Direct transport across the plasma membrane of mammalian cells of Leishmania HASPB as revealed by a CHO export mutant
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Direct transport across the plasma membrane of mammalian cells of Leishmania HASPB as revealed by a CHO export mutant. / Stegmayer, Carolin; Kehlenbach, Angelika; Tournaviti, Stella; Wegehingel, Sabine; Zehe, Christoph; Denny, Paul; Smith, Deborah F; Schwappach, Blanche; Nickel, Walter.
In: J CELL SCI, Vol. 118, No. Pt 3, 01.02.2005, p. 517-27.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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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 -