Key points are not available for this paper at this time.
The blue and green fluorescent proteins (BFP and GFP) have been fused at the N- and C-terminal ends, respectively, of the plasma membrane Ca2+ pump (PMCA) isoform 4xb (hPMCA4xb). The fusion protein was successfully expressed in yeast and purified by calmodulin affinity chromatography. Despite the presence of the fused autofluorescent proteins BFP-PMCA-GFP performed similarly to the wild-type enzyme with respect to Ca2+-ATPase activity and sensitivity to calmodulin activation. In the autoinhibited state BFP-PMCA-GFP exhibited a significant intramolecular fluorescence resonance energy transfer (FRET) consistent with the location of the fluorophores at an average distance of 45Å. The FRET intensity in BFP-PMCA-GFP decreased when the enzyme was activated either by Ca2+-calmodulin, partial proteolysis, or acidic lipids. Moreover, FRET decreased and became insensitive to calmodulin when hPMCA4xb was activated by mutation D170N in BFP-PMCA(D170N)-GFP. The results suggest that the ends of the PMCA are in close proximity in the autoinhibited conformation, and they separate or reorient when the PMCA achieves its final activated conformation. The blue and green fluorescent proteins (BFP and GFP) have been fused at the N- and C-terminal ends, respectively, of the plasma membrane Ca2+ pump (PMCA) isoform 4xb (hPMCA4xb). The fusion protein was successfully expressed in yeast and purified by calmodulin affinity chromatography. Despite the presence of the fused autofluorescent proteins BFP-PMCA-GFP performed similarly to the wild-type enzyme with respect to Ca2+-ATPase activity and sensitivity to calmodulin activation. In the autoinhibited state BFP-PMCA-GFP exhibited a significant intramolecular fluorescence resonance energy transfer (FRET) consistent with the location of the fluorophores at an average distance of 45Å. The FRET intensity in BFP-PMCA-GFP decreased when the enzyme was activated either by Ca2+-calmodulin, partial proteolysis, or acidic lipids. Moreover, FRET decreased and became insensitive to calmodulin when hPMCA4xb was activated by mutation D170N in BFP-PMCA(D170N)-GFP. The results suggest that the ends of the PMCA are in close proximity in the autoinhibited conformation, and they separate or reorient when the PMCA achieves its final activated conformation. The homeostasis of the intracellular Ca2+ is crucial for cell function. The Ca2+ ATPases from plasma membrane (PMCAs) 2The abbreviations used are: PMCA, plasma membrane Ca2+ pump; h4xb, plasma membrane Ca2+ pump derived from the human gene 4 (spliced form x at the splicing site A and spliced form b at the splicing site C); FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; BFP, blue fluorescent protein; PC, phosphatidylcholine; SERCA, sarcoplasmic-endoplasmic reticulum calcium ATPase; C12E10, polyoxyethylene-10-laurylether. participate in the modulation of Ca2+ signals and are responsible for the long term maintenance of the low concentration of intracellular Ca2+ (1Strehler E.E. Treiman M. Curr. Mol. Med. 2004; 4: 323-335Crossref PubMed Scopus (139) Google Scholar). The PMCAs belong to the P2-type ATPase superfamily of ion pumps and form an aspartyl phosphate intermediate during the transport cycle (2Axelsen K.B. Palmgren M.G. Biochim. Biophys. Acta. 1998; 1365: 37-45Crossref PubMed Scopus (200) Google Scholar). Another essential feature of these ATPases is their ability to switch between two different conformational states from E2 to E1 in the presence of the transported ion. Human PMCAs are encoded by four separate genes, and additional variants are generated via alternative splicing of primary gene transcripts. PMCA4 is found virtually in all human tissues, and the splice variant xb is the most studied isoform. Computer modeling and sequence comparisons indicate that the overall structure of the PMCAs closely resembles that of other P-ATPases. Following the domain organization proposed for the SERCA (3Toyoshima C. Inesi G. Annu. Rev. Biochem. 2004; 73: 269-292Crossref PubMed Scopus (305) Google Scholar, 4Lund Jensen A.M. Lykke-Moller Sorensen T. Olesen C. Moller J.V. Nissen P. EMBO J. 2006; 25: 2305-2314Crossref PubMed Scopus (171) Google Scholar), the PMCA would contain a transmembrane region of 10 segments (M1–M10) and three major catalytic domains exposed to the cytosol. The nucleotide-binding (N) and the phosphorylation (P) domains contain the ATP binding site and the aspartate residue that forms the acyl phosphate intermediate, respectively, whereas the actuator domain (A) plays an essential role in the long range transmission of the conformational changes occurring during the transport cycle. Despite the clear overall homology, certain amino acid segments of the PMCA protein are not found in SERCA. The existence of these segments in the PMCA molecule has been generally associated with the extensive regulatory mechanisms that are known to alter the function of the PMCA. Indeed, the major difference between the two calcium pumps is the long C-terminal segment (C region) of the PMCA following M10, and this region is involved in the so-called intrasteric inhibition of the pump (5Penniston J.T. Enyedi A. J. Membr. Biol. 1998; 165: 101-109Crossref PubMed Scopus (159) Google Scholar). This type of control of enzymatic activity by inhibitory sequences acting directly at the active site of the enzyme is characteristic of protein kinases and phosphatases in which the autoinhibitory sequences resemble their substrates (6Kobe B. Kemp B.E. Nature. 1999; 402: 373-376Crossref PubMed Scopus (170) Google Scholar). In analogy with activation mechanism of these enzymes, it is proposed that in the resting state the autoinhibitor interacts with the catalytic region maintaining the PMCA inhibited. Calmodulin is the main activator of the pump, but a variety of mechanisms, including proteolysis, binding of acidic lipids, and phosphorylation by protein kinases, have been shown to switch partially or totally the activity of the PMCA back on. In the hPMCA4xb isoform the C-terminal autoinhibitor region has been found to contain two domains. A sequence of 28 amino acids identified as domain “C” (residues 1086–1113) binds calmodulin and acidic lipids and is responsible for ∼80% of the inhibition. The binding of calmodulin to domain C enhances both the Ca2+ sensitivity and the turnover of the pump. Alternative splicing at the so-called alternative splice C modifies the calmodulin-binding autoinhibitory region leading to isoforms with different responsiveness toward calmodulin activation (7Enyedi A. Verma A.K. Heim R. Adamo H.P. Filoteo A.G. Strehler E.E. Penniston J.T. J. Biol. Chem. 1994; 269: 41-43Abstract Full Text PDF PubMed Google Scholar). Full inhibition has been found to require a segment further downstream the calmodulin binding site (8Verma A.K. Enyedi A. Filoteo A.G. Strehler E.E. Penniston J.T. J. Biol. Chem. 1996; 271: 3714-3718Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). This second inhibitory sequence, which has been called domain “I,” encompass residues 1114–1157. Phosphorylation by protein kinase C of domain I residues has been shown to result in a partial activation of the pump in the absence of calmodulin (9Enyedi A. Verma A.K. Filoteo A.G. Penniston J.T. J. Biol. Chem. 1996; 271: 32461-32467Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Cross-linking experiments using a synthetic peptide C28W made after the sequence of the autoinhibitory domain C of hPMCA4xb revealed that the N-terminal half of this sequence interacts with the N domain (Cys537–Thr544), whereas the C-terminal part interacts with the A domain (Ile206–Val271) of the catalytic core (10Falchetto R. Vorherr T. Brunner J. Carafoli E. J. Biol. Chem. 1991; 266: 2930-2936Abstract Full Text PDF PubMed Google Scholar, 11Falchetto R. Vorherr T. Carafoli E. Protein Sci. 1992; 1: 1613-1621Crossref PubMed Scopus (140) Google Scholar). It has been hypothesized that the binding of calmodulin to the C28W sequence in the pump induces a conformational change in the autoinhibitory domain that disrupts its interaction with the catalytic domain, thereby activating the pump. Consistently with this model it has been shown that the C-terminal segment of the hPMCA4xb is more accessible to proteases when the pump in an activated open state than in the inhibited closed state (12Padanyi R. Paszty K. Penheiter A.R. Filoteo A.G. Penniston J.T. Enyedi A. J. Biol. Chem. 2003; 278: 35798-35804Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Furthermore, the activating effect of mutations of Asp1080 has been suggested to arise from the impaired function of the region preceding the autoinhibitory sequence as a hinge between the regulatory and catalytic regions (13Paszty K. Penheiter A.R. Verma A.K. Padanyi R. Filoteo A.G. Penniston J.T. Enyedi A. J. Biol. Chem. 2002; 277: 36146-36151Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Although the concept that activation of the PMCA involves conformational changes associated with the removal of the autoinhibitory domain from the active site is widely accepted there have been no direct measurements of the rearrangements that take place in the PMCA during the activation. Moreover, the degree in which the C-terminal sequence downstream domain C participates in the conformational changes associated, which would occur during activation, is not clear at this time. In this study we have explored the possibility of obtaining a functional PMCA labeled with two autofluorescent proteins and its potential use as a reporter of conformational changes associated with the activity of the enzyme. Chemicals—Polyoxyethylene-10-laurylether from activator from ATP yeast synthetic yeast amino and for the of and all other from and yeast from was by was by the of and Biochem. J. PubMed Scopus Google that no was to the and of of the for for was by a in a G. Google using and A and C C a site for at its whereas A to the hPMCA4xb of a occurring the a was using the as and and C. The was by in a and using The purified was used in the with A using the for hPMCA4xb as The was with and and the of hPMCA4xb the of the K. R. Palmgren M.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the for a site for was at 4 of the of this a was by using and with and and back at the of A was by using the as and the of and sequences as and and a site for The was by in a and using The was with and the hPMCA4xb a site at The for BFP-PMCA-GFP was by the wild-type of with the and of and A. R. 1996; PubMed Scopus Google was used for with the a and the with the a was R. 1992; Scholar). The in yeast and for their ability to in the absence of amino acids and and of of the BFP-PMCA-GFP protein in at 28 in with after the an of 4 of was and the for yeast with C12E10, and the PMCA was purified by as Adamo H.P. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). of purified PMCA was from of the of the concentration the of BFP-PMCA-GFP was as and that of the labeled PMCAs as Protein protein concentration was by the of M. Biochem. PubMed Scopus Google using as a a of the of PMCA protein in the by using PMCA purified from as and the intensity of the was after the with of BFP-PMCA-GFP in the BFP-PMCA-GFP protein as by Inesi G. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google with a of purified BFP-PMCA-GFP was to of a of and The was for and the was partially by of The was at for and the by Ca2+-ATPase Ca2+-ATPase activity was from the of from at Biochim. Biophys. Acta. PubMed Scopus Google in of ATPase at 4 and and calmodulin to the Ca2+ and calmodulin in The was by the of of BFP-PMCA-GFP in and was by acid after this the of from ATP with time. The Ca2+ concentration in was using the and A. J. Google using the for and Ca2+ the Ca2+ activity of BFP-PMCA-GFP was in as for that the of The was by the protein with of The was at for more of was and the was for The by the was with of at and the by at for The Ca2+ was as Adamo H.P. 2006; PubMed Scopus Google Scholar). The at and with to 10 The in the at for and the was by the of The was by the a The by the was by in a The in the in the absence of ATP was from and Protein and as M. Penniston J.T. Biochim. Biophys. Acta. 1991; PubMed Scopus (34) Google Scholar). a to PubMed Scopus Google and revealed by or binding was by the at 4 in a of and The for with H.P. Penniston J.T. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, Filoteo A.G. Enyedi A. Verma A.K. Penniston J.T. Biochem. J. 1996; PubMed Scopus Google from and of of BFP-PMCA-GFP with was at in a of C12E10, PC, at and The was by the of and the at different as in the of BFP-PMCA-GFP was in of the as and the was by the of of different of the was and with of The protein was and in and the by in The was with and for fluorescence at in a fluorescence measurements performed in an using a The fluorescent C12E10, PC, at and The final was The was at The and at 4 to the from the measurements to for The of energy transfer was as the of via energy transfer to the and are the intensity of of the in the presence and in the absence of a more of the of and the of the the between and was The of the distance between the fluorophores was to the is the distance between the and and is the distance at which the energy is the of the the of the the of the and and the of the of Google Scholar). A distance of was used for the R. Curr. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, 1996; PubMed Scopus Google Scholar), and a was for distance have found that the N-terminal segment of the hPMCA4xb (residues is not for the activity of the PMCA H.P. PubMed Scopus Google Scholar). the was fused at the N-terminal segment of after The was fused after the C-terminal residue at residues downstream of the calmodulin A was between the PMCA and the fluorescent proteins to the that the fluorophores would a as for distance from the FRET from yeast with the the for the autofluorescent PMCA and by the C12E10, and the or PMCA proteins purified by following the used for the of the wild-type PMCA. shown in the of the purified proteins revealed the presence of major which to the in that BFP-PMCA-GFP was of The Ca2+-ATPase activity of BFP-PMCA-GFP with of Ca2+ a activity of at The of calmodulin the activity of BFP-PMCA-GFP and the concentration of Ca2+ for activity from to shown in activation of BFP-PMCA-GFP was at In the presence of Ca2+ the activity of the BFP-PMCA-GFP enzyme in the absence of calmodulin was of that at of This of activity is to that of the wild-type hPMCA4xb enzyme from Adamo H.P. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). results suggest that the inhibitory function of the C-terminal regulatory domain was the of the In results in that after in BFP-PMCA-GFP the of further the of the functional of the PMCA fusion The BFP-PMCA-GFP protein exhibited and at the that the fluorescent the fluorescence of and BFP-PMCA-GFP when the was at and that of at The fluorescence of BFP-PMCA-GFP was than that of at the and the fluorescence intensity in the region of the to the This was of the existence of energy transfer between the two fluorescent have shown that the purified with a T. J. Biol. Chem. Full Text PDF PubMed Google Scholar, T. G. A. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar, Biophys. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). between BFP-PMCA-GFP was FRET, the of was with of either in the presence of or that in the absence of Ca2+ the of of and to to that of the of the fluorescence at that at these energy was between and In the presence of Ca2+ the of the fluorescence was more in with the that the of the enzyme is by Ca2+ T. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The results shown in suggest at the concentration of BFP-PMCA-GFP used in FRET from the interaction between fluorophores to the The transfer by the of BFP-PMCA-GFP and at was a of is the average distance between and in the BFP-PMCA-GFP protein was It has been shown that a of the PMCA to to the removal of from both ends of the molecule H.P. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). the effect of the with the BFP-PMCA-GFP the of the at with the that the of the fluorescent proteins from the PMCA the between the of FRET with the of was with a for of the and a of BFP-PMCA-GFP was by the of revealed by whereas the of the for the of the BFP-PMCA-GFP was to a peptide of of and to other of the peptide not fluorescence the of BFP-PMCA-GFP to the peptide have involved the of the the all the fluorescence was in a peptide of to the The of the of the is in with the of FRET the of to after of following a to the of FRET the effect of the fluorescence of In the presence of 10 Ca2+ the not different from in the absence of the of 10 Ca2+ calmodulin decreased the intensity of fluorescence to This effect was by the removal of Ca2+ with of that the fluorescence intensity of the not change when it was to The of the intensity of FRET with the concentration of calmodulin was in more in The of FRET by of calmodulin in with the activation of the of the average distance between the fluorophores in the BFP-PMCA-GFP to It is known that acidic lipids the activation of the PMCA by calmodulin A.G. Enyedi A. Penniston J.T. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, P. R. Vorherr T. Carafoli E. J. Biochem. 1992; PubMed Scopus Google Scholar). shown in when BFP-PMCA-GFP was activated by the intensity of FRET was to that by the enzyme in the presence of Moreover, in the presence of the further of a effect the of a with the catalytic activity the PMCA is to conformational the most of was to which of the BFP-PMCA-GFP activation was by the in this we of the activating effect of mutation D170N Adamo H.P. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). shown in in the absence of Ca2+ FRET was in the than in the wild-type for an transfer of the of Ca2+ a significant change in the FRET is a used for the structure and of J. Biol. PubMed Scopus Google Scholar). the of the fluorophores is to protein function. of the at a of this was a BFP-PMCA-GFP two fluorescent protein generated the function the enzyme. The results by the fluorescent proteins at both ends of the PMCA, we have in a found that BFP-PMCA-GFP ATPase and Moreover, the Ca2+-ATPase activity of BFP-PMCA-GFP in the absence of calmodulin and its affinity for calmodulin to the for the wild-type enzyme Adamo H.P. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), that the fused proteins not the of the enzyme. The second in this study was the fluorophores would close in the BFP-PMCA-GFP molecule to This was the in the absence of and acidic lipids we significant FRET in The PMCA purified from human has been to form and the concentration of PMCA for between and T. J. Biol. Chem. Full Text PDF PubMed Google Scholar, T. G. A. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar, Biophys. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). FRET in BFP-PMCA-GFP both the interaction of fluorophores from the molecule and from associated PMCA at a concentration of protein we not significant FRET when the fluorescent proteins to different PMCA at low protein concentration the FRET we in BFP-PMCA-GFP was to the interaction of fluorophores from the The of energy transfer have been as distance between the J. Biol. PubMed Scopus Google Scholar). The of FRET in the BFP-PMCA-GFP protein that both would an average of the and would close in the both fluorophores than from as they are the fluorescent It that the distance has to with it is the average of the distance in all BFP-PMCA-GFP and it that both of the of homology, the N- and C-terminal ends of the PMCA with the of the structure of SERCA. the of results with the BFP-PMCA-GFP the ends of the PMCA would close to This the results of domain that the autoinhibitory domain C when at an N-terminal location H.P. Biochem. J. 1998; PubMed Scopus Google Scholar). In with the between the and in SERCA (3Toyoshima C. Inesi G. Annu. Rev. Biochem. 2004; 73: 269-292Crossref PubMed Scopus (305) Google Scholar, 4Lund Jensen A.M. Lykke-Moller Sorensen T. Olesen C. Moller J.V. Nissen P. EMBO J. 2006; 25: 2305-2314Crossref PubMed Scopus (171) Google Scholar), the intensity of FRET in BFP-PMCA-GFP not exhibited a significant change the of The absence of effect of Ca2+ indicate that the of the fluorophores in BFP-PMCA-GFP not change during the of E2 to E1 the of other domains in the the other the intensity of FRET decreased when was that a in the of the fluorophores place during the activation of the PMCA. This effect was partially when Ca2+ was and calmodulin from the enzyme. The of FRET and the BFP-PMCA-GFP activation with the concentration of calmodulin the that the conformational change by FRET was to the activation of the enzyme. the change in PMCA function activation, the distance change between the fluorophores to the catalytic different domains of the PMCA in conformational changes that not a change in the distance between the N- and C-terminal the change is an average of all the labeled the distance change activation by the presence of that not Furthermore, the changes in the purified protein with to the PMCA in the membrane It is known that the PMCA activated by acidic lipids. It has been proposed that the activation by acidic lipids is in part to their binding to the calmodulin-binding site the of calmodulin A.G. Enyedi A. Penniston J.T. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, P. R. Vorherr T. Carafoli E. J. Biochem. 1992; PubMed Scopus Google Scholar). Consistently with this BFP-PMCA-GFP low FRET to that by the enzyme activated by in the presence of This result that the activation by acidic lipids, that by involves a change in the of the N- and C-terminal of the Although the and proteins are to the PMCA is to at the N- and C-terminal The of the BFP-PMCA-GFP FRET with the of was consistent with the of the fluorescent proteins from the PMCA, and it the essential role of the PMCA in the two fluorescent proteins found that the of FRET with the of was of the FRET intensity with the removal of the from the BFP-PMCA-GFP the with a with the removal of the N-terminal region the The of FRET after the removal of from BFP-PMCA-GFP the interaction of a of and the C-terminal part of the pump In the C28W peptide the calmodulin-binding autoinhibitory domain is of the pump activated by but with than the C-terminal autoinhibitory sequence when it is part of the PMCA molecule A. Vorherr T. P. Filoteo A.G. Carafoli E. Penniston J.T. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The rearrangements of the PMCA molecule involved in the activation are not a model for the activation of the PMCA by calmodulin has been proposed A.R. Filoteo A.G. R. K. 2003; PubMed Scopus Google Scholar). to this model the leading to activation involves the binding of calmodulin to domain C of an inhibited of the pump, which has the C domain with the catalytic a conformational change in the autoinhibitor by the binding of calmodulin and the of the autoinhibitor from the catalytic and a further of the open conformation, which the pump in an activated state have shown that the mutation D170N the pump and it insensitive to further activation by calmodulin Adamo H.P. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). we found that exhibited FRET than the type in with the activity of D170N of the PMCA in the absence of The of FRET in a of the between the conformational change by FRET and the activation of the PMCA. FRET in was not by the in FRET is not to by the conformational changes during the activation have found the activity of the of of its C-terminal segment was by that the mutation not the interaction between the autoinhibitor and its site Adamo H.P. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). the FRET was insensitive to calmodulin the changes in FRET not to to the of the pump Following this of we that the changes in FRET in BFP-PMCA-GFP by the interaction between the N and C segments during the final activated state of the PMCA The results with suggest that the change in the location of the ends of the PMCA, occurring during activation, take place of the major autoinhibitory This is in with that the activated state a of the autoinhibitory domains from the catalytic The that the location of the ends of the PMCA molecule are to the activation state of the enzyme the that the amino acids segment the N the C has been to have a functional role (8Verma A.K. Enyedi A. Filoteo A.G. Strehler E.E. Penniston J.T. J. Biol. Chem. 1996; 271: 3714-3718Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, A. Verma A.K. Filoteo A.G. Penniston J.T. J. Biol. Chem. 1996; 271: 32461-32467Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, H.P. PubMed Scopus Google Scholar). of the type of BFP-PMCA-GFP a for more of the activation of PMCA. Furthermore, this of PMCA activation in its in by FRET In its to other PMCA isoforms to as to the in of the in their calmodulin from in M. Carafoli E. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar).
Corradi et al. (Fri,) studied this question.