Glu332 in the Nicastrin Ectodomain Is Essential for γ-Secretase Complex Maturation but Not for Its Activity

The γ-secretase complex is responsible for the proteolysis of integral membrane proteins. Nicastrin has been proposed to operate as the substrate receptor of the complex with the glutamate 332 (Glu333 in human) serving as the anionic binding site for the α-amino-terminal group of substrates. The putative binding site is located within the aminopeptidase-like domain of Nicastrin. The Glu332 is proposed to function as the counterpart of the exopeptidase Glu located in the active site of these peptidases. Although Glu332 could bind the α-amino-terminal group of substrates, we hypothesized, in analogy with M28-aminopeptidases, that other residues in the putative binding site of Nicastrin should participate in the interaction as well. Surprisingly, mutagenesis of these residues affected the in vivo processing of APP and Notch substrates only weakly. In addition, the E332Q mutation, which completely abolishes the anionic α-amino-terminal binding function, remained fully active. When we introduced the previously characterized E332A mutation, we found strongly decreased γ-secretase complex levels, but the remaining complex appeared as active as the wild-type complex. We confirmed in two independent in vitro assays that the specific enzymatic activity of the E332A mutant was comparable with that of the wild-type complex. Thus, Glu332 crucially affects complex maturation rather than substrate recognition. Moreover other Nicastrin mutants, designed to either impede or alter substantially the putative binding pocket, affected only marginally γ-secretase activity. Consequently, these studies indicate that the main role of the Glu332 is in the maturation and assembly of γ-secretase rather than in the recognition of the substrates. The γ-secretase complex is responsible for the proteolysis of integral membrane proteins. Nicastrin has been proposed to operate as the substrate receptor of the complex with the glutamate 332 (Glu333 in human) serving as the anionic binding site for the α-amino-terminal group of substrates. The putative binding site is located within the aminopeptidase-like domain of Nicastrin. The Glu332 is proposed to function as the counterpart of the exopeptidase Glu located in the active site of these peptidases. Although Glu332 could bind the α-amino-terminal group of substrates, we hypothesized, in analogy with M28-aminopeptidases, that other residues in the putative binding site of Nicastrin should participate in the interaction as well. Surprisingly, mutagenesis of these residues affected the in vivo processing of APP and Notch substrates only weakly. In addition, the E332Q mutation, which completely abolishes the anionic α-amino-terminal binding function, remained fully active. When we introduced the previously characterized E332A mutation, we found strongly decreased γ-secretase complex levels, but the remaining complex appeared as active as the wild-type complex. We confirmed in two independent in vitro assays that the specific enzymatic activity of the E332A mutant was comparable with that of the wild-type complex. Thus, Glu332 crucially affects complex maturation rather than substrate recognition. Moreover other Nicastrin mutants, designed to either impede or alter substantially the putative binding pocket, affected only marginally γ-secretase activity. Consequently, these studies indicate that the main role of the Glu332 is in the maturation and assembly of γ-secretase rather than in the recognition of the substrates. The γ-secretase is a multimeric membrane complex with aspartyl proteolytic activity. In contrast to other intramembrane proteases, the γ-secretase cleaves type I transmembrane proteins with broad specificity (1Kopan R. Ilagan M.X. Nat. Rev. Mol. Cell Biol. 2004; 5: 499-504Crossref PubMed Scopus (499) Google Scholar, 2Lleo A. Curr. Top. Med. Chem. 2008; 8: 9-16Crossref PubMed Scopus (69) Google Scholar). The size of the ectodomain determines whether a type I membrane protein is a γ-secretase substrate (3Struhl G. Adachi A. Mol. Cell. 2000; 6: 625-636Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). In addition to other substrates, this protease complex cleaves the COOH-terminal 99-amino acid fragment of the amyloid precursor protein (APP) 3The abbreviations used are: APP, amyloid precursor protein; Aβ, amyloid β peptide; AICD, APP intracellular domain; AP-like domain, aminopeptidase-like domain; CTF, COOH-terminal fragment; Nct–/– cells, Nicastrin knockout cells; MEF, mouse embryonic fibroblasts; NCT, Nicastrin; NICD, Notch intracellular domain; Ps, presenilin; BisTris, 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)propane-1,3-diol; MOPS, 4-morpholinepropanesulfonic acid; CHAPSO, 3-(3-cholamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; WT, wild type. within the membrane at different positions, to release the COOH-terminal intracellular domain (AICD) and COOH-terminal heterogeneous amyloid-β peptides (Aβ) (4De Strooper B. Saftig P. Craessaerts K. Vanderstichele H. Guhde G. Annaert W. Von Figura K. Van Leuven F. Nature. 1998; 391: 387-390Crossref PubMed Scopus (1560) Google Scholar, 5Annaert W. De Strooper B. Annu. Rev. Cell Dev. Biol. 2002; 18: 25-51Crossref PubMed Scopus (198) Google Scholar). Aβ peptides are the major component of amyloid deposits in the brains of patients with Alzheimer disease, and the longer Aβ42 peptide generation appears to be crucial in the Alzheimer amyloid cascade (6Hardy J. Selkoe D.J. Science. 2002; 297: 353-356Crossref PubMed Scopus (11121) Google Scholar, 7Walsh D.M. Selkoe D.J. Protein Pept. Lett. 2004; 11: 213-228Crossref PubMed Scopus (393) Google Scholar). Therefore, reduction of Aβ42 by specific inhibition of one of the key enzymes involved in its generation, the β- or the γ-secretase, is highly desirable. The γ-secretase complex is formed by presenilin (Ps), Nicastrin (Nct), anterior pharynx defective (Aph1), and presenilin enhancer-2 (Pen-2) (4De Strooper B. Saftig P. Craessaerts K. Vanderstichele H. Guhde G. Annaert W. Von Figura K. Van Leuven F. Nature. 1998; 391: 387-390Crossref PubMed Scopus (1560) Google Scholar, 8Goutte C. Hepler W. Mickey K.M. Priess J.R. Development. 2000; 127: 2481-2492Crossref PubMed Google Scholar, 9Yu G. Nishimura M. Arawaka S. Levitan D. Zhang L. Tandon A. Song Y.Q. Rogaeva E. Chen F. Kawarai T. Supala A. Levesque L. Yu H. Yang D.S. Holmes E. Milman P. Liang Y. Zhang D.M. Xu D.H. Sato C. Rogaev E. Smith M. Janus C. Zhang Y. Aebersold R. Farrer L.S. Sorbi S. Bruni A. Fraser P. St. George-Hyslop P. Nature. 2000; 407: 48-54Crossref PubMed Scopus (827) Google Scholar, 10Goutte C. Dev. Cell. 2002; 3: 6-7Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar, 11Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1699) Google Scholar). Ps is the catalytic subunit (reviewed in Ref. 12De Strooper B. Neuron. 2003; 38: 9-12Abstract Full Text Full Text PDF PubMed Scopus (840) Google Scholar) and Nct, a type 1 transmembrane protein with a large and highly glycosylated ectodomain (Nct-ECD), has been implicated in the initial recognition of substrates (13Shah S. Lee S.F. Tabuchi K. Hao Y.H. Yu C. LaPlant Q. Ball H. Dann 3rd, C.E. Sudhof T. Yu G. Cell. 2005; 122: 435-447Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar). Aph1 and Pen-2 are multipass transmembrane proteins that have been proposed to play a structural role in the assembly and maturation of the complex (14Shirotani K. Edbauer D. Kostka M. Steiner H. Haass C. J. Neurochem. 2004; 89: 1520-1527Crossref PubMed Scopus (59) Google Scholar, 15Prokop S. Shirotani K. Edbauer D. Haass C. Steiner H. J. Biol. Chem. 2004; 279: 23255-23261Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Although the sequence of events preceding full assembly and activation of the protease complex is controversial, the current hypothesis suggests that formation of the Nct-Aph1 subcomplex is the first step in γ-secretase assembly, followed by sequential incorporation of the Ps and Pen-2 subunits, respectively, or alternatively, incorporation of the Ps-Pen-2 subcomplex (16Hu Y. Fortini M.E. J. Cell Biol. 2003; 161: 685-690Crossref PubMed Scopus (127) Google Scholar, 17LaVoie M.J. Fraering P.C. Ostaszewski B.L. Ye W. Kimberly W.T. Wolfe M.S. Selkoe D.J. J. Biol. Chem. 2003; 278: 37213-37222Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 18Fraering P.C. LaVoie M.J. Ye W. Ostaszewski B.L. Kimberly W.T. Selkoe D.J. Wolfe M.S. Biochemistry. 2004; 43: 323-333Crossref PubMed Scopus (119) Google Scholar, 19Capell A. Beher D. Prokop S. Steiner H. Kaether C. Shearman M.S. Haass C. J. Biol. Chem. 2005; 280: 6471-6478Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Nct homology with the M28 aminopeptidase superfamily initially led to speculation that the proposed Nct aminopeptidase (AP)-like domain might possess catalytic activity or could serve as a binding domain for substrates (20Fagan R. Swindells M. Overington J. Weir M. Trends Biochem. Sci. 2001; 26: 213-214Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). More recently, demonstration that Nct acts as a substrate receptor (13Shah S. Lee S.F. Tabuchi K. Hao Y.H. Yu C. LaPlant Q. Ball H. Dann 3rd, C.E. Sudhof T. Yu G. Cell. 2005; 122: 435-447Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar) and is rate-limiting for access to the protease active site, suggests that this function could be exploited as a therapeutic target for Alzheimer disease. According to the prevailing Nct receptor model, the initial binding of substrates to the γ-secretase complex crucially depends on a salt interaction between human Nct-E333 (Glu332 in mouse) and the α-amino-terminal group of the substrate, which leads to translocation and docking of the substrate in the catalytic core (13Shah S. Lee S.F. Tabuchi K. Hao Y.H. Yu C. LaPlant Q. Ball H. Dann 3rd, C.E. Sudhof T. Yu G. Cell. 2005; 122: 435-447Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar). This model proposes that Nct-Glu333 is the counterpart of the Glu residue present within the exopeptidase motif of APs (21Vazeux G. Iturrioz X. Corvol P. Llorens-Cortes C. Biochem. J. 1998; 334: 407-413Crossref PubMed Scopus (71) Google Scholar, 22Luciani N. Marie-Claire C. Ruffet E. Beaumont A. Roques B.P. Fournie-Zaluski M.C. Biochemistry. 1998; 37: 686-692Crossref PubMed Scopus (101) Google Scholar), which is essential for the binding of the α-amino-terminal group of substrates and inhibitors. Furthermore, the model suggests that apart from Glu333 no other key structural determinants are involved in the Nct recognition mechanism, explaining the broad specificity of the γ-secretase. However, no further quantitative data are available to support this assumption. Indeed, functional and structural analysis of aminopeptidases, such as acidic and arginyl aminopeptidase LTA4H, has demonstrated that other residues in addition to the exopeptidase Glu are involved in substrate recognition (23Iturrioz X. Rozenfeld R. Michaud A. Corvol P. Llorens-Cortes C. Biochemistry. 2001; 40: 14440-14448Crossref PubMed Scopus (50) Google Scholar, 24Tholander F. Kull F. Ohlson E. Shafqat J. Thunnissen M.M. Haeggstrom J.Z. J. Biol. Chem. 2005; 280: 33477-33486Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). In particular, this has been demonstrated for the Streptomyces griseus aminopeptidase, one of the most similar of Nct R. A. D. Y. S. G. Biol. 2004; PubMed Scopus Google Scholar). The sequence between Nct and the M28 enzymes and the functional role of Glu in suggests a substrate binding for and this γ-secretase Thus, we that residues in the Nct AP-like domain, as in the aminopeptidases, participate in the interaction with the substrates. further the of Nct to γ-secretase function, we a structural model of the AP-like domain of Nct, and used this model to residues within the putative substrate binding site that play a role in substrate recognition. that the mouse and E332Q strongly complex assembly but complex activity as by specific that the substrate is by these Furthermore, in the putative binding γ-secretase complex but its specific the crucial of this domain in the maturation and assembly of the γ-secretase complex. mouse and APP and the of Nicastrin have been C. C. G. P. Craessaerts K. De Strooper B. Neuron. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, C. T. K. L. H. Saftig P. De Strooper B. J. Annaert W. J. Cell Biol. 2004; PubMed Scopus Google Scholar). from Cell and of Nct Cell from Nct–/– T. G. H. P.C. J. 2003; PubMed Google Scholar) and by large in Nct–/– a with either wild-type or mutant mouse Nct with with APP and with the human H. P. J. B. J. 1999; PubMed Scopus Google Scholar) as previously A. L. De Strooper B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (119) Google Scholar). was and for The was by and for the of and Aβ42 by specific to the used for of the of APP by and with the of Notch with the Notch F. H. L. P. B. K. J. R. J. F. S. K. H. E. A. W. H. Y. E. L. C. S. M. K. P. J. Nat. 2002; PubMed Scopus Google Scholar) for Cell with the for and of protein a in and to with a specific and to the of Notch was as previously M. K. A. N. Van C. P. K. Annaert W. De Strooper B. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar), with the that the on a at a of for Cell was as previously A. Van G. M. Craessaerts K. Annaert W. De Strooper B. J. Cell Sci. 2003; PubMed Scopus Google Scholar). and of APP with M. J. A. K. J. H. De Strooper B. J. Neurochem. PubMed Scopus Google Scholar). with to and in protease and on for 1 protein by at for was with the to the APP was in and to by the addition of In vitro activity was as previously N. S. S. M. S. N. Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) with in PIPES, 1 protease and on for 1 protein by at for 1 used in the in vitro or by with and In vitro with APP in PIPES, 1 and at In with for and with to of a in of PIPES, 1 and on or at followed by from and was at and substrate with and by the from and at the specific was and present in the membrane proteins with of in for at was with addition of and by with a the in this at in or The was used for analysis and of in the of residues that to the proposed substrate binding by Nct, and in the of structural we the between Nct and M28 with a to a model of the domain of In with the (20Fagan R. Swindells M. Overington J. Weir M. Trends Biochem. Sci. 2001; 26: 213-214Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), M.J. 2008; PubMed Scopus Google Scholar), 2003; PubMed Scopus Google Scholar, J. Mol. Biol. 1999; PubMed Scopus Google Scholar), and M.J. J. Mol. Biol. 2000; PubMed Scopus Google Scholar) of the M28 superfamily as proteins that structural homology with The aminopeptidase of S. griseus B. A. D. S. G. J. Mol. Biol. PubMed Scopus Google Scholar) and the human Sci. S. A. 2005; PubMed Scopus Google Scholar), two as structural and A. J. Mol. Biol. PubMed Scopus Google Scholar) was used to a Nct domain model to the sequence between Nct and M28 for the Nct domain, a with of In with (20Fagan R. Swindells M. Overington J. Weir M. Trends Biochem. Sci. 2001; 26: 213-214Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), residues involved in the catalytic of are in the putative substrate binding of the Nct Glu332 binding and in mouse Nct, which are in to residues and in is a residue that the as a involved in with the of the substrate, and the in this The other catalytic residues in in are by residues and in mouse the Nct model that is located to Glu332 and the of a salt between these residues Furthermore, the residue at in Nct suggests that could be of functional we and residues for further mutagenesis analysis of residues for griseus in the active site in the active site the active site in in substrate binding in in the active site in in C. binding binding binding in in Glu in to in the in a In addition, we used the of and human in complex with R. A. D. Y. S. G. Biol. 2004; PubMed Scopus Google Scholar, Sci. S. A. 2005; PubMed Scopus Google Scholar) to residues that could be involved in the recognition of substrates by In the bind to the two present in the active site of the is by interaction with catalytic residues and the and two other residues and R. A. D. Y. S. G. Biol. 2004; PubMed Scopus Google Scholar). the residue is by a that the catalytic the in the human the active site and with the of the Sci. S. A. 2005; PubMed Scopus Google Scholar). In Nct model, the the sequence that the motif is highly in and are completely in Nct model suggests that the the binding and that by a with Glu332 on this the residue was in mutagenesis the of the previously Glu332 residue (Glu333 in human was as a and the which is located in the pocket, was used as a a and in Cell the of the residues to the putative substrate binding site of Nct, we introduced acid that the of the domain but should substantially the binding of the in in most we the residues with We used sequence from the we that should impede substrate either by the anionic binding site or by residues at a to the or the Glu332 In we used Nct mouse to of wild-type Nct, which to to the different to γ-secretase We at two different We confirmed that Nct similar to in Nct wild-type cells, most the with the of the at Glu332 decreased Nct complex and its incorporation the γ-secretase complex We have previously that γ-secretase activity on of Nct A. Van G. M. Craessaerts K. Annaert W. De Strooper B. J. Cell Sci. 2003; PubMed Scopus Google Scholar) and the functional of decreased or of Nct is to in the of Nct Notch and APP to a whether of the Nct γ-secretase processing of APP or by the different mutant with or peptides to the or the of by or Although to or substrate most of the introduced only a or no on the processing of the substrates. In APP processing was only affected by the which was designed to substrate However, on the other processing was that the that this should substrate binding to Nicastrin a other mutants, and or the release of Aβ the in γ-secretase processing by the different are for with the of M. J. A. K. J. H. De Strooper B. J. Neurochem. PubMed Scopus Google and in only the E332A affected the activity of the complex. However, and in contrast to (13Shah S. Lee S.F. Tabuchi K. Hao Y.H. Yu C. LaPlant Q. Ball H. Dann 3rd, C.E. Sudhof T. Yu G. Cell. 2005; 122: 435-447Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar), we found that this mutant completely of either More the of the putative γ-secretase activity at that the anionic function of Nicastrin is for substrate E332A Glu332 is the key residue in the current hypothesis for the function of Nct, we in on this demonstrated by the E332A has a on the maturation of the γ-secretase complex and Although formation of the complex was the E332A Nct-Aph1 mutant present The of γ-secretase complex assembly was further by and Indeed, the E332A mutant of and Aph1 was The E332Q mutant on γ-secretase complex formation but to a than the E332A which is in with a in (14Shirotani K. Edbauer D. Kostka M. Steiner H. Haass C. J. Neurochem. 2004; 89: 1520-1527Crossref PubMed Scopus (59) Google Scholar). should be that the E332A and E332Q with the Nct-Aph1 subcomplex that the at the of the incorporation of the complex. as a the fully the complex maturation In addition, γ-secretase at the membrane in the E332A mutant and that the E332A mutant the as a E332A was indicate that the at 332 is essential for maturation of the γ-secretase complex. E332A but γ-secretase activity was in the E332A mutant which led to that the of activity could be by the in γ-secretase assembly and interaction between substrate and Therefore, we the specific activity of the E332A mutant complex. We the that appeared to processing of either APP or Notch in We in vitro activity assays the of γ-secretase complex in and that to the of The γ-secretase complex was in from and was as as the γ-secretase and Pen-2 addition of to the activity demonstrated that was on γ-secretase activity these in vitro assays that specific of the wild-type γ-secretase and the E332A mutant to and that the of E332A at the of complex and a complex is the mutant is as active as the we found a similar between of γ-secretase and activity of the and The similar specific of these to the wild-type complex further that these residues only participate in the γ-secretase complex Therefore, these studies that within the domain assembly of the γ-secretase complex but activity. However, substrate recognition by γ-secretase in the Thus, we to that in the in vitro assays the interaction between Nicastrin and the substrate to the active site of the this interaction no longer that we to γ-secretase activity Therefore, we and with the substrate and with a γ-secretase to the substrate to to a similar in the different We and at for We and from the and and γ-secretase present in the by In contrast to that the Glu332 γ-secretase activity to different to we previously in the the E332A led to the generation of a of which with the of that the of activity in the E332A mutant is only to the in the complex Furthermore, the E332Q mutation, which no on the of either Notch or APP, a similar specific activity as the are to with the that Nct as a substrate as this model on the anionic binding role of and was demonstrated by the that E332A Nct receptor the in the current with the E332A mutant with that that in the AP-like domain or the interaction and the activity of the γ-secretase G. Nishimura M. Arawaka S. Levitan D. Zhang L. Tandon A. Song Y.Q. Rogaeva E. Chen F. Kawarai T. Supala A. Levesque L. Yu H. Yang D.S. Holmes E. Milman P. Liang Y. Zhang D.M. Xu D.H. Sato C. Rogaev E. Smith M. Janus C. Zhang Y. Aebersold R. Farrer L.S. Sorbi S. Bruni A. Fraser P. St. George-Hyslop P. Nature. 2000; 407: 48-54Crossref PubMed Scopus (827) Google Scholar, F. Yu G. Arawaka S. Nishimura M. Kawarai T. Yu H. Tandon A. Supala A. Song Y.Q. Rogaeva E. Milman P. Sato C. Yu C. Janus C. Lee J. Song L. Zhang L. Fraser St. George-Hyslop Nat. Cell Biol. 2001; 3: PubMed Scopus Google Scholar, K. Edbauer D. A. J. Steiner H. Haass C. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, in of wild-type or or that and mutants, bind to the and Notch substrates, that the interaction between and the substrate that the of the the binding Glu residue is the other from of substrate and wild-type NCT, with the transmembrane of or the and that and of the (13Shah S. Lee S.F. Tabuchi K. Hao Y.H. Yu C. LaPlant Q. Ball H. Dann 3rd, C.E. Sudhof T. Yu G. Cell. 2005; 122: 435-447Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar). that the ectodomain of with the substrate but the interaction is by the of the transmembrane or other in the complex to substrate that the at 332 is for the assembly of the but for its that the recognition of the γ-secretase complex on the E332A Nicastrin we to whether in assembly of the γ-secretase was to a in the E332A Nct mutant by proteolytic has been that Nct is to Nct is that Nct a complex assembly K. Edbauer D. A. J. Steiner H. Haass C. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, we of to Nct and the E332A from and E332A in with of In with the and E332A the to different but a no between wild-type Nct and mutant structural data from the Nct that the glutamate is essential for the domain for this residue is in the receptor S. M. R. Science. 1999; PubMed Scopus Google Scholar), and in the of the human Sci. S. A. 2005; PubMed Scopus Google Scholar), the mutant to the E332Q in affects the activity but the of the Sci. S. A. 2005; PubMed Scopus Google Scholar). Thus, the role of the domain in Nct However, studies of the receptor have involved its domain in More the at the or at the of this domain, which are from the of the to the binding of the or the J. M. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Biol. 2003; PubMed Scopus Google Scholar). We that the Nct AP-like domain have in the of the γ-secretase as a binding for the presenilin or in which the the Glu332 residue to the assembly of the multimeric or the AP-like domain of could or in with other in Nct, in the the assembly of the γ-secretase complex. mutagenesis data of residues present in the putative substrate binding of Nct, to or substrate only or no in the processing of the substrates, with the of the previously Glu332 residue Furthermore, that E332A affects assembly and maturation of the γ-secretase complex. Although that the ectodomain of Nct in the enzymatic function of the γ-secretase that the previously proposed be as for the available data and a function in the intramembrane