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• W2004763373 abstract "The surface of the protozoan parasiteLeishmania is unusual in that it consists predominantly of glycosylphosphatidylinositol-anchored glycoconjugates and proteins. Additionally, a family of hydrophilic acylated surface proteins (HASPs) has been localized to the extracellular face of the plasma membrane in infective parasite stages. These surface polypeptides lack a recognizable endoplasmic reticulum secretory signal sequence, transmembrane spanning domain, or glycosylphosphatidylinositol-anchor consensus sequence, indicating that novel mechanisms are involved in their transport and localization. Here, we show that the N-terminal domain of HASPB contains primary structural information that directs both N-myristoylation and palmitoylation and is essential for correct localization of the protein to the plasma membrane. Furthermore, the N-terminal 18 amino acids of HASPB, encoding the dual acylation site, are sufficient to target the heterologousAequorea victoria green fluorescent protein to the cell surface of Leishmania. Mutagenesis of the predicted acylated residues confirms that modification by both myristate and palmitate is required for correct trafficking. These data suggest that HASPB is a representative of a novel class of proteins whose translocation onto the surface of eukaryotic cells is dependent upon a “non-classical” pathway involvingN-myristoylation/palmitoylation. Significantly, HASPB is also translocated on to the extracellular face of the plasma membrane of transfected mammalian cells, indicating that the export signal for HASPB is recognized by a higher eukaryotic export mechanism. The surface of the protozoan parasiteLeishmania is unusual in that it consists predominantly of glycosylphosphatidylinositol-anchored glycoconjugates and proteins. Additionally, a family of hydrophilic acylated surface proteins (HASPs) has been localized to the extracellular face of the plasma membrane in infective parasite stages. These surface polypeptides lack a recognizable endoplasmic reticulum secretory signal sequence, transmembrane spanning domain, or glycosylphosphatidylinositol-anchor consensus sequence, indicating that novel mechanisms are involved in their transport and localization. Here, we show that the N-terminal domain of HASPB contains primary structural information that directs both N-myristoylation and palmitoylation and is essential for correct localization of the protein to the plasma membrane. Furthermore, the N-terminal 18 amino acids of HASPB, encoding the dual acylation site, are sufficient to target the heterologousAequorea victoria green fluorescent protein to the cell surface of Leishmania. Mutagenesis of the predicted acylated residues confirms that modification by both myristate and palmitate is required for correct trafficking. These data suggest that HASPB is a representative of a novel class of proteins whose translocation onto the surface of eukaryotic cells is dependent upon a “non-classical” pathway involvingN-myristoylation/palmitoylation. Significantly, HASPB is also translocated on to the extracellular face of the plasma membrane of transfected mammalian cells, indicating that the export signal for HASPB is recognized by a higher eukaryotic export mechanism. glycosylphosphatidylinositol hydrophilic acylated surface protein lipophosphoglycan endoplasmic reticulum green fluorescent protein enhanced GFP concanavalin A detergent-resistant membranes Chinese hamster ovary bovine serum albumin phosphate-buffered saline 4′,6′-dianidino-2-phenylindole 1,4-piperazinediethanesulfonic acid human growth hormone Protozoan parasites of the genus Leishmania cause a spectrum of tropical and sub-tropical diseases termed the leishmaniases. Leishmania live as either extracellular, flagellated promastigotes in the digestive tract of their sandfly vector or as aflagellated amastigotes within the phagolysosomes of mammalian macrophages (1.Molyneux D. Killick-Kendrick R. Peters W. Killick-Kendrick R. The Leishmaniases in Biology and Medicine. 1. Academic Press, London1987: 122-168Google Scholar). Much research has focused on the unusually high levels of glycosylphosphatidylinositol (GPI)1-anchored surface molecules present in these organisms (2.Ferguson M.A. Brimacombe J.S. Cottaz S. Field R.A. Guther L.S. Homans S.W. McConville M.J. Mehlert A. Milne K.G. Ralton J.E. Roy Y.A. Schneider P. Zitzmann N. Parasitology. 1994; 108: S45-S54Crossref PubMed Scopus (62) Google Scholar), particularly the unique glycoconjugate, lipophosphoglycan (LPG), abundant in promastigotes (3.McConville M.J. Ferguson M.A.J. Biochem. J. 1993; 294: 305-324Crossref PubMed Scopus (798) Google Scholar), and the metalloprotease GP63 (or leishmanolysin) (4.Frommel T.O. Button L.L. Fujikura Y. McMaster W.R. Mol. Biochem. Parasitol. 1990; 38: 25-32Crossref PubMed Scopus (71) Google Scholar). The hydrophilic acylated surface proteins (HASPs; originally named GBP and GA/CP inLeishmania major) are a family of related surface molecules expressed only in infective parasite stages (5.Flinn H.M. Rangarajan D. Smith D.F. Mol. Biochem. Parasitol. 1994; 65: 259-270Crossref PubMed Scopus (63) Google Scholar, 6.Pimenta P.F.P. Da Silva P.P. Rangarajan D. Smith D.F. Sacks D.L. Exp. Parasitol. 1994; 79: 468-479Crossref PubMed Scopus (24) Google Scholar). These proteins are unusual in that they lack a “classical” endoplasmic reticulum (ER) secretory signal sequence, a GPI-anchor consensus, or membrane-spanning domains but are surface-localized (6.Pimenta P.F.P. Da Silva P.P. Rangarajan D. Smith D.F. Sacks D.L. Exp. Parasitol. 1994; 79: 468-479Crossref PubMed Scopus (24) Google Scholar), partition into the hydrophobic phase on Triton X-114 separation, and fractionate with lipid species (5.Flinn H.M. Rangarajan D. Smith D.F. Mol. Biochem. Parasitol. 1994; 65: 259-270Crossref PubMed Scopus (63) Google Scholar). The mechanism of transport and attachment to the cell surface is unclear, although it has been suggested that this may occur by virtue of a close association with LPG via an HASP repeat region with homology to the peptidoglycan binding domain of Staphylococcus aureusprotein A (6.Pimenta P.F.P. Da Silva P.P. Rangarajan D. Smith D.F. Sacks D.L. Exp. Parasitol. 1994; 79: 468-479Crossref PubMed Scopus (24) Google Scholar). More recent studies have not substantiated this hypothesis, as HASPs are still presented on the surface of infective metacyclic parasites in the absence of LPG (7.Rangarajan D. Gokool S. McCrossan M.V. Smith D.F. J. Cell Sci. 1995; 108: 3359-3366PubMed Google Scholar).Leishmania (and other trypanosomatids) possess a conventional eukaryotic secretory pathway (8.Clayton C. Hausler T. Blattner J. Microbiol. Rev. 1995; 59: 325-344Crossref PubMed Google Scholar, 9.Al-Qahtani A. Teilhet M. Mensa-Wilmot K. Biochem. J. 1998; 331: 521-529Crossref PubMed Scopus (30) Google Scholar) and require a signal sequence for translocation of secreted proteins into the ER (10.Tobin J.F. Wirth D.F. Mol. Biochem. Parasitol. 1993; 62: 243-248Crossref PubMed Scopus (7) Google Scholar). However, a number of specialized features are apparent in the secretory systems of Leishmania and related organisms, particularly the necessity to traffic large quantities of lipid-anchored molecules to the cell surface. In addition, and uniquely to these parasites, all exo- and endocytosis at the cell surface occurs via the flagellar pocket, an invagination of the plasma membrane at the base of the single flagellum (11.Webster P. Russell D.G. Parasitol. Today. 1993; 9: 201-205Abstract Full Text PDF PubMed Scopus (110) Google Scholar, 12.Overath P. Stierhof Y.-D. Wiese M. Trends Cell Biol. 1997; 7: 27-33Abstract Full Text PDF PubMed Scopus (121) Google Scholar).Absence of ER secretory signals within the HASP family would preclude ER transport through the classical secretory pathway, i.e.via translocation through a sec61p complex into the ER lumen. This suggestion is supported by the observation that HASPB is not glycosylated, despite the presence of several consensusN-glycosylation sites. 2T. M. Alce and D. F. Smith, unpublished data. 2T. M. Alce and D. F. Smith, unpublished data. These properties place HASPB among a group of eukaryotic proteins that lack an ER-targeting secretory signal but are still exported out of the cell, by so called “non-classical” transport (13.Kuchler K. Trends Cell Biol. 1993; 3: 421-426Abstract Full Text PDF PubMed Scopus (62) Google Scholar, 14.Cleves A.E. Curr. Biol. 1997; 7: R318-R320Abstract Full Text Full Text PDF PubMed Google Scholar). Examples of proteins undergoing non-classical export include fibroblast growth factors-1 and -2, interleukin-1, yeast a-factor, and galectin. Export of Saccharomyces cerevisiae a-factor is the best understood; this short, 12-amino acid lipoprotein is exported via an ATP binding cassette transporter (15.Kuchler K. Sterne R.E. Thorner J. EMBO J. 1989; 8: 3973-3984Crossref PubMed Scopus (323) Google Scholar). By contrast, galectin appears to be released from the plasma membrane of mammalian cells through vesicular budding (16.Mehul B. Hughes R.C. J. Cell Sci. 1997; 110: 1169-1178Crossref PubMed Google Scholar), possibly via a novel pathway involving a transporter (17.Cleves A.E. Cooper D.N.W. Barondes S.H. Kelly R.B. J. Cell Biol. 1996; 5: 1017-1026Crossref Scopus (185) Google Scholar). It is unclear at present whether the examples of “non-classically” exported proteins cited above use the same or different mechanisms, and it is highly possible that several auxiliary systems are present.Here we report that the 18-amino acid N-terminal region of HASPB is acylated in vivo and is sufficient to target a heterologous protein (Aequorea victoria green fluorescent protein, GFP) to the cell surface of Leishmania. In addition, we demonstrate that HASPB is also exported by mammalian cells, suggesting that this pathway is also present in higher eukaryotes.DISCUSSIONIn the absence of a secretory signal sequence, a GPI-anchor consensus, or transmembrane domain, the pathway and mechanisms that facilitate localization of the HASP family proteins to theLeishmania cell surface have been unknown to date. Here, a deletion strategy has identified the extreme N terminus of HASPB as being essential for surface presentation of the protein. The presence of N-myristoylation and palmitoylation motifs within these residues indicates that dual acylation may mediate membrane association and also potentially act as the cell surface anchor for HASPB.Two Signals Are Required for Plasma Membrane TargetingMetabolic labeling indicated that glycine 2 and cysteine 5 in the HASPB N-terminal region are required for myristoylation and palmitoylation. Co-translational myristoylation is required for post-translational palmitoylation to occur, in agreement with the hypothesis that a myristate moiety is required to bring the protein to a membrane-bound palmitoylacyltransferase (23.Resh M.D. Cell. Signal. 1996; 8: 403-412Crossref PubMed Scopus (196) Google Scholar). According to the two-signal model (23.Resh M.D. Cell. Signal. 1996; 8: 403-412Crossref PubMed Scopus (196) Google Scholar), such modifications would be sufficient to allow the stable association of HASPB with membranes previously observed (5.Flinn H.M. Rangarajan D. Smith D.F. Mol. Biochem. Parasitol. 1994; 65: 259-270Crossref PubMed Scopus (63) Google Scholar, 6.Pimenta P.F.P. Da Silva P.P. Rangarajan D. Smith D.F. Sacks D.L. Exp. Parasitol. 1994; 79: 468-479Crossref PubMed Scopus (24) Google Scholar). Many proteins have been shown to be targeted to the plasma membrane by dual acylation. The mechanism of targeting is unclear, although the identification of a Gα protein palmitoylacyltransferase activity in plasma membrane fractions (30.Dunphy J.T. Greentree W.K. Manahan C.L. Linder M.E. J. Biol. Chem. 1996; 271: 7154-7159Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) supports a model in which an N-myristoylated protein could become “trapped” in a lipid bilayer when it associates with this putative enzyme and is palmitoylated (Refs. 31.van't Hof W. Resh M. J. Cell Biol. 1997; 136: 1023-1035Crossref PubMed Scopus (123) Google Scholar and 32.Morales J. Fishburn C.S. Wilson P.T. Bourne H.R. Mol. Biol. Cell. 1998; 9: 1-14Crossref PubMed Scopus (81) Google Scholar and references therein).The requirement of dual acylation for stable membrane association and targeting to the plasma membrane was tested using a series of fusion proteins expressed in L. major, in which the N-terminal HASPB 18 amino acids were fused to the N terminus of GFP. Cell fractionation demonstrated that both myristate and palmitate were necessary for efficient membrane binding; myristate alone led to only partial association. Epifluorescent microscopic analyses were consistent with these observations, as the fusion protein required both myristoylation and palmitoylation signals to facilitate localization to the plasma membrane.Trafficking of HASP through the CellThe route dually acylated proteins take to the plasma membrane has only recently been investigated. Trafficking of theN-myristoylated/palmitoylated Src tyrosine kinase, Fyn, to the plasma membrane in mammalian cells is thought to occur very rapidly via a novel pathway (31.van't Hof W. Resh M. J. Cell Biol. 1997; 136: 1023-1035Crossref PubMed Scopus (123) Google Scholar). Another dually acylated Src protein, Lck, was found at the plasma membrane and in the Golgi by co-localization when expressed in certain mammalian cell lines (33.Bijlmakers M.-J. J.E. Isobe-Nakamura M. Ruddock L.J. Marsh M. J. Cell Biol. 1997; 137: 1029-1040Crossref PubMed Scopus (75) Google Scholar). These observations implicate the Golgi apparatus in Lck trafficking. Immunoelectron microscopy of L. major expressing full-length GENE10-tagged HASPB or the parental GFP construct, HASPB18::GFP, revealed the protein localizing primarily to the plasma membrane but with some Golgi-associated staining (only detectable by this highly sensitive method). Such data support an hypothesis in which the Golgi and by extrapolation the exocytic pathway are involved in both Src and HASP trafficking. Another particularly striking observation is that mutation of the HASP palmitoylation site in this chimera leads to the protein concentrating in an organelle stained by a Golgi-specific lipid marker. This supports the suggestion that palmitoylation ofN-myristoylated proteins occurs at a membranous structure possibly belonging to the exocytic pathway (33.Bijlmakers M.-J. J.E. Isobe-Nakamura M. Ruddock L.J. Marsh M. J. Cell Biol. 1997; 137: 1029-1040Crossref PubMed Scopus (75) Google Scholar). This situation is reminiscent of the trafficking of certain acylated Ras proteins that have recently been suggested to be targeted to the endomembrane network (including the Golgi) by virtue of prenylation. Further trafficking of these proteins to the plasma membrane is dependent upon subsequent palmitoylation (34.Choy E. Chiu V.K. Silletti J. Feoktistov M. Morimoto T. Michaelson D. Ivanov I.E. Philips M.R. Cell. 1999; 98: 69-80Abstract Full Text Full Text PDF PubMed Scopus (615) Google Scholar).Therefore, this study implicates the exocytic structures in the transport of HASP, despite the lack of an ER secretory signal. In addition, like other dually acylated proteins (31.van't Hof W. Resh M. J. Cell Biol. 1997; 136: 1023-1035Crossref PubMed Scopus (123) Google Scholar), the trafficking of HASPB in a mammalian system was unaffected by brefeldin A, a potent inhibitor of anterograde vesicular transport (data not shown). Our data, like that of Bijlmakers et al. (33.Bijlmakers M.-J. J.E. Isobe-Nakamura M. Ruddock L.J. Marsh M. J. Cell Biol. 1997; 137: 1029-1040Crossref PubMed Scopus (75) Google Scholar), imply that palmitoylation occurs in the region of the Golgi, with subsequent re-localization to the plasma membrane. This contrasts with the proposal that Fyn is targeted directly to and palmitoylated at the plasma membrane (31.van't Hof W. Resh M. J. Cell Biol. 1997; 136: 1023-1035Crossref PubMed Scopus (123) Google Scholar). However, van't Hof and Resh (31.van't Hof W. Resh M. J. Cell Biol. 1997; 136: 1023-1035Crossref PubMed Scopus (123) Google Scholar) detected a small quantity of Fyn in Golgi membrane fractions. This fraction could be analogous to the HASPB detected in the region of the Golgi apparatus and could explain the partial perinuclear localization of Fyn (31.van't Hof W. Resh M. J. Cell Biol. 1997; 136: 1023-1035Crossref PubMed Scopus (123) Google Scholar).Recently, several studies have suggested that proteins modified by saturated acyl groups are targeted to detergent-resistant membrane (DRM) rafts in mammalian cells (35.Melkonian K.A. Ostermeyer A.G. Chen J.Z. Roth M.G. Brown D.A. J. Biol. Chem. 1999; 274: 3910-3917Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar). These DRMs are implicated in protein sorting in the secretory pathway (36.Simons K. Ikonian E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8019) Google Scholar). In keeping with our trafficking model, it may be predicted thatN-myristoylated/palmitoylated proteins associate with DRMs on the cytoplasmic face of structures in the exocytic pathway and are subsequently transported to the plasma membrane. It is possible that any other machinery required for localization is brought to its protein target by association with these rafts. In the case of HASP, this machinery may include a palmitoylacyltransferase and some translocation channel, such as that discussed below, to allow the protein to reach the extracellular environment.HASP Appears to Be Localized to the Cell Surface by a Conserved, Novel PathwayDespite incomplete characterization of the mechanism, it is clear that N-myristoylation/palmitoylation is sufficient to traffic a large number of proteins to the plasma membrane (32.Morales J. Fishburn C.S. Wilson P.T. Bourne H.R. Mol. Biol. Cell. 1998; 9: 1-14Crossref PubMed Scopus (81) Google Scholar). Unlike these other examples, HASPB is surface-localized in L. major (5.Flinn H.M. Rangarajan D. Smith D.F. Mol. Biochem. Parasitol. 1994; 65: 259-270Crossref PubMed Scopus (63) Google Scholar, 6.Pimenta P.F.P. Da Silva P.P. Rangarajan D. Smith D.F. Sacks D.L. Exp. Parasitol. 1994; 79: 468-479Crossref PubMed Scopus (24) Google Scholar) despite lacking any identifiable signal that could lead to translocation by the exocytic pathway. Electron microscopic and biochemical analyses demonstrate that the N-terminal 18 amino acids of HASPB are sufficient to take a heterologous protein (GFP) to the cell surface. WhereasN-myristoylation/palmitoylation is presumably sufficient to transport HASP to the cytoplasmic face of the plasma membrane, some additional determinant within the 18-amino acid signal is presumed to facilitate translocation of a proportion of the protein across the membrane and into the extracellular environment. The N-terminal 10 amino acids of HASP, maintaining the acylation consensus, are sufficient only to take GFP as far as the flagellar pocket. The reasons for biochemically detecting only 20–30% of HASPB18::GFP at the cell surface are unclear and, as discussed above, could be due to either limited surface presentation of the protein or limits inherent within the assays developed.The mechanism that would take a lipid-anchored protein from one face of the plasma membrane to the other is unclear. However, one hypothesis that would fit our data is the “flippase” model (37.Higgins C.F. Gottesman M.M. Trends Biochem. Sci. 1992; 17: 18-21Abstract Full Text PDF PubMed Scopus (701) Google Scholar) in which a membrane-bound transporter protein allows translocation of the hydrophobic anchor through the lipid phase and the relatively hydrophilic polypeptide through an aqueous channel. Recent structural evidence indicates that such a model holds true for the P-glycoprotein family of transport molecules (38.Rosenberg M.F. Callaghan R. Ford R.F. Higgins C.F. J. Biol. Chem. 1997; 272: 10685-10694Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). Currently knowledge of the involvement of these proteins in the secretion of eukaryotic polypeptides is restricted to export of the yeast a -mating factor (15.Kuchler K. Sterne R.E. Thorner J. EMBO J. 1989; 8: 3973-3984Crossref PubMed Scopus (323) Google Scholar). A genetic complementation strategy may identify molecules involved in the transport of HASP to the cell surface.Whatever the mechanisms involved, the targeting and translocation of the protozoan protein, HASP, onto the surface of the plasma membrane is of general interest as it is conserved in higher eukaryotic cells. This suggests that HASP may represent the first in a family of non-classically exported, lipid-anchored surface proteins. Protozoan parasites of the genus Leishmania cause a spectrum of tropical and sub-tropical diseases termed the leishmaniases. Leishmania live as either extracellular, flagellated promastigotes in the digestive tract of their sandfly vector or as aflagellated amastigotes within the phagolysosomes of mammalian macrophages (1.Molyneux D. Killick-Kendrick R. Peters W. Killick-Kendrick R. The Leishmaniases in Biology and Medicine. 1. Academic Press, London1987: 122-168Google Scholar). Much research has focused on the unusually high levels of glycosylphosphatidylinositol (GPI)1-anchored surface molecules present in these organisms (2.Ferguson M.A. Brimacombe J.S. Cottaz S. Field R.A. Guther L.S. Homans S.W. McConville M.J. Mehlert A. Milne K.G. Ralton J.E. Roy Y.A. Schneider P. Zitzmann N. Parasitology. 1994; 108: S45-S54Crossref PubMed Scopus (62) Google Scholar), particularly the unique glycoconjugate, lipophosphoglycan (LPG), abundant in promastigotes (3.McConville M.J. Ferguson M.A.J. Biochem. J. 1993; 294: 305-324Crossref PubMed Scopus (798) Google Scholar), and the metalloprotease GP63 (or leishmanolysin) (4.Frommel T.O. Button L.L. Fujikura Y. McMaster W.R. Mol. Biochem. Parasitol. 1990; 38: 25-32Crossref PubMed Scopus (71) Google Scholar). The hydrophilic acylated surface proteins (HASPs; originally named GBP and GA/CP inLeishmania major) are a family of related surface molecules expressed only in infective parasite stages (5.Flinn H.M. Rangarajan D. Smith D.F. Mol. Biochem. Parasitol. 1994; 65: 259-270Crossref PubMed Scopus (63) Google Scholar, 6.Pimenta P.F.P. Da Silva P.P. Rangarajan D. Smith D.F. Sacks D.L. Exp. Parasitol. 1994; 79: 468-479Crossref PubMed Scopus (24) Google Scholar). These proteins are unusual in that they lack a “classical” endoplasmic reticulum (ER) secretory signal sequence, a GPI-anchor consensus, or membrane-spanning domains but are surface-localized (6.Pimenta P.F.P. Da Silva P.P. Rangarajan D. Smith D.F. Sacks D.L. Exp. Parasitol. 1994; 79: 468-479Crossref PubMed Scopus (24) Google Scholar), partition into the hydrophobic phase on Triton X-114 separation, and fractionate with lipid species (5.Flinn H.M. Rangarajan D. Smith D.F. Mol. Biochem. Parasitol. 1994; 65: 259-270Crossref PubMed Scopus (63) Google Scholar). The mechanism of transport and attachment to the cell surface is unclear, although it has been suggested that this may occur by virtue of a close association with LPG via an HASP repeat region with homology to the peptidoglycan binding domain of Staphylococcus aureusprotein A (6.Pimenta P.F.P. Da Silva P.P. Rangarajan D. Smith D.F. Sacks D.L. Exp. Parasitol. 1994; 79: 468-479Crossref PubMed Scopus (24) Google Scholar). More recent studies have not substantiated this hypothesis, as HASPs are still presented on the surface of infective metacyclic parasites in the absence of LPG (7.Rangarajan D. Gokool S. McCrossan M.V. Smith D.F. J. Cell Sci. 1995; 108: 3359-3366PubMed Google Scholar). Leishmania (and other trypanosomatids) possess a conventional eukaryotic secretory pathway (8.Clayton C. Hausler T. Blattner J. Microbiol. Rev. 1995; 59: 325-344Crossref PubMed Google Scholar, 9.Al-Qahtani A. Teilhet M. Mensa-Wilmot K. Biochem. J. 1998; 331: 521-529Crossref PubMed Scopus (30) Google Scholar) and require a signal sequence for translocation of secreted proteins into the ER (10.Tobin J.F. Wirth D.F. Mol. Biochem. Parasitol. 1993; 62: 243-248Crossref PubMed Scopus (7) Google Scholar). However, a number of specialized features are apparent in the secretory systems of Leishmania and related organisms, particularly the necessity to traffic large quantities of lipid-anchored molecules to the cell surface. In addition, and uniquely to these parasites, all exo- and endocytosis at the cell surface occurs via the flagellar pocket, an invagination of the plasma membrane at the base of the single flagellum (11.Webster P. Russell D.G. Parasitol. Today. 1993; 9: 201-205Abstract Full Text PDF PubMed Scopus (110) Google Scholar, 12.Overath P. Stierhof Y.-D. Wiese M. Trends Cell Biol. 1997; 7: 27-33Abstract Full Text PDF PubMed Scopus (121) Google Scholar). Absence of ER secretory signals within the HASP family would preclude ER transport through the classical secretory pathway, i.e.via translocation through a sec61p complex into the ER lumen. This suggestion is supported by the observation that HASPB is not glycosylated, despite the presence of several consensusN-glycosylation sites. 2T. M. Alce and D. F. Smith, unpublished data. 2T. M. Alce and D. F. Smith, unpublished data. These properties place HASPB among a group of eukaryotic proteins that lack an ER-targeting secretory signal but are still exported out of the cell, by so called “non-classical” transport (13.Kuchler K. Trends Cell Biol. 1993; 3: 421-426Abstract Full Text PDF PubMed Scopus (62) Google Scholar, 14.Cleves A.E. Curr. Biol. 1997; 7: R318-R320Abstract Full Text Full Text PDF PubMed Google Scholar). Examples of proteins undergoing non-classical export include fibroblast growth factors-1 and -2, interleukin-1, yeast a-factor, and galectin. Export of Saccharomyces cerevisiae a-factor is the best understood; this short, 12-amino acid lipoprotein is exported via an ATP binding cassette transporter (15.Kuchler K. Sterne R.E. Thorner J. EMBO J. 1989; 8: 3973-3984Crossref PubMed Scopus (323) Google Scholar). By contrast, galectin appears to be released from the plasma membrane of mammalian cells through vesicular budding (16.Mehul B. Hughes R.C. J. Cell Sci. 1997; 110: 1169-1178Crossref PubMed Google Scholar), possibly via a novel pathway involving a transporter (17.Cleves A.E. Cooper D.N.W. Barondes S.H. Kelly R.B. J. Cell Biol. 1996; 5: 1017-1026Crossref Scopus (185) Google Scholar). It is unclear at present whether the examples of “non-classically” exported proteins cited above use the same or different mechanisms, and it is highly possible that several auxiliary systems are present. Here we report that the 18-amino acid N-terminal region of HASPB is acylated in vivo and is sufficient to target a heterologous protein (Aequorea victoria green fluorescent protein, GFP) to the cell surface of Leishmania. In addition, we demonstrate that HASPB is also exported by mammalian cells, suggesting that this pathway is also present in higher eukaryotes. DISCUSSIONIn the absence of a secretory signal sequence, a GPI-anchor consensus, or transmembrane domain, the pathway and mechanisms that facilitate localization of the HASP family proteins to theLeishmania cell surface have been unknown to date. Here, a deletion strategy has identified the extreme N terminus of HASPB as being essential for surface presentation of the protein. The presence of N-myristoylation and palmitoylation motifs within these residues indicates that dual acylation may mediate membrane association and also potentially act as the cell surface anchor for HASPB.Two Signals Are Required for Plasma Membrane TargetingMetabolic labeling indicated that glycine 2 and cysteine 5 in the HASPB N-terminal region are required for myristoylation and palmitoylation. Co-translational myristoylation is required for post-translational palmitoylation to occur, in agreement with the hypothesis that a myristate moiety is required to bring the protein to a membrane-bound palmitoylacyltransferase (23.Resh M.D. Cell. Signal. 1996; 8: 403-412Crossref PubMed Scopus (196) Google Scholar). According to the two-signal model (23.Resh M.D. Cell. Signal. 1996; 8: 403-412Crossref PubMed Scopus (196) Google Scholar), such modifications would be sufficient to allow the stable association of HASPB with membranes previously observed (5.Flinn H.M. Rangarajan D. Smith D.F. Mol. Biochem. Parasitol. 1994; 65: 259-270Crossref PubMed Scopus (63) Google Scholar, 6.Pimenta P.F.P. Da Silva P.P. Rangarajan D. Smith D.F. Sacks D.L. Exp. Parasitol. 1994; 79: 468-479Crossref PubMed Scopus (24) Google Scholar). Many proteins have been shown to be targeted to the plasma membrane by dual acylation. The mechanism of targeting is unclear, although the identification of a Gα protein palmitoylacyltransferase activity in plasma membrane fractions (30.Dunphy J.T. Greentree W.K. Manahan C.L. Linder M.E. J. Biol. Chem. 1996; 271: 7154-7159Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) supports a model in which an N-myristoylated protein could become “trapped” in a lipid bilayer when it associates with this putative enzyme and is palmitoylated (Refs. 31.van't Hof W. Resh M. J. Cell Biol. 1997; 136: 1023-1035Crossref PubMed Scopus (123) Google Scholar and 32.Morales J. Fishburn C.S. Wilson P.T. Bourne H.R. Mol. Biol. Cell. 1998; 9: 1-14Crossref PubMed Scopus (81) Google Scholar and references therein).The requirement of dual acylation for stable membrane association and targeting to the plasma membrane was tested using a series of fusion proteins expressed in L. major, in which the N-terminal HASPB 18 amino acids were fused to the N terminus of GFP. Cell fractionation demonstrated that both myristate and palmitate were necessary for efficient membrane binding; myristate alone l" @default.
• W2004763373 created "2016-06-24" @default.
• W2004763373 creator A5010421361 @default.
• W2004763373 creator A5030633948 @default.
• W2004763373 creator A5047431371 @default.
• W2004763373 creator A5061440312 @default.
• W2004763373 creator A5084813588 @default.
• W2004763373 date "2000-04-01" @default.
• W2004763373 modified "2023-10-16" @default.
• W2004763373 title "Acylation-dependent Protein Export inLeishmania" @default.
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