of the full-length clone, we turned to Sf9 cells and the baculovirus system for expression. Figure 3.Cyclase activities of the recombinant guanylyl cyclase domain and of the holoenzyme. (A) Guanylyl and adenylyl cyclase activities in cell membranes of HEK293 cells transfected with the GC domain were measured with 75 μM MgGTP or MnATP, respectively. (B) Guanylyl and adenylyl cyclase activities in cell membranes from Sf9 cells infected with either the GC domain (black bars) or the GC holoenzyme (shaded bars) were measured with MgGTP or MnATP as indicated. In either expression system, cAMP formation with MgATP as a substrate was barely above background. Open bars are from vector-infected controls. Note the different scales of the ordinates indicating the much higher expression efficiency in Sf9 cells. Download figure Download PowerPoint Expression of the Paramecium GC domain in Sf9 cells was equally efficient whether the ciliate cDNA (stop codons removed) or the partially synthetic cDNA clone (see above) was used for infection. GC catalytic activities were ∼10-fold higher than in HEK293 cells (Figure 3B). The Km for MgGTP was 32 μM. AC activity was negligible using MgATP as a substrate, yet was substantial with Mn2+ as a divalent cation (Figure 3B). In a competition assay with 75 μM GTP and 1 mM ATP in the presence of 5 mM Mg2+ as the physiologically relevant divalent cation, GC activity was diminished by only 25%, i.e. the Paramecium cyclase domain truly is a GC. The expression of the GC domain was monitored by Western blotting using an antibody directed against the C1b-positioned region (Figure 4, lane 4). The calculated mass of the GC domain was 115 kDa. The slightly lower apparent molecular weight is probably due to the hydrophobic transmembrane regions, as similar effects have been reported for proteins with large transmembrane domains (Hauser et al., 1998; Mons et al., 1998). Variable glycosylation may account for the appearance of closely spaced bands. Figure 4.Proteolytic processing of full-length Paramecium guanylyl cyclase expressed in Sf9 cells. Membranes were probed with the anti ATPase antibody (lane 1 and 2) or with the anti-GC antibody (lanes 3–5). Lanes 2 and 3 are from cells infected with the 7.2 kb cDNA coding for the holoenzyme. In lane 4, the expression of a 3 kb cDNA coding only for the GC domain was tested. Lanes 1 and 5 are membranes from control infections. The specificity of the antibodies was ascertained by pre-blocking with the respective antigens and cross-blocking experiments (blots not shown). Download figure Download PowerPoint We were unable to express the full-length Paramecium GC gene in Sf9 cells as determined by Western blotting. Again we reasoned that this may have been due to the high A/T content of the gene, in particular to two stretches of seven and nine As in the ATPase-like region. Multiple As in an mRNA may constitute a signal for premature mRNA degradation, and slippage may occur during replication and transcription (Laken et al., 1997). Therefore, we resynthesized four DNA segments of the ATPase-like domain comprising 46% of this gene portion (nucleotides 1–436, 1012–1387, 1973–2363 and 3303–3991). The G/C content of the synthetic DNA was 56%; the amino acid sequence remained unchanged. Nevertheless, for reasons not yet understood, we were unable to express only the ATPase-like domain in Sf9 cells as determined by Western blot analysis. However, upon infection with the partially synthetic gene coding for the holoenzyme, expression in Sf9 cells was achieved (Figure 4, lanes 2 and 3), although we never detected a product corresponding to the full-length protein of 280 kDa. Using affinity-purified polyclonal antibodies directed either against a 20 kDa cytosolic portion of the P-type ATPase-like domain (L549–M718; anti-ATPase antibody), the 23 kDa C1b-positioned segment of the cyclase domain (K1811–A2007; anti-GC antibody) or a peptide antibody directed against the N-terminal hexadecapeptide R5–N20, we labeled specific bands of lower molecular mass in a Western blot. Because Sf9 membrane proteins up to 350 kDa were blotted successfully as visualized by Ponceau S staining, and because inclusion of a cocktail of protease inhibitors during cell lysis had no effect, the result most likely reflected protein processing in the Sf9 cells. The anti-GC antibody recognized proteins with an apparent molecular mass of mainly 85 kDa up to 100 kDa (Figure 4, lane 3). In the same membrane preparations, the anti-ATPase antibody and the N-terminal peptide antibody specifically detected a 120 kDa band (Figure 4, lane 2, and data not shown). Any unspecificity and cross-reactivity of the antibodies was excluded by respective pre-blocking experiments (data not shown). The proteolytic processing in Sf9 cells must have occurred in the area linking the ATPase-like and the GC domains because of the size of the two generated proteins and beause GC activity was obtained (Figure 3B). This conclusion was supported by the isolation of mRNA from infected Sf9 cells and the unequivocal demonstration by RT–PCR that a correct and uninterrupted mRNA existed (data not shown). In Sf9 cells infected with the gene for the holoenzyme, GC activity was ∼50% of that obtained with the GC domain expressed alone. All enzymatic properties of this proteolytically processed holoenzyme corresponded to those of the expressed GC domain. This finding indicated that the physical presence of the ATPase-like protein in the membrane did not affect GC activity per se. Localization of Paramecium GC Using anti-GC or anti-ATPase antibodies, we detected the GC immunocytochemically in cross-sectioned Paramecium using gold conjugates of F(ab)2 as a second antibody (Table I; Figure 5). Both primary antibodies labeled the same membranes, i.e. the GC was present in the ciliary membrane, in the complex formed by the cell membrane and the outer alveolar sac membrane (not resolvable) and in the inner alveolar sac membrane. Background without the first antibody was negligible (Table I). In semi-quantitative evaluations with both antibodies, we subtracted background labeling determined for probably irrelevant membranes such as outer mitochondrial and trichocyst membranes (Table I). The differences in the labeling intensity were probably caused by differential accessibilities or different affinities of the antigenic sites for the respective primary antibodies. Figure 5.Immunolocalization of the guanylyl cyclase domain in formaldehyde-fixed Paramecium with a rabbit anti-GC antibody followed by incubation with gold-labeled (5 nm) F(ab)2 from goat anti-rabbit antibodies. Note the label on the complex formed by the cell membrane and the outer alveolar sac membrane (CM/OASM), on the inner alveolar sac membrane (IASM), on a cilium cross-section (Ci) and on a 'grazing' section along the membrane of an emerging longitudinally cut cilium (upper right). Download figure Download PowerPoint Table 1. Immunogold labeling of Paramecium cellular structures with anti-GC antibody or anti-ATPase antibody Structures analyzed Au grains per 100 μm membrane length Anti-GC Anti-ATPase CM/OASM 37.9 4.8 IASM 34.5 12.0 CiM 17.6 7.2 TM 0.0 0.0 OMM 0.4 0.0 CM/OASM, cell membrane/outer alveolar sac membrane complex; IASM, inner alveolar sac membrane (facing cell center); CiM, ciliary membrane; TM, OMM, trichocyst or outer mitochondrial membranes. Chimeras between Paramecium GC and mammalian AC The GC domain is topologically identical to mammalian ACs. Therefore, we investigated whether a mammalian membrane anchor would be compatible with the GC catalytic domains derived from Paramecium. First, we replaced the mammalian C1a, C1b and C2a regions of a bovine type VII AC (Völkel et al., 1996) by the C1a-positioned, C1a,b-positioned and C2a-positioned regions of the Paramecium GC domain, i.e. using the mammalian AC as a scaffold for the Paramecium catalyst (Figure 6, constructs I and II). Secondly, we generated a construct with an inverse order of the C1a- and C2a-positioned regions from Paramecium because, as outlined above, these two regions seem to have been switched in the Paramecium GC domain (construct III). The constructs showed substantial and similar GC activity and a minor AC activity when assayed with MnATP (Figure 6). We conclude that the Paramecium GC catalytic center comprised of C1- and C2-positioned loops is expressed and active in the frame of a mammalian AC irrespective of the origin of the C1b region and, surprisingly, of the arrangement of the catalytic subdomains. Figure 6.Guanylyl cyclase and adenylyl cyclase activities of chimeras between the Paramecium GC domain and bovine type VII AC. Regions from the Paramecium GC are shaded, type VII AC domains are open boxes. Compatible restriction sites (MluI–AscI and SalI–XhoI) were introduced as shown and used for domain shuffling. The locations of the restriction sites in the Paramecium GC were: XhoI at 4765; AvrII at 5986; SalI at 6532; and AscI at the 3′ terminus; and in the bovine type VII AC: SalI at 1183; MluI at 2032; AvrII at 2350; and XhoI at 3022. GC activity was assayed with 75 μM GTP and 5 mM Mg2+, AC activity with 75 μM ATP and 2 mM Mn2+. Basal enzyme activities (infections with vector without insert) were subtracted. Download figure Download PowerPoint Tetrahymena guanylyl cyclase The heterologous expression of Paramecium GC holoenzyme in Sf9 cells did not reveal if and how the GC activity is regulated by the P-type ATPase-like domain. Therefore, we wished to clone a GC from the related ciliate Tetrahymena for sequence comparisons. A PCR with primers designed to recognize a protozoan C1a-positioned region and cDNA as a template yielded six different PCR products with a high degree of amino acid identity (Figure 7). One fragment was chosen arbitrarily as a probe to clone the corresponding full-length gene from a Tetrahymena gDNA library; 9.7 kb of genomic sequence was obtained by chromosome walking. The 3′ end of the ORF was completed by vectorette PCR (Gonzalez and Chan, 1993) and the introns were identified unambigously by PCR using Tetrahymena cDNA as a template. The protein encoded by the 8.4 kb ORF had exactly the same topology as the Paramecium GC: an N-terminal P-type ATPase-like domain of 168 kDa, followed by a 146 kDa GC domain with mammalian AC membrane topology. As in the Paramecium GC, the C1a- and C2a-positioned regions were inverted compared with mammalian ACs. The amino acids decisive for substrate specificity (E1808 and S1885 in C1a and R2734 in C2a) unequivocally identified the encoded protein as a GC. Interestingly, the P-type ATPase-like domain resembled the general ATPase consensus sequence much less than this region from t
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