Direct transport across the plasma membrane of mammalian cells of Leishmania HASPB as revealed by a CHO export mutant

TY - JOUR

T1 - Direct transport across the plasma membrane of mammalian cells of Leishmania HASPB as revealed by a CHO export mutant

AU - Stegmayer, Carolin

AU - Kehlenbach, Angelika

AU - Tournaviti, Stella

AU - Wegehingel, Sabine

AU - Zehe, Christoph

AU - Denny, Paul

AU - Smith, Deborah F

AU - Schwappach, Blanche

AU - Nickel, Walter

PY - 2005/2/1

Y1 - 2005/2/1

N2 - Leishmania HASPB is a lipoprotein that is exported to the extracellular space from both Leishmania parasites and mammalian cells via an unconventional secretory pathway. Exported HASPB remains anchored in the outer leaflet of the plasma membrane mediated by myristate and palmitate residues covalently attached to the N-terminal SH4 domain of HASPB. HASPB targeting to the plasma membrane depends on SH4 acylation that occurs at intracellular membranes. How acylated HASPB is targeted to the plasma membrane and, in particular, the subcellular site of HASPB membrane translocation is unknown. In order to address this issue, we screened for clonal CHO mutants that are incapable of exporting HASPB. A detailed characterization of such a CHO mutant cell line revealed that the expression level of the HASPB reporter molecule is unchanged compared to CHO wild-type cells; that it is both myristoylated and palmitoylated; and that it is mainly localized to the plasma membrane as judged by confocal microscopy and subcellular fractionation. However, based on a quantitative flow cytometry assay and a biochemical biotinylation assay of surface proteins, HASPB transport to the outer leaflet of the plasma membrane is largely reduced in this mutant. From these data, we conclude that the subcellular site of HASPB membrane translocation is the plasma membrane as the reporter molecule accumulates in this location when export is blocked. Thus, these results allow us to define a two-step process of HASPB cell surface biogenesis in which SH4 acylation of HASPB firstly mediates intracellular targeting to the plasma membrane. In a second step, the plasma membrane-resident machinery, which is apparently disrupted in the CHO mutant cell line, mediates membrane translocation of HASPB. Intriguingly, the angiogenic growth factor FGF-2, another protein secreted by unconventional means, is shown to be secreted normally from the HASPB export mutant cell line. These observations demonstrate that the export machinery component defective in the export mutant cell line functions specifically in the HASPB export pathway.

AB - Leishmania HASPB is a lipoprotein that is exported to the extracellular space from both Leishmania parasites and mammalian cells via an unconventional secretory pathway. Exported HASPB remains anchored in the outer leaflet of the plasma membrane mediated by myristate and palmitate residues covalently attached to the N-terminal SH4 domain of HASPB. HASPB targeting to the plasma membrane depends on SH4 acylation that occurs at intracellular membranes. How acylated HASPB is targeted to the plasma membrane and, in particular, the subcellular site of HASPB membrane translocation is unknown. In order to address this issue, we screened for clonal CHO mutants that are incapable of exporting HASPB. A detailed characterization of such a CHO mutant cell line revealed that the expression level of the HASPB reporter molecule is unchanged compared to CHO wild-type cells; that it is both myristoylated and palmitoylated; and that it is mainly localized to the plasma membrane as judged by confocal microscopy and subcellular fractionation. However, based on a quantitative flow cytometry assay and a biochemical biotinylation assay of surface proteins, HASPB transport to the outer leaflet of the plasma membrane is largely reduced in this mutant. From these data, we conclude that the subcellular site of HASPB membrane translocation is the plasma membrane as the reporter molecule accumulates in this location when export is blocked. Thus, these results allow us to define a two-step process of HASPB cell surface biogenesis in which SH4 acylation of HASPB firstly mediates intracellular targeting to the plasma membrane. In a second step, the plasma membrane-resident machinery, which is apparently disrupted in the CHO mutant cell line, mediates membrane translocation of HASPB. Intriguingly, the angiogenic growth factor FGF-2, another protein secreted by unconventional means, is shown to be secreted normally from the HASPB export mutant cell line. These observations demonstrate that the export machinery component defective in the export mutant cell line functions specifically in the HASPB export pathway.

KW - Acylation

KW - Animals

KW - Antigens, Protozoan/genetics

KW - Biotinylation

KW - CHO Cells

KW - Cell Membrane/chemistry

KW - Cricetinae

KW - Cricetulus

KW - Cytosol/chemistry

KW - Doxycycline/pharmacology

KW - Fatty Acids/metabolism

KW - Fibroblast Growth Factor 2/genetics

KW - Flow Cytometry

KW - Gene Expression/drug effects

KW - Green Fluorescent Proteins/genetics

KW - Intracellular Membranes/chemistry

KW - Leishmania/physiology

KW - Membrane Proteins/analysis

KW - Mutagenesis, Insertional

KW - Mutation

KW - Parasites/physiology

KW - Peptide Fragments/genetics

KW - Protein Transport

KW - Protozoan Proteins/genetics

KW - Recombinant Fusion Proteins/genetics

KW - Retroviridae/genetics

U2 - 10.1242/jcs.01645

DO - 10.1242/jcs.01645

M3 - SCORING: Journal article

C2 - 15657075

VL - 118

SP - 517

EP - 527

JO - J CELL SCI

JF - J CELL SCI

SN - 0021-9533

IS - Pt 3

ER -