Two Highly Conserved Glutamate Residues Critical for Type III Sodium-dependent Phosphate Transport Revealed by Uncoupling Transport Function from Retroviral Receptor Function
2002; Elsevier BV; Volume: 277; Issue: 45 Linguagem: Inglês
10.1074/jbc.m207096200
ISSN1083-351X
AutoresPernille Bøttger, Lene Pedersen,
Tópico(s)Genetic Syndromes and Imprinting
ResumoType III sodium-dependent phosphate (NaPi) cotransporters, Pit1 and Pit2, have been assigned housekeeping Pi transport functions and suggested involved in chondroblastic and osteoblastic mineralization and ectopic calcification. Both proteins exhibit dual function, thus, besides being transporters, they also serve as receptors for several gammaretroviruses. We here show that it is possible to uncouple transport and receptor functions of a type III NaPicotransporter and thus exploit the retroviral receptor function as a control for proper processing and folding of mutant proteins. Thus exchanging two putative transmembranic glutamate residues in human Pit2, Glu55 and Glu575, with glutamine or with lysine severely impaired or knocked out, respectively, Pitransport function, but left viral receptor function undisturbed. Both glutamates are conserved in type III NaPi cotransporters, in fungal NaPi cotransporters PHO-4 and Pho89, and in other known or putative phosphate permeases from a number of species and are the first residues shown to be critical for type III NaPicotransport. Their putative transmembranic positions together with the presented data are consistent with Glu55 and Glu575 being parts of a cation liganding site or playing roles in conformational changes associated with substrate transport. Finally, the results also show that Pit2 retroviral receptor function per se is not dependent on Pit2 Pitransport function. Type III sodium-dependent phosphate (NaPi) cotransporters, Pit1 and Pit2, have been assigned housekeeping Pi transport functions and suggested involved in chondroblastic and osteoblastic mineralization and ectopic calcification. Both proteins exhibit dual function, thus, besides being transporters, they also serve as receptors for several gammaretroviruses. We here show that it is possible to uncouple transport and receptor functions of a type III NaPicotransporter and thus exploit the retroviral receptor function as a control for proper processing and folding of mutant proteins. Thus exchanging two putative transmembranic glutamate residues in human Pit2, Glu55 and Glu575, with glutamine or with lysine severely impaired or knocked out, respectively, Pitransport function, but left viral receptor function undisturbed. Both glutamates are conserved in type III NaPi cotransporters, in fungal NaPi cotransporters PHO-4 and Pho89, and in other known or putative phosphate permeases from a number of species and are the first residues shown to be critical for type III NaPicotransport. Their putative transmembranic positions together with the presented data are consistent with Glu55 and Glu575 being parts of a cation liganding site or playing roles in conformational changes associated with substrate transport. Finally, the results also show that Pit2 retroviral receptor function per se is not dependent on Pit2 Pitransport function. inorganic phosphate sodium-dependent phosphate murine leukemia virus amphotropic MLV an MLV isolate related to AM-MLV QQQ, etc., abbreviations for mutant Pit2 proteins where the three letters indicate the identity of the amino acids in positions 55, 91, and 575, respectively transmembrane Chinese hamster ovary phosphate-buffered saline N-hydroxysulfosuccinimide Inorganic phosphate (Pi)1 is essential for cellular metabolism and skeletal mineralization. Moreover, it serves as the source of phosphate for organic cell constituents, e.g. nucleotides and a variety of phosphorylated metabolic intermediates. Two proteins that show the same transport characteristics as Pi uptake across the plasma membrane in animal cells have been identified (1Kavanaugh M.P. Miller D.G. Zhang W. Law W. Kozak S.L. Kabat D. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7071-7075Crossref PubMed Scopus (526) Google Scholar, 2Olah Z. Lehel C. Anderson W.B. Eiden M.V. Wilson C.A. J. Biol. Chem. 1994; 269: 25426-25431Abstract Full Text PDF PubMed Google Scholar, 3Wilson C.A. Eiden M.V. Anderson W.B. Lehel C. Olah Z. J. Virol. 1995; 69: 534-537Crossref PubMed Google Scholar), namely the sodium-dependent phosphate (NaPi) cotransporters, Pit1 (human Pit1 formerly GLVR1 (4O'Hara B. Johann S.V. Klinger H.P. Blair D.G. Rubinson H. Dunn K.J. Sass P. Vitek S.M. Robins T. Cell Growth Differ. 1990; 1: 119-127PubMed Google Scholar)) and Pit2 (human Pit2 formerly GLVR2 (5van Zeijl M. Johann S.V. Closs E. Cunningham J. Eddy R. Shows T.B. O'Hara B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1168-1172Crossref PubMed Scopus (240) Google Scholar)). Both proteins are characterized as type III NaPi cotransporters (6Kavanaugh M.P. Kabat D. Kidney Int. 1996; 49: 959-963Abstract Full Text PDF PubMed Scopus (159) Google Scholar) and show a broad tissue distribution being expressed in all investigated human tissues albeit at different levels (7Uckert W. Willimsky G. Pedersen F.S. Blankenstein T. Pedersen L. Hum. Gene Ther. 1998; 9: 2619-2627Crossref PubMed Google Scholar). Furthermore, low extracellular Pilevels result in up-regulated Pit1 and Pit2 expression in mammalian cells (1Kavanaugh M.P. Miller D.G. Zhang W. Law W. Kozak S.L. Kabat D. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7071-7075Crossref PubMed Scopus (526) Google Scholar, 8Chien M.L. O'Neill E. Garcia J.V. Virology. 1998; 240: 109-117Crossref PubMed Scopus (26) Google Scholar). These observations strongly suggest that the major cellular Pi demand in mammalian cells is handled by type III NaPi cotransporters (1Kavanaugh M.P. Miller D.G. Zhang W. Law W. Kozak S.L. Kabat D. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7071-7075Crossref PubMed Scopus (526) Google Scholar). However, recent results also point at type III transporters as playing specific roles in chondroblastic and osteoblastic mineralization (9Nielsen L.B. Pedersen F.S. Pedersen L. Bone. 2001; 28: 160-166Crossref PubMed Scopus (61) Google Scholar, 10Palmer G. Zhao J. Bonjour J. Hofstetter W. Caverzasio J. Bone. 1999; 24: 1-7Crossref PubMed Scopus (82) Google Scholar) as well as being critically involved in vascular calcification under hyperphosphatemic conditions, which are often present in diabetic patients and individuals with renal failure (11Jono S. McKee M.D. Murry C.E. Shioi A. Nishizawa Y. Mori K. Morii H. Giachelli C.M. Circ. Res. 2000; 87: e10-e17Crossref PubMed Google Scholar). The mechanisms underlying the bone-forming roles of type III NaPi transporters are presently not known. Recent results, however, showed that high extracellular Pi levels can induce expression of the gene for osteopontin and that the induction is dependent on Na+-dependent Pi uptake across the plasma membrane (12Beck Jr., G.R. Zerler B. Moran E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8352-8357Crossref PubMed Scopus (417) Google Scholar). Interestingly, osteopontin is involved in normal bone development and present in calcified arterial plaques (13Giachelli C.M. Am. J. Pathol. 1999; 154: 671-675Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar).Despite the important roles of type III NaPi cotransporters in cellular Pi uptake and increasing evidence for their critical roles in normal and pathologic calcification, nothing is known about what determines their transport function. The two known type III NaPi cotransporters, Pit1 and Pit2, show about 60% amino acid identity (5van Zeijl M. Johann S.V. Closs E. Cunningham J. Eddy R. Shows T.B. O'Hara B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1168-1172Crossref PubMed Scopus (240) Google Scholar), whereas orthologs of Pit1 and Pit2 exhibit more than 90% amino acid identity (5van Zeijl M. Johann S.V. Closs E. Cunningham J. Eddy R. Shows T.B. O'Hara B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1168-1172Crossref PubMed Scopus (240) Google Scholar, 14Johann S.V. Gibbons J.J. O'Hara B. J. Virol. 1992; 66: 1635-1640Crossref PubMed Google Scholar, 15Wilson C.A. Farrell K.B. Eiden M.V. J. Virol. 1994; 68: 7697-7703Crossref PubMed Google Scholar). A putative topological model for both proteins based on hydropathy plots predicts 10 transmembrane (TM) regions and 5 extracellular loops (5van Zeijl M. Johann S.V. Closs E. Cunningham J. Eddy R. Shows T.B. O'Hara B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1168-1172Crossref PubMed Scopus (240) Google Scholar, 14Johann S.V. Gibbons J.J. O'Hara B. J. Virol. 1992; 66: 1635-1640Crossref PubMed Google Scholar, 16Chien M.L. Foster J.L. Douglas J.L. Garcia J.V. J. Virol. 1997; 71: 4564-4570Crossref PubMed Google Scholar). No significant overall sequence similarity exists between type III cotransporters and members of the two other NaPi cotransport systems referred to as types I and II (1Kavanaugh M.P. Miller D.G. Zhang W. Law W. Kozak S.L. Kabat D. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7071-7075Crossref PubMed Scopus (526) Google Scholar).Both Pit1 and Pit2 were originally identified as receptors for retroviruses belonging to the gammaretrovirus genus, and they are currently the two cellular receptors targeted in human gene therapy trials employing retroviral vectors. The human gene SLC20A1encoding Pit1 was cloned as a receptor for gibbon ape leukemia virus (4O'Hara B. Johann S.V. Klinger H.P. Blair D.G. Rubinson H. Dunn K.J. Sass P. Vitek S.M. Robins T. Cell Growth Differ. 1990; 1: 119-127PubMed Google Scholar) and feline leukemia virus subgroup B (17Takeuchi Y. Vile R.G. Simpson G. O'Hara B. Collins M.K. Weiss R.A. J. Virol. 1992; 66: 1219-1222Crossref PubMed Google Scholar), and the Pit2-encoding gene SLC20A2 from human and rats was cloned as receptor for amphotropic murine leukemia virus (AM-MLV) (5van Zeijl M. Johann S.V. Closs E. Cunningham J. Eddy R. Shows T.B. O'Hara B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1168-1172Crossref PubMed Scopus (240) Google Scholar, 18Miller D.G. Edwards R.H. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 78-82Crossref PubMed Scopus (327) Google Scholar); an AM-MLV-related isolate, 10A1 MLV, utilizes both transporters as receptor (19Miller D.G. Miller A.D. J. Virol. 1994; 68: 8270-8276Crossref PubMed Google Scholar). In recent years, substantial insight into the receptor functions of Pit1 and Pit2 for their cognate viruses has been obtained (reviewed in Refs.20Overbaugh J. Miller A.D. Eiden M.V. Microbiol. Mol. Biol. Rev. 2001; 65: 371-389Crossref PubMed Scopus (155) Google Scholar and 21Sommerfelt M.A. J. Gen. Virol. 1999; 80: 3049-3064Crossref PubMed Scopus (67) Google Scholar).We speculated that the retroviral receptor function might provide a functional assay for proper processing and folding of mutant type III NaPi transporters with knocked out NaPitransport function thus allowing for identification of amino acids critical for transport function. We here show that it is possible to uncouple human Pit2 retroviral receptor function from NaPitransport function and to exploit the dual function of Pit2 as NaPi transporter and retroviral receptor for identification of amino acids critical for NaPi transport. Doing this, we identified two Pit2 glutamate residues Glu55 (E55) and Glu575 (E575) as critical for Na+-dependent Pi transport function. The glutamates are positioned in putative transmembranic domains and are conserved in type III NaPi cotransporters, in NaPi cotransporters PHO-4 from Neurospora crassa (22Versaw W.K. Metzenberg R.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3884-3887Crossref PubMed Scopus (70) Google Scholar) and Pho89 from Saccharomyces cerevisiae(23Martinez P. Persson B.L. Mol. Gen. Genet. 1998; 258: 628-638Crossref PubMed Scopus (154) Google Scholar), and in a number of known and putative phosphate permeases from other species (see on-line supplemental material) strongly suggesting that they also are critical for the physiological function of these transporters.DISCUSSIONType III NaPi cotransporters, Pit1 and Pit2, exhibit dual function. Here we have shown that it is possible to knock out Pi transport function of human Pit2, leaving its retroviral receptor function undisturbed. Thus, it is possible to exploit the retroviral receptor function of Pit2, and possibly of Pit1, as a control for processing and folding of mutant proteins with knocked out transport function. Doing this, we identified two glutamate residues, E55 and E575 in human Pit2, as critical for Pit2 Pitransport function. This is the first time amino acids critical for Pi transport of a type III NaPi cotransporter have been identified. The two glutamates are both highly conserved in type III NaPi cotransporters and in a number of known and putative phosphate permeases from other species (see supplemental material) strongly suggesting that they also are critical for the physiological function of these transporters.The strength of employing retroviral receptor function as a control for correct processing and folding of mutant Pit2 proteins rather than relying on protein expression in outer membranes is illustrated with the mutant QQQ. It exhibited severely impaired transport function, which could not be explained by the protein not being present in the oocyte outer membrane, but it did, however, also show significantly impaired receptor function. It is possible that impaired receptor function can be due to involvement in retroviral entry of the amino acids analyzed; however, in this case, it is not likely, because mutant proteins like QEE, EQE, QEQ, KEE, EEK, and KEK were fully functional receptors, rather we suggest that the QQQ mutant proteins in the plasma membrane exhibit disturbed processing and/or folding.The question arises whether viral receptor function will be applicable as control when studying involvement of extracellular loop positioned amino acids in transport function of type III transporters. It is in this connection noteworthy that the amino acids so far identified as critical for retroviral receptor function of Pit1 and Pit2 orthologs are those highly variable between Pit1 and Pit2 and the NaPi transporter PHO-4 from N. crassa(19Miller D.G. Miller A.D. J. Virol. 1994; 68: 8270-8276Crossref PubMed Google Scholar, 24Pedersen L. Johann S.V. van Zeijl M. Pedersen F.S. O'Hara B. J. Virol. 1995; 69: 2401-2405Crossref PubMed Google Scholar, 37Dreyer K. Pedersen F.S. Pedersen L. J. Virol. 2000; 74: 2926-2929Crossref PubMed Scopus (12) Google Scholar, 39Eiden M.V. Farrell K.B. Wilson C.A. J. Virol. 1996; 70: 1080-1085Crossref PubMed Google Scholar, 40Leverett B.D. Farrell K.B. Eiden M.V. Wilson C.A. J. Virol. 1998; 72: 4956-4961Crossref PubMed Google Scholar, 41Lundorf M.D. Pedersen F.S. O'Hara B. Pedersen L. J. Virol. 1999; 73: 3169-3175Crossref PubMed Google Scholar, 42Pedersen L. van Zeijl M. Johann S.V. O'Hara B. J. Virol. 1997; 71: 7619-7622Crossref PubMed Google Scholar, 43Tailor C.S. Nouri A. Kabat D. J. Virol. 2000; 74: 237-244Crossref PubMed Scopus (23) Google Scholar), whereas the amino acids critical for NaPi uptake are expected to be found among the residues conserved in Pit1, Pit2, and related proteins from other species as shown in the present work. In line with this, we have recently shown that a Pit1 mutant with abolished receptor functions showed no impairment in Pi transport function as analyzed inXenopus oocytes. 2M. D. Lundorf, P. Bøttger, and L. Pedersen, unpublished data. Thus, it is possible that there in general is no overlap between Pit sequences critical for Pi transport function and Pit sequences critical for receptor function.We do not know the exact role of Pit2 residues E55 and E575 in Pi transport. However, the observation that changing either E55 or E575 for lysine was sufficient to knock out Pitransport function leaving receptor function undisturbed is indeed in agreement with these residues being involved in NaPitransport function, rather than fulfilling structural roles. It is possible that E55 and E575 are involved in function-dependent conformational changes of the transporter. Our data are, however, also consistent with these glutamates being parts of a cation liganding site created by residues from different transmembrane regions as has been implicated for negatively charged residues of the melbiose permease ofEscherichia coli, the rabbit Na+/dicarboxylate cotransporter NaDC-1, Na+/H+ exchangers, thec subunit of the Propionigenium modestumF1/F0-ATP synthase and Na+,K+-ATPases (29Dibrov P. Fliegel L. FEBS Lett. 1998; 424: 1-5Crossref PubMed Scopus (72) Google Scholar, 30Griffith D.A. Pajor A.M. Biochemistry. 1999; 38: 7524-7531Crossref PubMed Scopus (27) Google Scholar, 31Pedersen P.A. Rasmussen J.H. Nielsen J.M. Jorgensen P.L. FEBS Lett. 1997; 400: 206-210Crossref PubMed Scopus (56) Google Scholar, 32Poolman B. Knol J. van der Does C. Henderson P.J. Liang W.J. Leblanc G. Pourcher T. Mus-Veteau I. Mol. Microbiol. 1996; 19: 911-922Crossref PubMed Scopus (134) Google Scholar, 33Vilsen B. Biochemistry. 1995; 34: 1455-1463Crossref PubMed Scopus (86) Google Scholar, 44Kaim G. Wehrle F. Gerike U. Dimroth P. Biochemistry. 1997; 36: 9185-9194Crossref PubMed Scopus (91) Google Scholar). However, a Na+-liganding site may contain only one acidic residue, the other residues being, e.g. glutamine and serine (44Kaim G. Wehrle F. Gerike U. Dimroth P. Biochemistry. 1997; 36: 9185-9194Crossref PubMed Scopus (91) Google Scholar). In this context it is worthy to note that at least some of the mutant Pit2 proteins with Gln for Glu substitutions in only one of the positions E55 or E575 (mutants QEE, EEQ, and EQQ) still supported low levels of NaPi uptake (Fig. 1 A).In recent years, a number of transport proteins have been identified that have dual function as transporters and retroviral receptors (4O'Hara B. Johann S.V. Klinger H.P. Blair D.G. Rubinson H. Dunn K.J. Sass P. Vitek S.M. Robins T. Cell Growth Differ. 1990; 1: 119-127PubMed Google Scholar, 5van Zeijl M. Johann S.V. Closs E. Cunningham J. Eddy R. Shows T.B. O'Hara B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1168-1172Crossref PubMed Scopus (240) Google Scholar,18Miller D.G. Edwards R.H. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 78-82Crossref PubMed Scopus (327) Google Scholar, 45Albritton L.M. Tseng L. Scadden D. Cunningham J.M. Cell. 1989; 57: 659-666Abstract Full Text PDF PubMed Scopus (552) Google Scholar, 46Rasko J.E. Battini J.L. Gottschalk R.J. Mazo I. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2129-2134Crossref PubMed Scopus (186) Google Scholar, 47Tailor C.S. Nouri A. Zhao Y. Takeuchi Y. Kabat D. J. Virol. 1999; 73: 4470-4474Crossref PubMed Google Scholar, 48Tailor C.S. Willett B.J. Kabat D. J. Virol. 1999; 73: 6500-6505Crossref PubMed Google Scholar). For murine CAT1, which is a Na+-dependent cationic amino acid transporter, it has also been shown that a glutamate residue in a putative transmembrane region was critical for the transport function but not for retroviral receptor function of the protein (49Wang H. Kavanaugh M.P. Kabat D. Virology. 1994; 202: 1058-1060Crossref PubMed Scopus (26) Google Scholar). Based on the data presented here and that of Wang et al. (49Wang H. Kavanaugh M.P. Kabat D. Virology. 1994; 202: 1058-1060Crossref PubMed Scopus (26) Google Scholar), we suggest that for proteins exhibiting dual function as transporters and retroviral receptors, their viral receptor function might in general be exploited for identification of amino acid residues critical for transport function. Using a transient transfection-infection assay, which is a fast and reliable method, for analyzing the viral receptor function as described here, indeed makes it a highly feasible approach to analyze processing/overall topology of mutant proteins by investigating their receptor function.The presented data also have implications for understanding the role of Pit2 in retroviral entry. Addition of sulfhydryl reagents to cells was previously shown to impair Pit2 Pi transport and AM-MLV infection but not virus binding (50Rodrigues P. Heard J.M. J. Virol. 1999; 73: 3789-3799Crossref PubMed Google Scholar). According to the authors, these data suggest that Pi-induced conformational changes in Pit2 are involved in AM-MLV entry via Pit2 (50Rodrigues P. Heard J.M. J. Virol. 1999; 73: 3789-3799Crossref PubMed Google Scholar). Our data are not in direct conflict with this interpretation, but they do show that viral receptor function is not dependent on Pi transport functionper se. Indeed, comparison of the data in Figs. 1 and 2reveals that there is no correlation between Na+-dependent Pi transport function and retroviral receptor function of Pit2. We suggest that further important insight into both functions of the type III NaPitransporters can be achieved by combining studies on transport and receptor functions as presented here. Inorganic phosphate (Pi)1 is essential for cellular metabolism and skeletal mineralization. Moreover, it serves as the source of phosphate for organic cell constituents, e.g. nucleotides and a variety of phosphorylated metabolic intermediates. Two proteins that show the same transport characteristics as Pi uptake across the plasma membrane in animal cells have been identified (1Kavanaugh M.P. Miller D.G. Zhang W. Law W. Kozak S.L. Kabat D. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7071-7075Crossref PubMed Scopus (526) Google Scholar, 2Olah Z. Lehel C. Anderson W.B. Eiden M.V. Wilson C.A. J. Biol. Chem. 1994; 269: 25426-25431Abstract Full Text PDF PubMed Google Scholar, 3Wilson C.A. Eiden M.V. Anderson W.B. Lehel C. Olah Z. J. Virol. 1995; 69: 534-537Crossref PubMed Google Scholar), namely the sodium-dependent phosphate (NaPi) cotransporters, Pit1 (human Pit1 formerly GLVR1 (4O'Hara B. Johann S.V. Klinger H.P. Blair D.G. Rubinson H. Dunn K.J. Sass P. Vitek S.M. Robins T. Cell Growth Differ. 1990; 1: 119-127PubMed Google Scholar)) and Pit2 (human Pit2 formerly GLVR2 (5van Zeijl M. Johann S.V. Closs E. Cunningham J. Eddy R. Shows T.B. O'Hara B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1168-1172Crossref PubMed Scopus (240) Google Scholar)). Both proteins are characterized as type III NaPi cotransporters (6Kavanaugh M.P. Kabat D. Kidney Int. 1996; 49: 959-963Abstract Full Text PDF PubMed Scopus (159) Google Scholar) and show a broad tissue distribution being expressed in all investigated human tissues albeit at different levels (7Uckert W. Willimsky G. Pedersen F.S. Blankenstein T. Pedersen L. Hum. Gene Ther. 1998; 9: 2619-2627Crossref PubMed Google Scholar). Furthermore, low extracellular Pilevels result in up-regulated Pit1 and Pit2 expression in mammalian cells (1Kavanaugh M.P. Miller D.G. Zhang W. Law W. Kozak S.L. Kabat D. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7071-7075Crossref PubMed Scopus (526) Google Scholar, 8Chien M.L. O'Neill E. Garcia J.V. Virology. 1998; 240: 109-117Crossref PubMed Scopus (26) Google Scholar). These observations strongly suggest that the major cellular Pi demand in mammalian cells is handled by type III NaPi cotransporters (1Kavanaugh M.P. Miller D.G. Zhang W. Law W. Kozak S.L. Kabat D. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7071-7075Crossref PubMed Scopus (526) Google Scholar). However, recent results also point at type III transporters as playing specific roles in chondroblastic and osteoblastic mineralization (9Nielsen L.B. Pedersen F.S. Pedersen L. Bone. 2001; 28: 160-166Crossref PubMed Scopus (61) Google Scholar, 10Palmer G. Zhao J. Bonjour J. Hofstetter W. Caverzasio J. Bone. 1999; 24: 1-7Crossref PubMed Scopus (82) Google Scholar) as well as being critically involved in vascular calcification under hyperphosphatemic conditions, which are often present in diabetic patients and individuals with renal failure (11Jono S. McKee M.D. Murry C.E. Shioi A. Nishizawa Y. Mori K. Morii H. Giachelli C.M. Circ. Res. 2000; 87: e10-e17Crossref PubMed Google Scholar). The mechanisms underlying the bone-forming roles of type III NaPi transporters are presently not known. Recent results, however, showed that high extracellular Pi levels can induce expression of the gene for osteopontin and that the induction is dependent on Na+-dependent Pi uptake across the plasma membrane (12Beck Jr., G.R. Zerler B. Moran E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8352-8357Crossref PubMed Scopus (417) Google Scholar). Interestingly, osteopontin is involved in normal bone development and present in calcified arterial plaques (13Giachelli C.M. Am. J. Pathol. 1999; 154: 671-675Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Despite the important roles of type III NaPi cotransporters in cellular Pi uptake and increasing evidence for their critical roles in normal and pathologic calcification, nothing is known about what determines their transport function. The two known type III NaPi cotransporters, Pit1 and Pit2, show about 60% amino acid identity (5van Zeijl M. Johann S.V. Closs E. Cunningham J. Eddy R. Shows T.B. O'Hara B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1168-1172Crossref PubMed Scopus (240) Google Scholar), whereas orthologs of Pit1 and Pit2 exhibit more than 90% amino acid identity (5van Zeijl M. Johann S.V. Closs E. Cunningham J. Eddy R. Shows T.B. O'Hara B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1168-1172Crossref PubMed Scopus (240) Google Scholar, 14Johann S.V. Gibbons J.J. O'Hara B. J. Virol. 1992; 66: 1635-1640Crossref PubMed Google Scholar, 15Wilson C.A. Farrell K.B. Eiden M.V. J. Virol. 1994; 68: 7697-7703Crossref PubMed Google Scholar). A putative topological model for both proteins based on hydropathy plots predicts 10 transmembrane (TM) regions and 5 extracellular loops (5van Zeijl M. Johann S.V. Closs E. Cunningham J. Eddy R. Shows T.B. O'Hara B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1168-1172Crossref PubMed Scopus (240) Google Scholar, 14Johann S.V. Gibbons J.J. O'Hara B. J. Virol. 1992; 66: 1635-1640Crossref PubMed Google Scholar, 16Chien M.L. Foster J.L. Douglas J.L. Garcia J.V. J. Virol. 1997; 71: 4564-4570Crossref PubMed Google Scholar). No significant overall sequence similarity exists between type III cotransporters and members of the two other NaPi cotransport systems referred to as types I and II (1Kavanaugh M.P. Miller D.G. Zhang W. Law W. Kozak S.L. Kabat D. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7071-7075Crossref PubMed Scopus (526) Google Scholar). Both Pit1 and Pit2 were originally identified as receptors for retroviruses belonging to the gammaretrovirus genus, and they are currently the two cellular receptors targeted in human gene therapy trials employing retroviral vectors. The human gene SLC20A1encoding Pit1 was cloned as a receptor for gibbon ape leukemia virus (4O'Hara B. Johann S.V. Klinger H.P. Blair D.G. Rubinson H. Dunn K.J. Sass P. Vitek S.M. Robins T. Cell Growth Differ. 1990; 1: 119-127PubMed Google Scholar) and feline leukemia virus subgroup B (17Takeuchi Y. Vile R.G. Simpson G. O'Hara B. Collins M.K. Weiss R.A. J. Virol. 1992; 66: 1219-1222Crossref PubMed Google Scholar), and the Pit2-encoding gene SLC20A2 from human and rats was cloned as receptor for amphotropic murine leukemia virus (AM-MLV) (5van Zeijl M. Johann S.V. Closs E. Cunningham J. Eddy R. Shows T.B. O'Hara B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1168-1172Crossref PubMed Scopus (240) Google Scholar, 18Miller D.G. Edwards R.H. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 78-82Crossref PubMed Scopus (327) Google Scholar); an AM-MLV-related isolate, 10A1 MLV, utilizes both transporters as receptor (19Miller D.G. Miller A.D. J. Virol. 1994; 68: 8270-8276Crossref PubMed Google Scholar). In recent years, substantial insight into the receptor functions of Pit1 and Pit2 for their cognate viruses has been obtained (reviewed in Refs.20Overbaugh J. Miller A.D. Eiden M.V. Microbiol. Mol. Biol. Rev. 2001; 65: 371-389Crossref PubMed Scopus (155) Google Scholar and 21Sommerfelt M.A. J. Gen. Virol. 1999; 80: 3049-3064Crossref PubMed Scopus (67) Google Scholar). We speculated that the retroviral receptor function might provide a functional assay for proper processing and folding of mutant type III NaPi transporters with knocked out NaPitransport function thus allowing for identification of amino acids critical for transport function. We here show that it is possible to uncouple human Pit2 retroviral receptor function from NaPitransport function and to exploit the dual function of Pit2 as NaPi transporter and retroviral receptor for identification of amino acids critical for NaPi transport. Doing this, we identified two Pit2 glutamate residues Glu55 (E55) and Glu575 (E575) as critical for Na+-dependent Pi transport function. The glutamates are positioned in putative transmembranic domains and are conserved in type III NaPi cotransporters, in NaPi cotransporters PHO-4 from Neurospora crassa (22Versaw W.K. Metzenberg R.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3884-3887Crossref PubMed Scopus (70) Google Scholar) and Pho89 from Saccharomyces cerevisiae(23Martinez P. Persson B.L. Mol. Gen. Genet. 1998; 258: 628-638Crossref PubMed Scopus (154) Google Scholar), and in a number of known and putative phosphate permeases from other species (see on-line supplemental material) strongly suggesting that they also are critical for the physiological function of these transporters. DISCUSSIONType III NaPi cotransporters, Pit1 and Pit2, exhibit dual function. Here we have shown that it is possible to knock out Pi transport function of human Pit2, leaving its retroviral receptor function undisturbed. Thus, it is possible to exploit the retroviral receptor function of Pit2, and possibly of Pit1, as a control for processing and folding of mutant proteins with knocked out transport function. Doing this, we identified two glutamate residues, E55 and E575 in human Pit2, as critical for Pit2 Pitransport function. This is the first time amino acids critical for Pi transport of a type III NaPi cotransporter have been identified. The two glutamates are both highly conserved in type III NaPi cotransporters and in a number of known and putative phosphate permeases from other species (see supplemental material) strongly suggesting that they also are critical for the physiological function of these transporters.The strength of employing retroviral receptor function as a control for correct processing and folding of mutant Pit2 proteins rather than relying on protein expression in outer membranes is illustrated with the mutant QQQ. It exhibited severely impaired transport function, which could not be explained by the protein not being present in the oocyte outer membrane, but it did, however, also show significantly impaired receptor function. It is possible that impaired receptor function can be due to involvement in retroviral entry of the amino acids analyzed; however, in this case, it is not likely, because mutant proteins like QEE, EQE, QEQ, KEE, EEK, and KEK were fully functional receptors, rather we suggest that the QQQ mutant proteins in the plasma membrane exhibit disturbed processing and/or folding.The question arises whether viral receptor function will be applicable as control when studying involvement of extracellular loop positioned amino acids in transport function of type III transporters. It is in this connection noteworthy that the amino acids so far identified as critical for retroviral receptor function of Pit1 and Pit2 orthologs are those highly variable between Pit1 and Pit2 and the NaPi transporter PHO-4 from N. crassa(19Miller D.G. Miller A.D. J. Virol. 1994; 68: 8270-8276Crossref PubMed Google Scholar, 24Pedersen L. Johann S.V. van Zeijl M. Pedersen F.S. O'Hara B. J. Virol. 1995; 69: 2401-2405Crossref PubMed Google Scholar, 37Dreyer K. Pedersen F.S. Pedersen L. J. Virol. 2000; 74: 2926-2929Crossref PubMed Scopus (12) Google Scholar, 39Eiden M.V. Farrell K.B. Wilson C.A. J. Virol. 1996; 70: 1080-1085Crossref PubMed Google Scholar, 40Leverett B.D. Farrell K.B. Eiden M.V. Wilson C.A. J. Virol. 1998; 72: 4956-4961Crossref PubMed Google Scholar, 41Lundorf M.D. Pedersen F.S. O'Hara B. Pedersen L. J. Virol. 1999; 73: 3169-3175Crossref PubMed Google Scholar, 42Pedersen L. van Zeijl M. Johann S.V. O'Hara B. J. Virol. 1997; 71: 7619-7622Crossref PubMed Google Scholar, 43Tailor C.S. Nouri A. Kabat D. J. Virol. 2000; 74: 237-244Crossref PubMed Scopus (23) Google Scholar), whereas the amino acids critical for NaPi uptake are expected to be found among the residues conserved in Pit1, Pit2, and related proteins from other species as shown in the present work. In line with this, we have recently shown that a Pit1 mutant with abolished receptor functions showed no impairment in Pi transport function as analyzed inXenopus oocytes. 2M. D. Lundorf, P. Bøttger, and L. Pedersen, unpublished data. Thus, it is possible that there in general is no overlap between Pit sequences critical for Pi transport function and Pit sequences critical for receptor function.We do not know the exact role of Pit2 residues E55 and E575 in Pi transport. However, the observation that changing either E55 or E575 for lysine was sufficient to knock out Pitransport function leaving receptor function undisturbed is indeed in agreement with these residues being involved in NaPitransport function, rather than fulfilling structural roles. It is possible that E55 and E575 are involved in function-dependent conformational changes of the transporter. Our data are, however, also consistent with these glutamates being parts of a cation liganding site created by residues from different transmembrane regions as has been implicated for negatively charged residues of the melbiose permease ofEscherichia coli, the rabbit Na+/dicarboxylate cotransporter NaDC-1, Na+/H+ exchangers, thec subunit of the Propionigenium modestumF1/F0-ATP synthase and Na+,K+-ATPases (29Dibrov P. Fliegel L. FEBS Lett. 1998; 424: 1-5Crossref PubMed Scopus (72) Google Scholar, 30Griffith D.A. Pajor A.M. Biochemistry. 1999; 38: 7524-7531Crossref PubMed Scopus (27) Google Scholar, 31Pedersen P.A. Rasmussen J.H. Nielsen J.M. Jorgensen P.L. FEBS Lett. 1997; 400: 206-210Crossref PubMed Scopus (56) Google Scholar, 32Poolman B. Knol J. van der Does C. Henderson P.J. Liang W.J. Leblanc G. Pourcher T. Mus-Veteau I. Mol. Microbiol. 1996; 19: 911-922Crossref PubMed Scopus (134) Google Scholar, 33Vilsen B. Biochemistry. 1995; 34: 1455-1463Crossref PubMed Scopus (86) Google Scholar, 44Kaim G. Wehrle F. Gerike U. Dimroth P. Biochemistry. 1997; 36: 9185-9194Crossref PubMed Scopus (91) Google Scholar). However, a Na+-liganding site may contain only one acidic residue, the other residues being, e.g. glutamine and serine (44Kaim G. Wehrle F. Gerike U. Dimroth P. Biochemistry. 1997; 36: 9185-9194Crossref PubMed Scopus (91) Google Scholar). In this context it is worthy to note that at least some of the mutant Pit2 proteins with Gln for Glu substitutions in only one of the positions E55 or E575 (mutants QEE, EEQ, and EQQ) still supported low levels of NaPi uptake (Fig. 1 A).In recent years, a number of transport proteins have been identified that have dual function as transporters and retroviral receptors (4O'Hara B. Johann S.V. Klinger H.P. Blair D.G. Rubinson H. Dunn K.J. Sass P. Vitek S.M. Robins T. Cell Growth Differ. 1990; 1: 119-127PubMed Google Scholar, 5van Zeijl M. Johann S.V. Closs E. Cunningham J. Eddy R. Shows T.B. O'Hara B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1168-1172Crossref PubMed Scopus (240) Google Scholar,18Miller D.G. Edwards R.H. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 78-82Crossref PubMed Scopus (327) Google Scholar, 45Albritton L.M. Tseng L. Scadden D. Cunningham J.M. Cell. 1989; 57: 659-666Abstract Full Text PDF PubMed Scopus (552) Google Scholar, 46Rasko J.E. Battini J.L. Gottschalk R.J. Mazo I. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2129-2134Crossref PubMed Scopus (186) Google Scholar, 47Tailor C.S. Nouri A. Zhao Y. Takeuchi Y. Kabat D. J. Virol. 1999; 73: 4470-4474Crossref PubMed Google Scholar, 48Tailor C.S. Willett B.J. Kabat D. J. Virol. 1999; 73: 6500-6505Crossref PubMed Google Scholar). For murine CAT1, which is a Na+-dependent cationic amino acid transporter, it has also been shown that a glutamate residue in a putative transmembrane region was critical for the transport function but not for retroviral receptor function of the protein (49Wang H. Kavanaugh M.P. Kabat D. Virology. 1994; 202: 1058-1060Crossref PubMed Scopus (26) Google Scholar). Based on the data presented here and that of Wang et al. (49Wang H. Kavanaugh M.P. Kabat D. Virology. 1994; 202: 1058-1060Crossref PubMed Scopus (26) Google Scholar), we suggest that for proteins exhibiting dual function as transporters and retroviral receptors, their viral receptor function might in general be exploited for identification of amino acid residues critical for transport function. Using a transient transfection-infection assay, which is a fast and reliable method, for analyzing the viral receptor function as described here, indeed makes it a highly feasible approach to analyze processing/overall topology of mutant proteins by investigating their receptor function.The presented data also have implications for understanding the role of Pit2 in retroviral entry. Addition of sulfhydryl reagents to cells was previously shown to impair Pit2 Pi transport and AM-MLV infection but not virus binding (50Rodrigues P. Heard J.M. J. Virol. 1999; 73: 3789-3799Crossref PubMed Google Scholar). According to the authors, these data suggest that Pi-induced conformational changes in Pit2 are involved in AM-MLV entry via Pit2 (50Rodrigues P. Heard J.M. J. Virol. 1999; 73: 3789-3799Crossref PubMed Google Scholar). Our data are not in direct conflict with this interpretation, but they do show that viral receptor function is not dependent on Pi transport functionper se. Indeed, comparison of the data in Figs. 1 and 2reveals that there is no correlation between Na+-dependent Pi transport function and retroviral receptor function of Pit2. We suggest that further important insight into both functions of the type III NaPitransporters can be achieved by combining studies on transport and receptor functions as presented here. Type III NaPi cotransporters, Pit1 and Pit2, exhibit dual function. Here we have shown that it is possible to knock out Pi transport function of human Pit2, leaving its retroviral receptor function undisturbed. Thus, it is possible to exploit the retroviral receptor function of Pit2, and possibly of Pit1, as a control for processing and folding of mutant proteins with knocked out transport function. Doing this, we identified two glutamate residues, E55 and E575 in human Pit2, as critical for Pit2 Pitransport function. This is the first time amino acids critical for Pi transport of a type III NaPi cotransporter have been identified. The two glutamates are both highly conserved in type III NaPi cotransporters and in a number of known and putative phosphate permeases from other species (see supplemental material) strongly suggesting that they also are critical for the physiological function of these transporters. The strength of employing retroviral receptor function as a control for correct processing and folding of mutant Pit2 proteins rather than relying on protein expression in outer membranes is illustrated with the mutant QQQ. It exhibited severely impaired transport function, which could not be explained by the protein not being present in the oocyte outer membrane, but it did, however, also show significantly impaired receptor function. It is possible that impaired receptor function can be due to involvement in retroviral entry of the amino acids analyzed; however, in this case, it is not likely, because mutant proteins like QEE, EQE, QEQ, KEE, EEK, and KEK were fully functional receptors, rather we suggest that the QQQ mutant proteins in the plasma membrane exhibit disturbed processing and/or folding. The question arises whether viral receptor function will be applicable as control when studying involvement of extracellular loop positioned amino acids in transport function of type III transporters. It is in this connection noteworthy that the amino acids so far identified as critical for retroviral receptor function of Pit1 and Pit2 orthologs are those highly variable between Pit1 and Pit2 and the NaPi transporter PHO-4 from N. crassa(19Miller D.G. Miller A.D. J. Virol. 1994; 68: 8270-8276Crossref PubMed Google Scholar, 24Pedersen L. Johann S.V. van Zeijl M. Pedersen F.S. O'Hara B. J. Virol. 1995; 69: 2401-2405Crossref PubMed Google Scholar, 37Dreyer K. Pedersen F.S. Pedersen L. J. Virol. 2000; 74: 2926-2929Crossref PubMed Scopus (12) Google Scholar, 39Eiden M.V. Farrell K.B. Wilson C.A. J. Virol. 1996; 70: 1080-1085Crossref PubMed Google Scholar, 40Leverett B.D. Farrell K.B. Eiden M.V. Wilson C.A. J. Virol. 1998; 72: 4956-4961Crossref PubMed Google Scholar, 41Lundorf M.D. Pedersen F.S. O'Hara B. Pedersen L. J. Virol. 1999; 73: 3169-3175Crossref PubMed Google Scholar, 42Pedersen L. van Zeijl M. Johann S.V. O'Hara B. J. Virol. 1997; 71: 7619-7622Crossref PubMed Google Scholar, 43Tailor C.S. Nouri A. Kabat D. J. Virol. 2000; 74: 237-244Crossref PubMed Scopus (23) Google Scholar), whereas the amino acids critical for NaPi uptake are expected to be found among the residues conserved in Pit1, Pit2, and related proteins from other species as shown in the present work. In line with this, we have recently shown that a Pit1 mutant with abolished receptor functions showed no impairment in Pi transport function as analyzed inXenopus oocytes. 2M. D. Lundorf, P. Bøttger, and L. Pedersen, unpublished data. Thus, it is possible that there in general is no overlap between Pit sequences critical for Pi transport function and Pit sequences critical for receptor function. We do not know the exact role of Pit2 residues E55 and E575 in Pi transport. However, the observation that changing either E55 or E575 for lysine was sufficient to knock out Pitransport function leaving receptor function undisturbed is indeed in agreement with these residues being involved in NaPitransport function, rather than fulfilling structural roles. It is possible that E55 and E575 are involved in function-dependent conformational changes of the transporter. Our data are, however, also consistent with these glutamates being parts of a cation liganding site created by residues from different transmembrane regions as has been implicated for negatively charged residues of the melbiose permease ofEscherichia coli, the rabbit Na+/dicarboxylate cotransporter NaDC-1, Na+/H+ exchangers, thec subunit of the Propionigenium modestumF1/F0-ATP synthase and Na+,K+-ATPases (29Dibrov P. Fliegel L. FEBS Lett. 1998; 424: 1-5Crossref PubMed Scopus (72) Google Scholar, 30Griffith D.A. Pajor A.M. Biochemistry. 1999; 38: 7524-7531Crossref PubMed Scopus (27) Google Scholar, 31Pedersen P.A. Rasmussen J.H. Nielsen J.M. Jorgensen P.L. FEBS Lett. 1997; 400: 206-210Crossref PubMed Scopus (56) Google Scholar, 32Poolman B. Knol J. van der Does C. Henderson P.J. Liang W.J. Leblanc G. Pourcher T. Mus-Veteau I. Mol. Microbiol. 1996; 19: 911-922Crossref PubMed Scopus (134) Google Scholar, 33Vilsen B. Biochemistry. 1995; 34: 1455-1463Crossref PubMed Scopus (86) Google Scholar, 44Kaim G. Wehrle F. Gerike U. Dimroth P. Biochemistry. 1997; 36: 9185-9194Crossref PubMed Scopus (91) Google Scholar). However, a Na+-liganding site may contain only one acidic residue, the other residues being, e.g. glutamine and serine (44Kaim G. Wehrle F. Gerike U. Dimroth P. Biochemistry. 1997; 36: 9185-9194Crossref PubMed Scopus (91) Google Scholar). In this context it is worthy to note that at least some of the mutant Pit2 proteins with Gln for Glu substitutions in only one of the positions E55 or E575 (mutants QEE, EEQ, and EQQ) still supported low levels of NaPi uptake (Fig. 1 A). In recent years, a number of transport proteins have been identified that have dual function as transporters and retroviral receptors (4O'Hara B. Johann S.V. Klinger H.P. Blair D.G. Rubinson H. Dunn K.J. Sass P. Vitek S.M. Robins T. Cell Growth Differ. 1990; 1: 119-127PubMed Google Scholar, 5van Zeijl M. Johann S.V. Closs E. Cunningham J. Eddy R. Shows T.B. O'Hara B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1168-1172Crossref PubMed Scopus (240) Google Scholar,18Miller D.G. Edwards R.H. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 78-82Crossref PubMed Scopus (327) Google Scholar, 45Albritton L.M. Tseng L. Scadden D. Cunningham J.M. Cell. 1989; 57: 659-666Abstract Full Text PDF PubMed Scopus (552) Google Scholar, 46Rasko J.E. Battini J.L. Gottschalk R.J. Mazo I. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2129-2134Crossref PubMed Scopus (186) Google Scholar, 47Tailor C.S. Nouri A. Zhao Y. Takeuchi Y. Kabat D. J. Virol. 1999; 73: 4470-4474Crossref PubMed Google Scholar, 48Tailor C.S. Willett B.J. Kabat D. J. Virol. 1999; 73: 6500-6505Crossref PubMed Google Scholar). For murine CAT1, which is a Na+-dependent cationic amino acid transporter, it has also been shown that a glutamate residue in a putative transmembrane region was critical for the transport function but not for retroviral receptor function of the protein (49Wang H. Kavanaugh M.P. Kabat D. Virology. 1994; 202: 1058-1060Crossref PubMed Scopus (26) Google Scholar). Based on the data presented here and that of Wang et al. (49Wang H. Kavanaugh M.P. Kabat D. Virology. 1994; 202: 1058-1060Crossref PubMed Scopus (26) Google Scholar), we suggest that for proteins exhibiting dual function as transporters and retroviral receptors, their viral receptor function might in general be exploited for identification of amino acid residues critical for transport function. Using a transient transfection-infection assay, which is a fast and reliable method, for analyzing the viral receptor function as described here, indeed makes it a highly feasible approach to analyze processing/overall topology of mutant proteins by investigating their receptor function. The presented data also have implications for understanding the role of Pit2 in retroviral entry. Addition of sulfhydryl reagents to cells was previously shown to impair Pit2 Pi transport and AM-MLV infection but not virus binding (50Rodrigues P. Heard J.M. J. Virol. 1999; 73: 3789-3799Crossref PubMed Google Scholar). According to the authors, these data suggest that Pi-induced conformational changes in Pit2 are involved in AM-MLV entry via Pit2 (50Rodrigues P. Heard J.M. J. Virol. 1999; 73: 3789-3799Crossref PubMed Google Scholar). Our data are not in direct conflict with this interpretation, but they do show that viral receptor function is not dependent on Pi transport functionper se. Indeed, comparison of the data in Figs. 1 and 2reveals that there is no correlation between Na+-dependent Pi transport function and retroviral receptor function of Pit2. We suggest that further important insight into both functions of the type III NaPitransporters can be achieved by combining studies on transport and receptor functions as presented here. We thank Drs. Bryan O'Hara for pOJ74, Maribeth V. Eiden for the PA317GBN cell line, Victor Garcia for human Pit2-specific antibody, and Jan Egebjerg Jensen for use of hisX. laevis oocyte facilities. We thank Dr. Bente Vilsen for helpful discussions and suggestions. Supplementary Material Download .pdf (.19 MB) Help with pdf files Download .pdf (.19 MB) Help with pdf files
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