Lipidation by the Host Prenyltransferase Machinery Facilitates Membrane Localization of Legionella pneumophila Effector Proteins

Abstract

The intracellular human pathogen Legionella pneumophila translocates multiple proteins in the host cytosol known as effectors, which subvert host cellular processes to create a membrane-bound organelle that supports bacterial replication. It was observed that several Legionella effectors encode a prototypical eukaryotic prenylation CAAX motif (where C represents a cysteine residue and A denotes an aliphatic amino acid). These bacterial motifs mediated posttranslational modification of effector proteins resulting in the addition of either a farnesyl or geranylgeranyl isoprenyl lipid moiety to the cysteine residue of the CAAX tetrapeptide. Lipidation enhanced membrane affinity for most Legionella CAAX motif proteins and facilitated the localization of these effector proteins to host organelles. Host farnesyltransferase and class I geranylgeranyltransferase were both involved in the lipidation of the Legionella CAAX motif proteins. Perturbation of the host prenylation machinery during infection adversely affected the remodeling of the Legionella-containing vacuole. Thus, these data indicate that Legionella utilize the host prenylation machinery to facilitate targeting of effector proteins to membrane-bound organelles during intracellular infection. The intracellular human pathogen Legionella pneumophila translocates multiple proteins in the host cytosol known as effectors, which subvert host cellular processes to create a membrane-bound organelle that supports bacterial replication. It was observed that several Legionella effectors encode a prototypical eukaryotic prenylation CAAX motif (where C represents a cysteine residue and A denotes an aliphatic amino acid). These bacterial motifs mediated posttranslational modification of effector proteins resulting in the addition of either a farnesyl or geranylgeranyl isoprenyl lipid moiety to the cysteine residue of the CAAX tetrapeptide. Lipidation enhanced membrane affinity for most Legionella CAAX motif proteins and facilitated the localization of these effector proteins to host organelles. Host farnesyltransferase and class I geranylgeranyltransferase were both involved in the lipidation of the Legionella CAAX motif proteins. Perturbation of the host prenylation machinery during infection adversely affected the remodeling of the Legionella-containing vacuole. Thus, these data indicate that Legionella utilize the host prenylation machinery to facilitate targeting of effector proteins to membrane-bound organelles during intracellular infection. IntroductionWhen inhaled, the human pathogen Legionella pneumophila can infect and replicate in a specialized vacuolar compartment within alveolar macrophages, causing a severe pneumonia known as Legionnaires' disease (1Fraser D.W. Tsai T.R. Orenstein W. Parkin W.E. Beecham H.J. Sharrar R.G. Harris J. Mallison G.F. Martin S.M. McDade J.E. Shepard C.C. Brachman P.S. N. Engl. J. Med. 1977; 297: 1189-1197Crossref PubMed Scopus (1281) Google Scholar, 2McDade J.E. Shepard C.C. Fraser D.W. Tsai T.R. Redus M.A. Dowdle W.R. N. Engl. J. Med. 1977; 297: 1197-1203Crossref PubMed Scopus (886) Google Scholar). Upon phagocytosis, Legionella delays endocytic maturation of the vacuole in which it resides (3Horwitz M.A. J. Exp. Med. 1983; 158: 1319-1331Crossref PubMed Scopus (489) Google Scholar, 4Horwitz M.A. J. Exp. Med. 1983; 158: 2108-2126Crossref PubMed Scopus (503) Google Scholar) and promotes the recruitment and fusion of early secretory vesicles to the vacuolar membrane (5Kagan J.C. Stein M.P. Pypaert M. Roy C.R. J. Exp. Med. 2004; 199: 1201-1211Crossref PubMed Scopus (231) Google Scholar, 6Kagan J.C. Roy C.R. Nat. Cell Biol. 2002; 4: 945-954Crossref PubMed Scopus (356) Google Scholar, 7Tilney L.G. Harb O.S. Connelly P.S. Robinson C.G. Roy C.R. J. Cell Sci. 2001; 114: 4637-4650PubMed Google Scholar). Ubiquitinated proteins are detected on the mature Legionella-containing vacuole (8Ivanov S.S. Roy C.R. Cell Microbiol. 2009; 11: 261-278Crossref PubMed Scopus (51) Google Scholar, 9Dorer M.S. Kirton D. Bader J.S. Isberg R.R. PLoS Pathog. 2006; 2: e34Crossref PubMed Scopus (171) Google Scholar), and ER-derived membranes surround and fuse with the Legionella-containing vacuole (10Swanson M.S. Isberg R.R. Infect Immun. 1995; 63: 3609-3620Crossref PubMed Google Scholar). Bacterial replication commences after biogenesis of this specialized vacuole is completed. Successful establishment of a vacuole that supports bacterial replication is mediated by the collective action of bacterial “effector” proteins (11Ninio S. Roy C.R. Trends Microbiol. 2007; 15: 372-380Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 12Burstein D. Zusman T. Degtyar E. Viner R. Segal G. Pupko T. PLoS Pathog. 2009; 5: e1000508Crossref PubMed Scopus (180) Google Scholar, 13Franco I.S. Shuman H.A. Charpentier X. Cell Microbiol. 2009; 11: 1435-1443Crossref PubMed Scopus (86) Google Scholar) that are translocated into the host cytosol by Legionella via a specialized type IV secretion system called Dot/Icm (14Vogel J.P. Andrews H.L. Wong S.K. Isberg R.R. Science. 1998; 279: 873-876Crossref PubMed Scopus (585) Google Scholar, 15Segal G. Purcell M. Shuman H.A. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 1669-1674Crossref PubMed Scopus (450) Google Scholar). The translocated effectors subvert different host processes to conduct vacuole remodeling (16Ensminger A.W. Isberg R.R. Curr. Opin. Microbiol. 2009; 12: 67-73Crossref PubMed Scopus (115) Google Scholar). Mutants lacking a functional Dot/Icm apparatus fail to deliver effectors into the host cytosol and are defective for intracellular replication. Dot/Icm mutants are avirulent in animal models of disease (17Archer K.A. Roy C.R. Infect. Immun. 2006; 74: 3325-3333Crossref PubMed Scopus (109) Google Scholar, 18Wiater L.A. Dunn K. Maxfield F.R. Shuman H.A. Infect. Immun. 1998; 66: 4450-4460Crossref PubMed Google Scholar, 19Marra A. Blander S.J. Horwitz M.A. Shuman H.A. Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 9607-9611Crossref PubMed Scopus (235) Google Scholar). Although the functions of most of the over 200 Legionella effectors identified so far remain largely unknown, many of the bacterial effectors have been found to modulate host functions at the cytosolic face of host membrane compartments (20Nagai H. Kagan J.C. Zhu X. Kahn R.A. Roy C.R. Science. 2002; 295: 679-682Crossref PubMed Scopus (445) Google Scholar, 21Machner M.P. Isberg R.R. Dev. Cell. 2006; 11: 47-56Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 22Murata T. Delprato A. Ingmundson A. Toomre D.K. Lambright D.G. Roy C.R. Nat. Cell Biol. 2006; 8: 971-977Crossref PubMed Scopus (286) Google Scholar). The accumulation of the host small GTPase Rab1 at the Legionella vacuole is controlled by the effector protein DrrA (SidM), which functions as a guanine nucleotide exchange factor (21Machner M.P. Isberg R.R. Dev. Cell. 2006; 11: 47-56Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 22Murata T. Delprato A. Ingmundson A. Toomre D.K. Lambright D.G. Roy C.R. Nat. Cell Biol. 2006; 8: 971-977Crossref PubMed Scopus (286) Google Scholar, 23Ingmundson A. Delprato A. Lambright D.G. Roy C.R. Nature. 2007; 450: 365-369Crossref PubMed Scopus (265) Google Scholar, 24Schoebel S. Oesterlin L.K. Blankenfeldt W. Goody R.S. Itzen A. Mol. Cell. 2009; 36: 1060-1072Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The protein LepB has GTPase-activating protein activity capable of inactivating Rab1 by promoting GTP hydrolysis (23Ingmundson A. Delprato A. Lambright D.G. Roy C.R. Nature. 2007; 450: 365-369Crossref PubMed Scopus (265) Google Scholar). How effector proteins target host membranes remains an important question. For the effector proteins SidG, YlfA, and YlfB, there is evidence that hydrophobic domains that insert in membranes mediate protein association with host membranes (25Campodonico E.M. Chesnel L. Roy C.R. Mol. Microbiol. 2005; 56: 918-933Crossref PubMed Scopus (109) Google Scholar). Recent evidence suggests that specific protein-protein interactions are important for the recruitment of the effector PieA to the Legionella-containing vacuoles (26Ninio S. Celli J. Roy C.R. PLoS Pathog. 2009; 5: e1000278Crossref PubMed Scopus (56) Google Scholar). Conversely, the effectors DrrA and SidC interact with the lipid phosphatidylinositol 4-phosphate using a domain that is important for host membrane localization (27Brombacher E. Urwyler S. Ragaz C. Weber S.S. Kami K. Overduin M. Hilbi H. J. Biol. Chem. 2009; 284: 4846-4856Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 28Ragaz C. Pietsch H. Urwyler S. Tiaden A. Weber S.S. Hilbi H. Cell Microbiol. 2008; 10: 2416-2433Crossref PubMed Scopus (162) Google Scholar).To further define the mechanisms used for membrane localization of Legionella effector proteins, we have been investigating the role of posttranslational modification by host enzymes might play in this process. Incorporation of lipid moiety in a polypeptide increases its hydrophobicity and can result in peripheral association of the modified protein with the membrane lipid bilayer (29Resh M.D. Nat. Chem. Biol. 2006; 2: 584-590Crossref PubMed Scopus (427) Google Scholar). Proteins containing a C-terminal CAAX tetrapeptide motif, where C represents cysteine and A is an aliphatic amino acid, can be lipidated by a class of enzymes known as prenyltransferases. Prenyltransferases use isoprenoid derivatives as their substrates and fall into three categories, depending upon their substrate specificity. Farnesyltransferase (FTase) 4The abbreviations used are: FTasefarnesyltransferaseGGTasegeranylgeranyltransferaseMOImultiplicity of infectionPNSpostnuclear supernatantCS mutantcysteine to serine mutantKEKK147AAAAK147A/E148A/K149A/K150Aalk-farnesol-1(2E,6E,10E)-3,7,11-trimethyl-12-(prop-2-yn-1-yloxy)dodeca-2,6,10-trien-1-olCMPCAAX motif protein. and class I geranylgeranyltransferase (GGTase-I), which share a common subunit, append farnesyl (C15) and geranylgeranyl (C20) isoprenoids, respectively, to the cysteine residue of a CAAX motif (30Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1725) Google Scholar, 31Schafer W.R. Rine J. Annu. Rev. Genet. 1992; 26: 209-237Crossref PubMed Scopus (343) Google Scholar). Conversely, class II GGTase enzyme has been shown primarily to target dicysteine (-CC- or -CXC-) motifs present in the Rab family of small GTPases (32Leung K.F. Baron R. Seabra M.C. J. Lipid Res. 2006; 47: 467-475Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Prenylated substrates undergo sequential processing by the Rce1 (Ras-converting enzyme 1) endopeptidase and Icmt (isoprenylcysteine-O-carboxyl methyltransferase), which cleave the -AAX tripeptide (33Kim E. Ambroziak P. Otto J.C. Taylor B. Ashby M. Shannon K. Casey P.J. Young S.G. J. Biol. Chem. 1999; 274: 8383-8390Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 34Boyartchuk V.L. Ashby M.N. Rine J. Science. 1997; 275: 1796-1800Crossref PubMed Scopus (304) Google Scholar) and methylate the prenylated cysteine residue (35Dai Q. Choy E. Chiu V. Romano J. Slivka S.R. Steitz S.A. Michaelis S. Philips M.R. J. Biol. Chem. 1998; 273: 15030-15034Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 36Hrycyna C.A. Sapperstein S.K. Clarke S. Michaelis S. EMBO J. 1991; 10: 1699-1709Crossref PubMed Scopus (190) Google Scholar), respectively. Together these modifications facilitate membrane association of prenylated proteins. The best characterized eukaryotic family of CAAX motif-containing proteins is the Ras superfamily of small GTPases, which function as GDP/GTP-controlled switches to regulate diverse cellular processes at the interface of different membrane compartments (37Wright L.P. Philips M.R. J. Lipid Res. 2006; 47: 883-891Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). Although prenylation is required for membrane targeting of Ras GTPases (38Willumsen B.M. Christensen A. Hubbert N.L. Papageorge A.G. Lowy D.R. Nature. 1984; 310: 583-586Crossref PubMed Scopus (392) Google Scholar, 39Gutierrez L. Magee A.I. Marshall C.J. Hancock J.F. EMBO J. 1989; 8: 1093-1098Crossref PubMed Scopus (328) Google Scholar, P.J. C.J. J.E. Proc. Natl. Acad. Sci. U.S.A. 1989; PubMed Scopus Google Scholar), it is for membrane association or accumulation at specific membrane organelles. CAAX motif proteins encode motifs in to at specific cellular compartments J.F. H. Marshall C.J. Cell. 63: Full Text PDF PubMed Scopus Google Scholar). and cysteine that can be to the localization A. M. P.J. C. M. J. H. A. Science. 2005; PubMed Scopus Google Scholar). Conversely, a of of the CAAX motif, which a that can interact with of in the membrane to a of association with different cellular membranes on the of the bilayer interface J.F. H. Marshall C.J. Cell. 63: Full Text PDF PubMed Scopus Google Scholar, T. J. J. A. S. Science. 2008; PubMed Scopus Google Scholar, T. M. L. J. Philips M. T. A. S. Science. 2006; PubMed Scopus Google Scholar). within these domains J.F. H. Marshall C.J. Cell. 63: Full Text PDF PubMed Scopus Google Scholar). this we that of the eukaryotic prenylation machinery is a that is used for the targeting of Legionella effectors to host membrane-bound we that L. pneumophila a family of effectors containing the CAAX motif that use the host lipidation machinery to mediate membrane evidence that at different effectors the CAAX motif for membrane localization this lipidation by host prenyltransferases. specific prenylation it was that of the Legionella for either or several of the effectors be modified by either enzyme The that the Legionella CAAX motifs both enzymes enzyme modification for eukaryotic substrates is and can undergo lipidation by both and P. H.L. J. Biol. Chem. 275: Full Text Full Text PDF PubMed Scopus Google Scholar, P. L. W.R. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google CAAX motif substrate substrate to prenylation in a that the CAAX motif is a of this by a cytosolic protein membrane a polypeptide that a domain membrane affinity by a CAAX either the CAAX motif protein localization and protein For of the CAAX motif its to J.F. H. Marshall C.J. Cell. 63: Full Text PDF PubMed Scopus Google Scholar). Legionella on the CAAX motif for localization and membrane For most Legionella localization was to the of a CAAX The proteins and were in that these proteins localization in the of a functional CAAX motif, which suggests that and encode and for these proteins the CAAX motif play a role in function and The and of processing might to the localization of Legionella translocated with host membranes by a that required both an CAAX motif and functional host was detected in the membrane at with the with the that prenylation is required for membrane and affinity for membranes by with proteins or eukaryotic proteins, the CAAX motif in with either an of amino or cysteine which can be J.F. H. Marshall C.J. Cell. 63: Full Text PDF PubMed Scopus Google Scholar). Although Legionella CAAX cysteine several proteins a amino and For these the of in the C with protein and which have and the C respectively, at the containing at the with protein an important for organelle of the protein. the of amino to in membrane localization the protein the membrane to the that multiple membrane membrane targeting of bacterial effectors is and was in the pathogen cysteine effector which an and motif E. S. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). Upon in the a the lipidation motif that is modified by the host membrane localization E. S. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, J.E. J. Biol. Chem. 2009; 284: Full Text Full Text PDF PubMed Scopus Google Scholar). the effector has been shown to have a CAAX is both and to facilitate membrane and a lacking both lipidation modifications was cytosolic Magee A.I. P. J. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). of host lipidation by bacterial a is by the that host and are required for of vacuoles containing these enzymes are the targeting of bacterial to the host membranes where their effector prenylation is a posttranslational of prenylated is to protein and of prenylation at the of infection have far on lipidation of the translocated bacterial effectors the of host prenylated proteins. the that prenylated host proteins to remodeling of the Legionella vacuole be this we have identified and characterized a family of Legionella CAAX motif-containing proteins that utilize the host prenylation machinery to with host organelles. These data further the of identified substrates of the Dot/Icm apparatus and a in targeting Legionella effector proteins to membrane-bound organelles. IntroductionWhen inhaled, the human pathogen Legionella pneumophila can infect and replicate in a specialized vacuolar compartment within alveolar macrophages, causing a severe pneumonia known as Legionnaires' disease (1Fraser D.W. Tsai T.R. Orenstein W. Parkin W.E. Beecham H.J. Sharrar R.G. Harris J. Mallison G.F. Martin S.M. McDade J.E. Shepard C.C. Brachman P.S. N. Engl. J. Med. 1977; 297: 1189-1197Crossref PubMed Scopus (1281) Google Scholar, 2McDade J.E. Shepard C.C. Fraser D.W. Tsai T.R. Redus M.A. Dowdle W.R. N. Engl. J. Med. 1977; 297: 1197-1203Crossref PubMed Scopus (886) Google Scholar). Upon phagocytosis, Legionella delays endocytic maturation of the vacuole in which it resides (3Horwitz M.A. J. Exp. Med. 1983; 158: 1319-1331Crossref PubMed Scopus (489) Google Scholar, 4Horwitz M.A. J. Exp. Med. 1983; 158: 2108-2126Crossref PubMed Scopus (503) Google Scholar) and promotes the recruitment and fusion of early secretory vesicles to the vacuolar membrane (5Kagan J.C. Stein M.P. Pypaert M. Roy C.R. J. Exp. Med. 2004; 199: 1201-1211Crossref PubMed Scopus (231) Google Scholar, 6Kagan J.C. Roy C.R. Nat. Cell Biol. 2002; 4: 945-954Crossref PubMed Scopus (356) Google Scholar, 7Tilney L.G. Harb O.S. Connelly P.S. Robinson C.G. Roy C.R. J. Cell Sci. 2001; 114: 4637-4650PubMed Google Scholar). Ubiquitinated proteins are detected on the mature Legionella-containing vacuole (8Ivanov S.S. Roy C.R. Cell Microbiol. 2009; 11: 261-278Crossref PubMed Scopus (51) Google Scholar, 9Dorer M.S. Kirton D. Bader J.S. Isberg R.R. PLoS Pathog. 2006; 2: e34Crossref PubMed Scopus (171) Google Scholar), and ER-derived membranes surround and fuse with the Legionella-containing vacuole (10Swanson M.S. Isberg R.R. Infect Immun. 1995; 63: 3609-3620Crossref PubMed Google Scholar). Bacterial replication commences after biogenesis of this specialized vacuole is completed. Successful establishment of a vacuole that supports bacterial replication is mediated by the collective action of bacterial “effector” proteins (11Ninio S. Roy C.R. Trends Microbiol. 2007; 15: 372-380Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 12Burstein D. Zusman T. Degtyar E. Viner R. Segal G. Pupko T. PLoS Pathog. 2009; 5: e1000508Crossref PubMed Scopus (180) Google Scholar, 13Franco I.S. Shuman H.A. Charpentier X. Cell Microbiol. 2009; 11: 1435-1443Crossref PubMed Scopus (86) Google Scholar) that are translocated into the host cytosol by Legionella via a specialized type IV secretion system called Dot/Icm (14Vogel J.P. Andrews H.L. Wong S.K. Isberg R.R. Science. 1998; 279: 873-876Crossref PubMed Scopus (585) Google Scholar, 15Segal G. Purcell M. Shuman H.A. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 1669-1674Crossref PubMed Scopus (450) Google Scholar). The translocated effectors subvert different host processes to conduct vacuole remodeling (16Ensminger A.W. Isberg R.R. Curr. Opin. Microbiol. 2009; 12: 67-73Crossref PubMed Scopus (115) Google Scholar). Mutants lacking a functional Dot/Icm apparatus fail to deliver effectors into the host cytosol and are defective for intracellular replication. Dot/Icm mutants are avirulent in animal models of disease (17Archer K.A. Roy C.R. Infect. Immun. 2006; 74: 3325-3333Crossref PubMed Scopus (109) Google Scholar, 18Wiater L.A. Dunn K. Maxfield F.R. Shuman H.A. Infect. Immun. 1998; 66: 4450-4460Crossref PubMed Google Scholar, 19Marra A. Blander S.J. Horwitz M.A. Shuman H.A. Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 9607-9611Crossref PubMed Scopus (235) Google Scholar). Although the functions of most of the over 200 Legionella effectors identified so far remain largely unknown, many of the bacterial effectors have been found to modulate host functions at the cytosolic face of host membrane compartments (20Nagai H. Kagan J.C. Zhu X. Kahn R.A. Roy C.R. Science. 2002; 295: 679-682Crossref PubMed Scopus (445) Google Scholar, 21Machner M.P. Isberg R.R. Dev. Cell. 2006; 11: 47-56Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 22Murata T. Delprato A. Ingmundson A. Toomre D.K. Lambright D.G. Roy C.R. Nat. Cell Biol. 2006; 8: 971-977Crossref PubMed Scopus (286) Google Scholar). The accumulation of the host small GTPase Rab1 at the Legionella vacuole is controlled by the effector protein DrrA (SidM), which functions as a guanine nucleotide exchange factor (21Machner M.P. Isberg R.R. Dev. Cell. 2006; 11: 47-56Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 22Murata T. Delprato A. Ingmundson A. Toomre D.K. Lambright D.G. Roy C.R. Nat. Cell Biol. 2006; 8: 971-977Crossref PubMed Scopus (286) Google Scholar, 23Ingmundson A. Delprato A. Lambright D.G. Roy C.R. Nature. 2007; 450: 365-369Crossref PubMed Scopus (265) Google Scholar, 24Schoebel S. Oesterlin L.K. Blankenfeldt W. Goody R.S. Itzen A. Mol. Cell. 2009; 36: 1060-1072Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The protein LepB has GTPase-activating protein activity capable of inactivating Rab1 by promoting GTP hydrolysis (23Ingmundson A. Delprato A. Lambright D.G. Roy C.R. Nature. 2007; 450: 365-369Crossref PubMed Scopus (265) Google Scholar). How effector proteins target host membranes remains an important question. For the effector proteins SidG, YlfA, and YlfB, there is evidence that hydrophobic domains that insert in membranes mediate protein association with host membranes (25Campodonico E.M. Chesnel L. Roy C.R. Mol. Microbiol. 2005; 56: 918-933Crossref PubMed Scopus (109) Google Scholar). Recent evidence suggests that specific protein-protein interactions are important for the recruitment of the effector PieA to the Legionella-containing vacuoles (26Ninio S. Celli J. Roy C.R. PLoS Pathog. 2009; 5: e1000278Crossref PubMed Scopus (56) Google Scholar). Conversely, the effectors DrrA and SidC interact with the lipid phosphatidylinositol 4-phosphate using a domain that is important for host membrane localization (27Brombacher E. Urwyler S. Ragaz C. Weber S.S. Kami K. Overduin M. Hilbi H. J. Biol. Chem. 2009; 284: 4846-4856Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 28Ragaz C. Pietsch H. Urwyler S. Tiaden A. Weber S.S. Hilbi H. Cell Microbiol. 2008; 10: 2416-2433Crossref PubMed Scopus (162) Google Scholar).To further define the mechanisms used for membrane localization of Legionella effector proteins, we have been investigating the role of posttranslational modification by host enzymes might play in this process. Incorporation of lipid moiety in a polypeptide increases its hydrophobicity and can result in peripheral association of the modified protein with the membrane lipid bilayer (29Resh M.D. Nat. Chem. Biol. 2006; 2: 584-590Crossref PubMed Scopus (427) Google Scholar). Proteins containing a C-terminal CAAX tetrapeptide motif, where C represents cysteine and A is an aliphatic amino acid, can be lipidated by a class of enzymes known as prenyltransferases. Prenyltransferases use isoprenoid derivatives as their substrates and fall into three categories, depending upon their substrate specificity. Farnesyltransferase (FTase) 4The abbreviations used are: FTasefarnesyltransferaseGGTasegeranylgeranyltransferaseMOImultiplicity of infectionPNSpostnuclear supernatantCS mutantcysteine to serine mutantKEKK147AAAAK147A/E148A/K149A/K150Aalk-farnesol-1(2E,6E,10E)-3,7,11-trimethyl-12-(prop-2-yn-1-yloxy)dodeca-2,6,10-trien-1-olCMPCAAX motif protein. and class I geranylgeranyltransferase (GGTase-I), which share a common subunit, append farnesyl (C15) and geranylgeranyl (C20) isoprenoids, respectively, to the cysteine residue of a CAAX motif (30Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1725) Google Scholar, 31Schafer W.R. Rine J. Annu. Rev. Genet. 1992; 26: 209-237Crossref PubMed Scopus (343) Google Scholar). Conversely, class II GGTase enzyme has been shown primarily to target dicysteine (-CC- or -CXC-) motifs present in the Rab family of small GTPases (32Leung K.F. Baron R. Seabra M.C. J. Lipid Res. 2006; 47: 467-475Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Prenylated substrates undergo sequential processing by the Rce1 (Ras-converting enzyme 1) endopeptidase and Icmt (isoprenylcysteine-O-carboxyl methyltransferase), which cleave the -AAX tripeptide (33Kim E. Ambroziak P. Otto J.C. Taylor B. Ashby M. Shannon K. Casey P.J. Young S.G. J. Biol. Chem. 1999; 274: 8383-8390Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 34Boyartchuk V.L. Ashby M.N. Rine J. Science. 1997; 275: 1796-1800Crossref PubMed Scopus (304) Google Scholar) and methylate the prenylated cysteine residue (35Dai Q. Choy E. Chiu V. Romano J. Slivka S.R. Steitz S.A. Michaelis S. Philips M.R. J. Biol. Chem. 1998; 273: 15030-15034Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 36Hrycyna C.A. Sapperstein S.K. Clarke S. Michaelis S. EMBO J. 1991; 10: 1699-1709Crossref PubMed Scopus (190) Google Scholar), respectively. Together these modifications facilitate membrane association of prenylated proteins. The best characterized eukaryotic family of CAAX motif-containing proteins is the Ras superfamily of small GTPases, which function as GDP/GTP-controlled switches to regulate diverse cellular processes at the interface of different membrane compartments (37Wright L.P. Philips M.R. J. Lipid Res. 2006; 47: 883-891Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). Although prenylation is required for membrane targeting of Ras GTPases (38Willumsen B.M. Christensen A. Hubbert N.L. Papageorge A.G. Lowy D.R. Nature. 1984; 310: 583-586Crossref PubMed Scopus (392) Google Scholar, 39Gutierrez L. Magee A.I. Marshall C.J. Hancock J.F. EMBO J. 1989; 8: 1093-1098Crossref PubMed Scopus (328) Google Scholar, P.J. C.J. J.E. Proc. Natl. Acad. Sci. U.S.A. 1989; PubMed Scopus Google Scholar), it is for membrane association or accumulation at specific membrane organelles. CAAX motif proteins encode motifs in to at specific cellular compartments J.F. H. Marshall C.J. Cell. 63: Full Text PDF PubMed Scopus Google Scholar). and cysteine that can be to the localization A. M. P.J. C. M. J. H. A. Science. 2005; PubMed Scopus Google Scholar). Conversely, a of of the CAAX motif, which a that can interact with of in the membrane to a of association with different cellular membranes on the of the bilayer interface J.F. H. Marshall C.J. Cell. 63: Full Text PDF PubMed Scopus Google Scholar, T. J. J. A. S. Science. 2008; PubMed Scopus Google Scholar, T. M. L. J. Philips M. T. A. S. Science. 2006; PubMed Scopus Google Scholar). within these domains J.F. H. Marshall C.J. Cell. 63: Full Text PDF PubMed Scopus Google Scholar). this we that of the eukaryotic prenylation machinery is a that is used for the targeting of Legionella effectors to host membrane-bound