Acylation-dependent Protein Export inLeishmania

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 the lipophosphoglycan in promastigotes M.J. PubMed and the PubMed Scholar). The hydrophilic acylated surface proteins and are a family of surface molecules in infective parasite D. 1994; PubMed D. 1994; PubMed Scholar). These proteins are unusual in that lack a endoplasmic reticulum secretory signal sequence, a or are D. 1994; PubMed the on and D. 1994; PubMed Scholar). The of transport and to the cell surface is it has been that by of a to the domain of A D. 1994; PubMed Scholar). as are on the surface of infective parasites in the of D. S. 108: a eukaryotic secretory pathway PubMed A. PubMed and a signal for translocation of proteins the PubMed Scholar). a of are in the secretory of Leishmania and the to of molecules to the cell and to these and the cell surface the of the plasma membrane the of the P. PubMed P. PubMed of secretory within the family transport the secretory translocation a the is by the that HASPB is the of and D. and D. These HASPB a of eukaryotic proteins that lack secretory signal are of the by “non-classical” transport PubMed PubMed Scholar). of proteins export growth and and of is the acid is PubMed Scholar). to the plasma membrane of mammalian cells PubMed a novel pathway a Scholar). is present the of proteins the or and it is that are we that the acid N-terminal of HASPB is acylated in and is sufficient to target a protein victoria green fluorescent to the cell surface of Leishmania. we that HASPB is also by mammalian cells, that pathway is also present in higher the of a secretory signal sequence, a or transmembrane domain, the pathway and mechanisms that localization of the family proteins to cell surface been to Here, a has the of HASPB as essential for surface of the The of N-myristoylation and palmitoylation within these residues that dual acylation membrane and also as the cell surface for for that and in the HASPB N-terminal are required for and is required for palmitoylation to in the that a myristate is required to the protein to a PubMed Scholar). to the PubMed sufficient to the of HASPB membranes D. 1994; PubMed D. 1994; PubMed Scholar). proteins been to to the plasma membrane by dual The of is the of a protein in plasma membrane PubMed a in protein in a it and is W. PubMed and PubMed and of dual acylation for membrane and to the plasma membrane a of proteins in in the N-terminal HASPB 18 amino acids to the of that both myristate and palmitate for membrane myristate to these as the protein required both and palmitoylation to localization to the plasma of the acylated proteins to the plasma membrane has been of to the plasma membrane in mammalian cells is to a novel pathway W. PubMed Scholar). acylated the plasma membrane and in the by in mammalian cell J.E. PubMed Scholar). These the in trafficking. of HASPB or the GFP the protein to the plasma membrane by data in the and by the pathway are involved in both and trafficking. is that of the palmitoylation in to the protein in by a the that palmitoylation proteins a to the pathway J.E. PubMed Scholar). is of the of acylated proteins that been to to the the by of of these proteins to the plasma membrane is dependent upon palmitoylation D. PubMed the in the transport of the lack of secretory acylated proteins W. PubMed the of HASPB in a mammalian by a of transport that of J.E. PubMed that palmitoylation in the of the to the plasma membrane. the that is to and the plasma membrane W. PubMed Scholar). and W. PubMed a of in membrane to the HASPB in the of the and the localization of W. PubMed that proteins by are to detergent-resistant membrane in mammalian cells PubMed Scholar). These are in protein in the secretory pathway PubMed Scholar). it predicted proteins on the face of in the pathway and are to the plasma membrane. is that required for localization is to protein target by these the of a and translocation as that to the protein to the extracellular to to the by a of the it is that is sufficient to a of proteins to the plasma membrane PubMed Scholar). these HASPB is in D. 1994; PubMed D. 1994; PubMed signal that to translocation by the and that the N-terminal 18 amino acids of HASPB are sufficient to a protein to the cell is sufficient to transport to the face of the plasma within the acid signal is to translocation of a of the protein the membrane and the extracellular The N-terminal amino acids of the acylation are sufficient to GFP as as the The for of the cell surface are as to either surface of the protein or within the that a protein face of the plasma membrane to the is that data is the PubMed in a protein translocation of the the and the hydrophilic structural that a for the family of transport molecules R. PubMed Scholar). of the of these proteins in the of eukaryotic polypeptides is to export of the a PubMed Scholar). A molecules involved in the transport of to the cell the mechanisms the and translocation of the protozoan onto the surface of the plasma membrane is of as it is in higher eukaryotic that the in a family of 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 the lipophosphoglycan in promastigotes M.J. PubMed and the PubMed Scholar). The hydrophilic acylated surface proteins and are a family of surface molecules in infective parasite D. 1994; PubMed D. 1994; PubMed Scholar). These proteins are unusual in that lack a endoplasmic reticulum secretory signal sequence, a or are D. 1994; PubMed the on and D. 1994; PubMed Scholar). The of transport and to the cell surface is it has been that by of a to the domain of A D. 1994; PubMed Scholar). as are on the surface of infective parasites in the of D. S. 108: Scholar). Leishmania a eukaryotic secretory pathway PubMed A. PubMed and a signal for translocation of proteins the PubMed Scholar). a of are in the secretory of Leishmania and the to of molecules to the cell and to these and the cell surface the of the plasma membrane the of the P. PubMed P. PubMed Scholar). of secretory within the family transport the secretory translocation a the is by the that HASPB is the of and D. and D. These HASPB a of eukaryotic proteins that lack secretory signal are of the by “non-classical” transport PubMed PubMed Scholar). of proteins export growth and and of is the acid is PubMed Scholar). to the plasma membrane of mammalian cells PubMed a novel pathway a Scholar). is present the of proteins the or and it is that are we that the acid N-terminal of HASPB is acylated in and is sufficient to target a protein victoria green fluorescent to the cell surface of Leishmania. we that HASPB is also by mammalian cells, that pathway is also present in higher the of a secretory signal sequence, a or transmembrane domain, the pathway and mechanisms that localization of the family proteins to cell surface been to Here, a has the of HASPB as essential for surface of the The of N-myristoylation and palmitoylation within these residues that dual acylation membrane and also as the cell surface for for that and in the HASPB N-terminal are required for and is required for palmitoylation to in the that a myristate is required to the protein to a PubMed Scholar). to the PubMed sufficient to the of HASPB membranes D. 1994; PubMed D. 1994; PubMed Scholar). proteins been to to the plasma membrane by dual The of is the of a protein in plasma membrane PubMed a in protein in a it and is W. PubMed and PubMed and of dual acylation for membrane and to the plasma membrane a of proteins in in the N-terminal HASPB 18 amino acids to the of that both myristate and palmitate for membrane myristate to these as the protein required both and palmitoylation to localization to the plasma of the acylated proteins to the plasma membrane has been of to the plasma membrane in mammalian cells is to a novel pathway W. PubMed Scholar). acylated the plasma membrane and in the by in mammalian cell J.E. PubMed Scholar). These the in trafficking. of HASPB or the GFP the protein to the plasma membrane by data in the and by the pathway are involved in both and trafficking. is that of the palmitoylation in to the protein in by a the that palmitoylation proteins a to the pathway J.E. PubMed Scholar). is of the of acylated proteins that been to to the the by of of these proteins to the plasma membrane is dependent upon palmitoylation D. PubMed the in the transport of the lack of secretory acylated proteins W. PubMed the of HASPB in a mammalian by a of transport that of J.E. PubMed that palmitoylation in the of the to the plasma membrane. the that is to and the plasma membrane W. PubMed Scholar). and W. PubMed a of in membrane to the HASPB in the of the and the localization of W. PubMed that proteins by are to detergent-resistant membrane in mammalian cells PubMed Scholar). These are in protein in the secretory pathway PubMed Scholar). it predicted proteins on the face of in the pathway and are to the plasma membrane. is that required for localization is to protein target by these the of a and translocation as that to the protein to the extracellular to to the by a of the it is that is sufficient to a of proteins to the plasma membrane PubMed Scholar). these HASPB is in D. 1994; PubMed D. 1994; PubMed signal that to translocation by the and that the N-terminal 18 amino acids of HASPB are sufficient to a protein to the cell is sufficient to transport to the face of the plasma within the acid signal is to translocation of a of the protein the membrane and the extracellular The N-terminal amino acids of the acylation are sufficient to GFP as as the The for of the cell surface are as to either surface of the protein or within the that a protein face of the plasma membrane to the is that data is the PubMed in a protein translocation of the the and the hydrophilic structural that a for the family of transport molecules R. PubMed Scholar). of the of these proteins in the of eukaryotic polypeptides is to export of the a PubMed Scholar). A molecules involved in the transport of to the cell the mechanisms the and translocation of the protozoan onto the surface of the plasma membrane is of as it is in higher eukaryotic that the in a family of surface proteins. the of a secretory signal sequence, a or transmembrane domain, the pathway and mechanisms that localization of the family proteins to cell surface been to Here, a has the of HASPB as essential for surface of the The of N-myristoylation and palmitoylation within these residues that dual acylation membrane and also as the cell surface for for that and in the HASPB N-terminal are required for and is required for palmitoylation to in the that a myristate is required to the protein to a PubMed Scholar). to the PubMed sufficient to the of HASPB membranes D. 1994; PubMed D. 1994; PubMed Scholar). proteins been to to the plasma membrane by dual The of is the of a protein in plasma membrane PubMed a in protein in a it and is W. PubMed and PubMed and of dual acylation for membrane and to the plasma membrane a of proteins in in the N-terminal HASPB 18 amino acids to the of that both myristate and palmitate for membrane myristate to these as the protein required both and palmitoylation to localization to the plasma of the acylated proteins to the plasma membrane has been of to the plasma membrane in mammalian cells is to a novel pathway W. PubMed Scholar). acylated the plasma membrane and in the by in mammalian cell J.E. PubMed Scholar). These the in trafficking. of HASPB or the GFP the protein to the plasma membrane by data in the and by the pathway are involved in both and trafficking. is that of the palmitoylation in to the protein in by a the that palmitoylation proteins a to the pathway J.E. PubMed Scholar). is of the of acylated proteins that been to to the the by of of these proteins to the plasma membrane is dependent upon palmitoylation D. PubMed the in the transport of the lack of secretory acylated proteins W. PubMed the of HASPB in a mammalian by a of transport that of J.E. PubMed that palmitoylation in the of the to the plasma membrane. the that is to and the plasma membrane W. PubMed Scholar). and W. PubMed a of in membrane to the HASPB in the of the and the localization of W. PubMed that proteins by are to detergent-resistant membrane in mammalian cells PubMed Scholar). These are in protein in the secretory pathway PubMed Scholar). it predicted proteins on the face of in the pathway and are to the plasma membrane. is that required for localization is to protein target by these the of a and translocation as that to the protein to the extracellular to to the by a of the it is that is sufficient to a of proteins to the plasma membrane PubMed Scholar). these HASPB is in D. 1994; PubMed D. 1994; PubMed signal that to translocation by the and that the N-terminal 18 amino acids of HASPB are sufficient to a protein to the cell is sufficient to transport to the face of the plasma within the acid signal is to translocation of a of the protein the membrane and the extracellular The N-terminal amino acids of the acylation are sufficient to GFP as as the The for of the cell surface are as to either surface of the protein or within the that a protein face of the plasma membrane to the is that data is the PubMed in a protein translocation of the the and the hydrophilic structural that a for the family of transport molecules R. PubMed Scholar). of the of these proteins in the of eukaryotic polypeptides is to export of the a PubMed Scholar). A molecules involved in the transport of to the cell the mechanisms the and translocation of the protozoan onto the surface of the plasma membrane is of as it is in higher eukaryotic that the in a family of surface proteins. for that and in the HASPB N-terminal are required for and is required for palmitoylation to in the that a myristate is required to the protein to a PubMed Scholar). to the PubMed sufficient to the of HASPB membranes D. 1994; PubMed D. 1994; PubMed Scholar). proteins been to to the plasma membrane by dual The of is the of a protein in plasma membrane PubMed a in protein in a it and is W. PubMed and PubMed and of dual acylation for membrane and to the plasma membrane a of proteins in in the N-terminal HASPB 18 amino acids to the of that both myristate and palmitate for membrane myristate to these as the protein required both and palmitoylation to localization to the plasma of the acylated proteins to the plasma membrane has been of to the plasma membrane in mammalian cells is to a novel pathway W. PubMed Scholar). acylated the plasma membrane and in the by in mammalian cell J.E. PubMed Scholar). These the in trafficking. of HASPB or the GFP the protein to the plasma membrane by data in the and by the pathway are involved in both and trafficking. is that of the palmitoylation in to the protein in by a the that palmitoylation proteins a to the pathway J.E. PubMed Scholar). is of the of acylated proteins that been to to the the by of of these proteins to the plasma membrane is dependent upon palmitoylation D. PubMed the in the transport of the lack of secretory acylated proteins W. PubMed the of HASPB in a mammalian by a of transport that of J.E. PubMed that palmitoylation in the of the to the plasma membrane. the that is to and the plasma membrane W. PubMed Scholar). and W. PubMed a of in membrane to the HASPB in the of the and the localization of W. PubMed that proteins by are to detergent-resistant membrane in mammalian cells PubMed Scholar). These are in protein in the secretory pathway PubMed Scholar). it predicted proteins on the face of in the pathway and are to the plasma membrane. is that required for localization is to protein target by these the of a and translocation as that to the protein to the extracellular to to the by a of the it is that is sufficient to a of proteins to the plasma membrane PubMed Scholar). these HASPB is in D. 1994; PubMed D. 1994; PubMed signal that to translocation by the and that the N-terminal 18 amino acids of HASPB are sufficient to a protein to the cell is sufficient to transport to the face of the plasma within the acid signal is to translocation of a of the protein the membrane and the extracellular The N-terminal amino acids of the acylation are sufficient to GFP as as the The for of the cell surface are as to either surface of the protein or within the that a protein face of the plasma membrane to the is that data is the PubMed in a protein translocation of the the and the hydrophilic structural that a for the family of transport molecules R. PubMed Scholar). of the of these proteins in the of eukaryotic polypeptides is to export of the a PubMed Scholar). A molecules involved in the transport of to the cell the mechanisms the and translocation of the protozoan onto the surface of the plasma membrane is of as it is in higher eukaryotic that the in a family of surface proteins. for that and in the HASPB N-terminal are required for and is required for palmitoylation to in the that a myristate is required to the protein to a PubMed Scholar). to the PubMed sufficient to the of HASPB membranes D. 1994; PubMed D. 1994; PubMed Scholar). proteins been to to the plasma membrane by dual The of is the of a protein in plasma membrane PubMed a in protein in a it and is W. PubMed and PubMed and of dual acylation for membrane and to the plasma membrane a of proteins in in the N-terminal HASPB 18 amino acids to the of that both myristate and palmitate for membrane myristate to these as the protein required both and palmitoylation to localization to the plasma membrane. that and in the HASPB N-terminal are required for and is required for palmitoylation to in the that a myristate is required to the protein to a PubMed Scholar). to the PubMed sufficient to the of HASPB membranes D. 1994; PubMed D. 1994; PubMed Scholar). proteins been to to the plasma membrane by dual The of is the of a protein in plasma membrane PubMed a in protein in a it and is W. PubMed and PubMed and The of dual acylation for membrane and to the plasma membrane a of proteins in in the N-terminal HASPB 18 amino acids to the of that both myristate and palmitate for membrane myristate to these as the protein required both and palmitoylation to localization to the plasma membrane. of the acylated proteins to the plasma membrane has been of to the plasma membrane in mammalian cells is to a novel pathway W. PubMed Scholar). acylated the plasma membrane and in the by in mammalian cell J.E. PubMed Scholar). These the in trafficking. of HASPB or the GFP the protein to the plasma membrane by data in the and by the pathway are involved in both and trafficking. is that of the palmitoylation in to the protein in by a the that palmitoylation proteins a to the pathway J.E. PubMed Scholar). is of the of acylated proteins that been to to the the by of of these proteins to the plasma membrane is dependent upon palmitoylation D. PubMed the in the transport of the lack of secretory acylated proteins W. PubMed the of HASPB in a mammalian by a of transport that of J.E. PubMed that palmitoylation in the of the to the plasma membrane. the that is to and the plasma membrane W. PubMed Scholar). and W. PubMed a of in membrane to the HASPB in the of the and the localization of W. PubMed that proteins by are to detergent-resistant membrane in mammalian cells PubMed Scholar). These are in protein in the secretory pathway PubMed Scholar). it predicted proteins on the face of in the pathway and are to the plasma membrane. is that required for localization is to protein target by these the of a and translocation as that to the protein to the extracellular The acylated proteins to the plasma membrane has been of to the plasma membrane in mammalian cells is to a novel pathway W. PubMed Scholar). acylated the plasma membrane and in the by in mammalian cell J.E. PubMed Scholar). These the in trafficking. of HASPB or the GFP the protein to the plasma membrane by data in the and by the pathway are involved in both and trafficking. is that of the palmitoylation in to the protein in by a the that palmitoylation proteins a to the pathway J.E. PubMed Scholar). is of the of acylated proteins that been to to the the by of of these proteins to the plasma membrane is dependent upon palmitoylation D. PubMed Scholar). the in the transport of the lack of secretory acylated proteins W. PubMed the of HASPB in a mammalian by a of transport that of J.E. PubMed that palmitoylation in the of the to the plasma membrane. the that is to and the plasma membrane W. PubMed Scholar). and W. PubMed a of in membrane to the HASPB in the of the and the localization of W. PubMed Scholar). that proteins by are to detergent-resistant membrane in mammalian cells PubMed Scholar). These are in protein in the secretory pathway PubMed Scholar). it predicted proteins on the face of in the pathway and are to the plasma membrane. is that required for localization is to protein target by these the of a and translocation as that to the protein to the extracellular to to the by a of the it is that is sufficient to a of proteins to the plasma membrane PubMed Scholar). these HASPB is in D. 1994; PubMed D. 1994; PubMed signal that to translocation by the and that the N-terminal 18 amino acids of HASPB are sufficient to a protein to the cell is sufficient to transport to the face of the plasma within the acid signal is to translocation of a of the protein the membrane and the extracellular The N-terminal amino acids of the acylation are sufficient to GFP as as the The for of the cell surface are as to either surface of the protein or within the that a protein face of the plasma membrane to the is that data is the PubMed in a protein translocation of the the and the hydrophilic structural that a for the family of transport molecules R. PubMed Scholar). of the of these proteins in the of eukaryotic polypeptides is to export of the a PubMed Scholar). A molecules involved in the transport of to the cell the mechanisms the and translocation of the protozoan onto the surface of the plasma membrane is of as it is in higher eukaryotic that the in a family of surface proteins. of the it is that is sufficient to a of proteins to the plasma membrane PubMed Scholar). these HASPB is in D. 1994; PubMed D. 1994; PubMed signal that to translocation by the and that the N-terminal 18 amino acids of HASPB are sufficient to a protein to the cell is sufficient to transport to the face of the plasma within the acid signal is to translocation of a of the protein the membrane and the extracellular The N-terminal amino acids of the acylation are sufficient to GFP as as the The for of the cell surface are as to either surface of the protein or within the The that a protein face of the plasma membrane to the is that data is the PubMed in a protein translocation of the the and the hydrophilic structural that a for the family of transport molecules R. PubMed Scholar). of the of these proteins in the of eukaryotic polypeptides is to export of the a PubMed Scholar). A molecules involved in the transport of to the cell the mechanisms the and translocation of the protozoan onto the surface of the plasma membrane is of as it is in higher eukaryotic that the in a family of surface proteins. and for and of the and for