IIGP1, an Interferon-γ-inducible 47-kDa GTPase of the Mouse, Showing Cooperative Enzymatic Activity and GTP-dependent Multimerization

Abstract

IIGP1 belongs to a well defined family of 47-kDa GTPases whose members are present at low resting levels in mouse cells but are strongly induced transcriptionally by interferons and are implicated in cell-autonomous resistance to intracellular pathogens. Recombinant IIGP1 was expressed in Escherichia coli and purified to homogeneity. Here we present a detailed biochemical characterization of IIGP1 using various biochemical and biophysical methods. IIGP1 binds to GTP and GDP with dissociation constants in the micromolar range with at least 10 times higher affinity for GDP than for GTP. IIGP1 hydrolyzes GTP to GDP, and the GTPase activity is concentration-dependent with a GTP turnover rate of 2 min–1 under saturating protein concentrations. Functional interaction between IIGP1 molecules is shown by nucleotide-dependent oligomerization in vitro. Both cooperative hydrolysis of GTP and GTP-dependent oligomerization are blocked in a mutant form of IIGP1 modified at the C terminus. IIGP1 shares micromolar nucleotide affinities and oligomerization-dependent hydrolytic activity with the 67-kDa GTPase hGBP1 (induced by type I and type II interferons), with the antiviral Mx proteins (interferon type I-induced) and with the paradigm of the self-activating large GTPases, the dynamins, with which Mx proteins show homology. The higher relative affinity for GDP and the relatively low GTPase activity distinguish IIGP1, but this study clearly adds IIGP1 and thus the p47 GTPases to the small group of cooperative GTPase families that appear to characterize the development of intracellular resistance during the interferon response to infection. The present analysis provides essential parameters to understand the molecular mechanism by which IIGP1 participates in this complex resistance program. IIGP1 belongs to a well defined family of 47-kDa GTPases whose members are present at low resting levels in mouse cells but are strongly induced transcriptionally by interferons and are implicated in cell-autonomous resistance to intracellular pathogens. Recombinant IIGP1 was expressed in Escherichia coli and purified to homogeneity. Here we present a detailed biochemical characterization of IIGP1 using various biochemical and biophysical methods. IIGP1 binds to GTP and GDP with dissociation constants in the micromolar range with at least 10 times higher affinity for GDP than for GTP. IIGP1 hydrolyzes GTP to GDP, and the GTPase activity is concentration-dependent with a GTP turnover rate of 2 min–1 under saturating protein concentrations. Functional interaction between IIGP1 molecules is shown by nucleotide-dependent oligomerization in vitro. Both cooperative hydrolysis of GTP and GTP-dependent oligomerization are blocked in a mutant form of IIGP1 modified at the C terminus. IIGP1 shares micromolar nucleotide affinities and oligomerization-dependent hydrolytic activity with the 67-kDa GTPase hGBP1 (induced by type I and type II interferons), with the antiviral Mx proteins (interferon type I-induced) and with the paradigm of the self-activating large GTPases, the dynamins, with which Mx proteins show homology. The higher relative affinity for GDP and the relatively low GTPase activity distinguish IIGP1, but this study clearly adds IIGP1 and thus the p47 GTPases to the small group of cooperative GTPase families that appear to characterize the development of intracellular resistance during the interferon response to infection. The present analysis provides essential parameters to understand the molecular mechanism by which IIGP1 participates in this complex resistance program. Interferons are immunomodulatory cytokines induced either directly by virus infection or by immune and inflammatory stimuli. Type I interferons are secreted by virus-infected tissue cells and by certain activated immune cells such as dendritic cells. Type II or γ-interferon is secreted largely by activated effectors of the innate and adaptive immune systems (natural killer cells, macrophages, and T lymphocytes). Interferons stimulate the transcription of several hundred genes belonging to partially overlapping sets (1Boehm U. Klamp T. Groot M. Howard J.C. Annu. Rev. Immunol. 1997; 15: 749-795Crossref PubMed Scopus (2505) Google Scholar). This complex cellular response resolves into a series of programs contributing in different ways to mechanisms of organismal and cellular resistance to pathogens, and this generalization holds for three described families of interferon-inducible GTPases. The antiviral Mx proteins induced exclusively by type I interferons were the first to be characterized (2Haller O. Kochs G. Traffic. 2002; 3: 710-717Crossref PubMed Scopus (376) Google Scholar). More recently, members of two further families, the p65 (guanylate-binding proteins (GBPs) 1The abbreviations used are: GBP, guanylate-binding protein; mant, 2′,3′-O-N-methylanthraniloyl; GTPγS, guanosine 5′-3-O-(thio)triphosphate; mGTPγS, mant-GTPγS; Gpp(NH)p, guanosine 5′-(β,γ-imido)-triphosphate; mGpp(NH)p, mant-Gpp(NH)p; HPLC, high pressure liquid chromatography; mGDP, mant-GDP; GST, glutathione S-transferase.) and the p47 GTPases (3Boehm U. Guethlein L. Klamp T. Ozbek K. Schaub A. Futterer A. Pfeffer K. Howard J.C. J. Immunol. 1998; 161: 6715-6723PubMed Google Scholar), both induced preferentially by interferon-γ have also been shown to contribute to pathogen resistance (4Anderson S.L. Carton J.M. Lou J. Xing L. Rubin B.Y. Virology. 1999; 256: 8-14Crossref PubMed Scopus (220) Google Scholar, 5Carlow D.A. Teh S.J. Teh H.S. J. Immunol. 1998; 161: 2348-2355PubMed Google Scholar, 6Collazo C.M. Yap G.S. Sempowski G.D. Lusby K.C. Tessarollo L. Woude G.F. Sher A. Taylor G.A. J. Exp. Med. 2001; 194: 181-188Crossref PubMed Scopus (272) Google Scholar, 7Halonen S.K. Taylor G.A. Weiss L.M. Infect. Immun. 2001; 69: 5573-5576Crossref PubMed Scopus (115) Google Scholar, 8Riemer C. Queck I. Simon D. Kurth R. Baier M. J. Virol. 2000; 74: 10245-10248Crossref PubMed Scopus (86) Google Scholar, 9Taylor G.A. Collazo C.M. Yap G.S. Nguyen K. Gregorio T.A. Taylor L.S. Eagleson B. Secrest L. Southon E.A. Reid S.W. Tessarollo L. Bray M. McVicar D.W. Komschlies K.L. Young H.A. Biron C.A. Sher A. Vande Woude G.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 751-755Crossref PubMed Scopus (216) Google Scholar). Presently, with the possible exception of MxA (2Haller O. Kochs G. Traffic. 2002; 3: 710-717Crossref PubMed Scopus (376) Google Scholar), no explanation is available to connect the biochemical activity of these GTPases with their adaptive function in pathogen resistance. A member of the p65 GTPases from human cells, hGBP1, has been shown to block viral replication and endothelial cell proliferation (4Anderson S.L. Carton J.M. Lou J. Xing L. Rubin B.Y. Virology. 1999; 256: 8-14Crossref PubMed Scopus (220) Google Scholar, 10Guenzi E. Topolt K. Cornali E. Lubeseder-Martellato C. Jorg A. Matzen K. Zietz C. Kremmer E. Nappi F. Schwemmle M. Hohenadl C. Barillari G. Tschachler E. Monini P. Ensoli B. Sturzl M. EMBO J. 2001; 20: 5568-5577Crossref PubMed Scopus (152) Google Scholar) and has been subjected to a detailed biochemical and structural analysis (11Praefcke G.J. Geyer M. Schwemmle M. Robert K.H. Herrmann C. J. Mol. Biol. 1999; 292: 321-332Crossref PubMed Scopus (99) Google Scholar, 12Prakash B. Renault L. Praefcke G.J. Herrmann C. Wittinghofer A. EMBO J. 2000; 19: 4555-4564Crossref PubMed Scopus (120) Google Scholar, 13Prakash B. Praefcke G.J. Renault L. Wittinghofer A. Herrmann C. Nature. 2000; 403: 567-571Crossref PubMed Scopus (256) Google Scholar). hGBP1 binds to GTP, GDP and GMP with equal affinity and hydrolyzes GTP to GDP and GMP. The GTPase activity of hGBP1 is stimulated at higher protein concentrations, and the presence of GTP or Gpp(NH)p favors the formation of oligomers. The nucleotide-free and the Gpp(NH)p-bound structures reveal many similarities and differences from other GTP-binding proteins. The high intrinsic GTPase activity, the cooperative hydrolytic behavior, and structural properties have suggested that hGBP1, representing the p65 GTPases, may be a functional and possibly also structural model for the family of large GTPases including Mx and dynamin (2Haller O. Kochs G. Traffic. 2002; 3: 710-717Crossref PubMed Scopus (376) Google Scholar, 14Danino D. Hinshaw J.E. Curr. Opin. Cell Biol. 2001; 13: 454-460Crossref PubMed Scopus (161) Google Scholar). In addition to cooperative GTPase activity, all of these GTPase families share the property of self-assembly into oligomers and higher order multimers, sometimes on specific templates such as lipid membranes in the case of J. 2002; PubMed Scopus Google Scholar, B. A. Nature. 2001; PubMed Scopus Google Scholar) and viral in the case of MxA (2Haller O. Kochs G. Traffic. 2002; 3: 710-717Crossref PubMed Scopus (376) Google Scholar). The of GTP to these structures that have been with function and favors further self-assembly under J. 2002; PubMed Scopus Google Scholar, B. A. J. Biol. 2002; PubMed Scopus Google Scholar, M. K. A. K. A. J. Biol. PubMed Google Scholar, G. M. U. O. J. Biol. 2002; PubMed Scopus Google Scholar). A and with to is function as GTPases or as S.L. PubMed Scopus Google Scholar). In of the structural between the protein families, the of this have for the function of dynamin but also for the of of interferon-inducible Mx and the p65 The that the p47 GTPases to this IIGP1, with which this study is is a member of the p47 GTPase The mouse p47 GTPases described form a family with no to other GTPases the GTP-binding (3Boehm U. Guethlein L. Klamp T. Ozbek K. Schaub A. Futterer A. Pfeffer K. Howard J.C. J. Immunol. 1998; 161: 6715-6723PubMed Google Scholar). The p47 GTPases have all three GTP-binding and clearly the p65 hGBP1, the is by a different (11Praefcke G.J. Geyer M. Schwemmle M. Robert K.H. Herrmann C. J. Mol. Biol. 1999; 292: 321-332Crossref PubMed Scopus (99) Google Scholar). members of the p47 GTPase family have been shown to specific resistance to intracellular pathogens. the p47 GTPase have to of virus but of virus D.A. Teh S.J. Teh H.S. J. Immunol. 1998; 161: 2348-2355PubMed Google Scholar). with of other p47 GTPases or are all to in the also to infection C.M. Yap G.S. Sempowski G.D. Lusby K.C. Tessarollo L. Woude G.F. Sher A. Taylor G.A. J. Exp. Med. 2001; 194: 181-188Crossref PubMed Scopus (272) Google Scholar, 7Halonen S.K. Taylor G.A. Weiss L.M. Infect. Immun. 2001; 69: 5573-5576Crossref PubMed Scopus (115) Google Scholar, 9Taylor G.A. Collazo C.M. Yap G.S. Nguyen K. Gregorio T.A. Taylor L.S. Eagleson B. Secrest L. Southon E.A. Reid S.W. Tessarollo L. Bray M. McVicar D.W. Komschlies K.L. Young H.A. Biron C.A. Sher A. Vande Woude G.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 751-755Crossref PubMed Scopus (216) Google Scholar). In of a structural analysis of IIGP1, we present a analysis of nucleotide and hydrolysis by the purified IIGP1 is in the of and has a relatively low affinity for in the micromolar The protein has higher affinity for GDP than for GTP, a higher dissociation rate for GTP. IIGP1 has low GTPase activity that is at higher of the cooperative nucleotide-free or in the presence of GDP, IIGP1 as a but in the presence of GTP or with the GTP In these properties IIGP1 in the functional as Mx and GBP, the other large interferon-inducible GTPases. the properties of IIGP1 as a to understand the mechanism of of this GTPase and function in cell-autonomous pathogen resistance. A of IIGP1 the and was into and into Escherichia coli were at to A of and IIGP1 was expressed as a protein with at The cells were and with and were using a at a pressure of The was purified in a affinity with 2 The was by of the protein with from on the at The IIGP1 was with and was by The protein was further subjected to on a in 2 2 The were by and the IIGP1 were to by a with a were in liquid and at The of IIGP1 was by at with the of on the of and PubMed Scopus Google Scholar). of the IIGP1 protein are described in this which at the C as shown in I. three proteins were and from the glutathione affinity by of the with as described and the at the terminus. The protein from a from the by a by the addition of has as a of the IIGP1 in a were to the nucleotide properties of of these on the of were used as properties to of The in of the to proteins has been used for the study of nucleotide with GTPases. The used in study and the and were and purified by as described in C. J. Wittinghofer A. 1998; PubMed Scopus Google The of the was by The of the was by using as described were by the addition of and 10 The formation of and is a as I. J. Biol. 1999; PubMed Scopus Google Scholar). were with 2 at at least a of protein to nucleotide were for The of the in of the protein to the was was at and a In a function be to the the rate A of the of protein was the of the the rate and the the dissociation rate The are from the of and protein was The were at and the was at The in the addition of the protein was and at the were The dissociation were by a function to the as described in C. Mol. Biol. PubMed Scopus Google was used to the of to The of was the addition of nucleotide into a cell the The were using the the of the the dissociation and the as described in T. S. PubMed Scopus Google of GTP and protein were and at in different were and subjected to analysis for GTP and were under on a with with a with in in at a rate of 2 were at in a The concentration-dependent GTPase activity of the three of IIGP1 protein was at a GTP of and at a range of protein concentrations. was using a molecular with a The was a of the was a using a into a The protein or protein with different in 2 was by a The of for was by from which the was The was from the which was from T using the and the using the of for proteins. The was by and A to show oligomerization was also in a the and was at and at the and were expressed as proteins in E. coli cells. the of The were to the of proteins by the of of and cell A large of the expressed protein was under the as shown in a was and was purified as described and the of the protein was by with on the The protein was further purified by The purified protein was by and to be parameters of for were at in of protein the for the in the of to was from a and at the for the in were a of constants were from to the C. Mol. Biol. PubMed Scopus Google The for nucleotide is in the micromolar and higher affinity than mGpp(NH)p, of GTP, in with the other of we in the addition of to and in no in the dissociation rate of be by in the presence of IIGP1 in in the presence of GDP to be to the of GTP hydrolysis of for the of and and GTP to by different in a The of GDP and with were by in order to possible of the group in the the for the addition of GDP to The is in a from which the of the is by The of the to the of GDP was the of GDP and as shown in the The parameters the a for this interaction of and of of In is from the The from II that the the affinity of GDP and for of of nucleotide to IIGP1 were by was used to for in the of GTP by was II and no to the turnover of GTP by IIGP1 The IIGP1 protein was used in a large the nucleotide in order to for in A function was to the the to the rate The rate constants for the of were The of the the rate and the the dissociation rate was also in a by the addition of nucleotide to This to a dissociation of and to a of as shown in the in and dissociation rate constants and for the of and to in a The of different to using are with from in The three different The nucleotide affinities by for the IIGP1 protein and for the two at the C are shown in is that the and the rate constants are for all three that the C is in nucleotide interaction with IIGP1 The rate constants are for the different but the dissociation rate constants The for for the higher affinity with and mGTPγS, as in the described the from three different and from the three of IIGP1, the dissociation for GDP is for GTP is In all GTPases, the of the nucleotide to the protein and the rate of of GTP to GDP by hydrolysis are their functional D.A. F. Nature. PubMed Scopus Google Scholar). a detailed analysis of the of GTP hydrolysis by The rate of hydrolysis and formation was by The activity of the three of IIGP1 and at was GTP from to 2 was nucleotide and that were of IIGP1 hydrolyzes GTP to GDP and to GMP. A for the of GTP hydrolysis by is shown in The hydrolysis of GTP to GDP with IIGP1 at is The rate for GTP hydrolysis was from the are by were to be essential for the GTPase GTPase activity was in the presence of 10 in of the GDP to IIGP1 be directly by the hydrolysis of GTP by IIGP1 was by the addition of that are under which the and nucleotide The activity of IIGP1 at different protein and nucleotide was as shown in The was to a of specific activity a range of protein a small in was with protein functional between IIGP1 In order to the the of the specific GTPase activity on IIGP1 was at a GTP of as shown in The specific rate of GTP hydrolysis by IIGP1 was to 2 min–1 and 2 GTP was The in activity of the protein with in protein in the case of and a cooperative mechanism of GTP the GTP hydrolysis rate of modified protein protein the at or to the rate of min–1 GTPases that show cooperative activity have also been shown to form oligomers in the presence of in O. Kochs G. Traffic. 2002; 3: 710-717Crossref PubMed Scopus (376) Google Scholar). The oligomerization of was using at at protein of and nucleotide GTP, and of The in the of IIGP1 from the analysis by was for of the sets with and the oligomerization of with GTP and A in was the addition of GTP and but to a the addition of higher molecular were with the or in the presence of GDP, the is which to the molecular of The in formation between and Gpp(NH)p-bound be to their in nucleotide shown in IIGP1 modified by the addition of a no cooperative GTPase was of to the also blocked the formation of nucleotide oligomers. the no oligomerization in the presence of GTP and Gpp(NH)p the for in the presence of GTP with nucleotide-free The of in the presence of GTP at thus a oligomerization was possible in the presence of GTP or Gpp(NH)p, and in the presence of GDP, the of these oligomers was with to GTP In order to show directly the between GTP hydrolysis and we used in a from which for nucleotide analysis were The of the oligomerization in a of GTP and is shown in The in was by a with hydrolysis of GTP. This oligomerization to which to a The in is to in the but to GTP hydrolysis and of the oligomers. The of GDP GTP-dependent oligomerization the relatively high affinity of GDP for The in the of the IIGP1 oligomers is with the of GTP hydrolysis the nucleotide and protein to the between the two The of the oligomers was in the using to the of the this we also to the of to shown in the high of IIGP1 the addition of GTP in the presence of with the of GTP hydrolysis by GTPases cellular in and by their to and GTP Wittinghofer A. 2001; PubMed Scopus Google Scholar). affinities are described for a of GTPases, a of which are and in B. 2000; PubMed Scopus Google Scholar, J. Biol. PubMed Google Scholar, 2002; PubMed Scopus Google Scholar, J. R. J. R. Wittinghofer A. PubMed Scopus Google Scholar, C. Wittinghofer A. PubMed Scopus Google Scholar, Schwemmle M. Herrmann C. Wittinghofer A. P. J. Biol. PubMed Scopus Google Scholar, G. J. Biol. PubMed Google Scholar, M. Herrmann C. P. J. Biol. PubMed Scopus Google Scholar). IIGP1 has a micromolar and is with the large GTPases has a higher affinity for GDP than for nucleotide affinities in GTPases B. 2000; PubMed Scopus Google M. Herrmann C. P. J. Biol. PubMed Scopus Google high GTP GDP hGBP1, IIGP1 in a to dissociation and GDP and and GDP is by the and 2002; PubMed Scopus Google Scholar, D. J. Nature. 1998; PubMed Scopus Google Scholar, P. L. J. D. PubMed Scopus Google Scholar). IIGP1, the nucleotide dissociation rate constants are for GTP and for This for the as with GTPases, or the cellular of GTP and GDP J. C. PubMed Scopus Google Scholar), the of the GDP and GTP of IIGP1 be in of GDP, but well a nucleotide of the high dissociation In to these from of IIGP1, a of nucleotide from the purified p47 for which no nucleotide parameters are GTP G.A. R. S. E. Vande Woude G.F. J. Biol. 1997; PubMed Scopus Google Scholar). may that the of and is in by interaction that the relative affinities for no interaction have been described for of the p47 GTPases. GTPase activity has been shown to be for the of all GTPases. to the GTPases, the from the of GTP to hydrolysis is for cellular and is for has a low intrinsic hydrolytic rate functional is by the turnover rate by a of by K. L. A. F. Wittinghofer A. 1997; PubMed Scopus Google Scholar). In the of large GTPases by a mechanism in which GTPase activity is with The of has been shown to be essential for function and is nucleotide-dependent J. 2002; PubMed Scopus Google Scholar, B. J. 1999; PubMed Scopus Google Scholar, P. Hinshaw J.E. Cell Biol. 2001; 3: PubMed Scopus Google Scholar). The Mx the p47 GTPases, are induced by interferons and function in pathogen also show in GTPase activity (2Haller O. Kochs G. Traffic. 2002; 3: 710-717Crossref PubMed Scopus (376) Google Scholar, M. K. A. K. A. J. Biol. PubMed Google Scholar, G. M. U. O. J. Biol. 2002; PubMed Scopus Google Scholar). is that of Mx may a in viral resistance the p65 GTPase hGBP1 also concentration-dependent GTPase activity and nucleotide-dependent oligomerization (11Praefcke G.J. Geyer M. Schwemmle M. Robert K.H. Herrmann C. J. Mol. Biol. 1999; 292: 321-332Crossref PubMed Scopus (99) Google Scholar, 13Prakash B. Praefcke G.J. Renault L. Wittinghofer A. Herrmann C. Nature. 2000; 403: 567-571Crossref PubMed Scopus (256) Google Scholar), in this case is no that these properties in The dynamins, Mx and the or no proteins Mx or between families, appear thus to share structural similarities have been and intrinsic their function interaction with the D. Hinshaw J.E. Curr. Opin. Cell Biol. 2001; 13: 454-460Crossref PubMed Scopus (161) Google Scholar). show that IIGP1 several biochemical properties that the dynamins, Mx and hGBP1 relatively low affinity for cooperative GTPase activity, and nucleotide-dependent IIGP1 clearly from in higher affinity for GDP than for GTP, high GTP dissociation and low GTPase activity min–1 under saturating IIGP1 GTPase activity may be to GTPases or a further interaction these with and is to the rate of GTP In the case of IIGP1, of the protein with intracellular membranes may contribute to a further of GTPase activity, as for B. A. Nature. 2001; PubMed Scopus Google Scholar). The cooperative activity and GTP-dependent oligomerization of IIGP1 are to be essential for function in is no for IIGP1 oligomers under in GTPase activity oligomerization has also been described for the small GTPase and a functional for the of the has been B. J. Biol. 2001; PubMed Scopus Google Scholar). In this the formation of oligomers is of the presence of a property that from the large GTPases. The protein modified at the C GTP-dependent oligomerization concentration-dependent GTPase activity, thus on the of the C in the formation of oligomers. of the in on nucleotide that the with oligomerization than or the GTP The properties of the that oligomerization a in of GTPase The other modified form of IIGP1 to be the the of the and the of GTP hydrolysis R. Wittinghofer A. PubMed Scopus Google Scholar). small GTPases in the has been shown in the presence of a which the the In the is of the to in the of has been (11Praefcke G.J. Geyer M. Schwemmle M. Robert K.H. Herrmann C. J. Mol. Biol. 1999; 292: 321-332Crossref PubMed Scopus (99) Google Scholar). In the of this be by the that hGBP1 have the we were to of to by these two of to IIGP1 is in the that is GTP is and of these is that binds to the is representing the A from these the and paradigm be that be in by as for in the The in or the be in the family of large GTPases. IIGP1 has in the of all members of the family of large GTPases, the dynamins, and hGBP1, have a as in IIGP1, that the group a different mechanism from that of the small GTPases. The of the p47 GTPase mechanism is further by the presence of a in of at the of the in three members of the and (3Boehm U. Guethlein L. Klamp T. Ozbek K. Schaub A. Futterer A. Pfeffer K. Howard J.C. J. Immunol. 1998; 161: 6715-6723PubMed Google Scholar). this which is to this of p47 GTPases L. J. Mol. Biol. 2002; PubMed Scopus Google Scholar), has been shown to have GTPase activity G.A. M. D.A. Woude G.F. J. Biol. PubMed Scopus (99) Google Scholar). be to the mechanism of the p47 GTPases. IIGP1 is in the of the and J. U. J. Immunol. 2002; PubMed Scopus Google Scholar), and is at least on C. and J. C. Both and Mx proteins with lipid which by the formation of oligomers (2Haller O. Kochs G. Traffic. 2002; 3: 710-717Crossref PubMed Scopus (376) Google Scholar, B. A. J. Biol. 2002; PubMed Scopus Google Scholar). hGBP1 a and J.E. Mol. Biol. 2000; PubMed Scopus Google Scholar). The interaction of all of the large GTPase families with membranes may that the interferon-inducible members of these their function of of or that are by intracellular pathogens. A of the mechanism by which p47 GTPases with intracellular into the nucleotide properties and of the purified proteins but a detailed of their properties and in is that with other proteins IIGP1 in the of may that the with intracellular membranes may also be the and of in the in properties of the protein B. S. P. M. 1997; PubMed Scopus Google Scholar, J. 1998; PubMed Scopus Google Scholar). of the protein or of IIGP1 in in the cell further in the of oligomerization and GTPase activity as well as for the function of the protein in