C Stegmayer
Direct transport across the plasma membrane of mammalian cells of Leishmania HASPB as revealed by a CHO export mutant
Stegmayer, C; Kehlenbach, A; Tournaviti, S; Wegehingel, S; Zehe, C; Denny, P; Smith, DF; Schwappach, B; Nickel, W
Authors
A Kehlenbach
S Tournaviti
S Wegehingel
C Zehe
Professor Paul Denny p.w.denny@durham.ac.uk
Professor
DF Smith
B Schwappach
W Nickel
Abstract
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.
Citation
Stegmayer, C., Kehlenbach, A., Tournaviti, S., Wegehingel, S., Zehe, C., Denny, P., …Nickel, W. (2005). Direct transport across the plasma membrane of mammalian cells of Leishmania HASPB as revealed by a CHO export mutant. Journal of Cell Science, 118(3), 517-527. https://doi.org/10.1242/jcs.01645
Journal Article Type | Article |
---|---|
Publication Date | Feb 1, 2005 |
Deposit Date | May 11, 2009 |
Publicly Available Date | May 11, 2009 |
Journal | Journal of Cell Science |
Print ISSN | 0021-9533 |
Electronic ISSN | 1477-9137 |
Publisher | The Company of Biologists |
Peer Reviewed | Peer Reviewed |
Volume | 118 |
Issue | 3 |
Pages | 517-527 |
DOI | https://doi.org/10.1242/jcs.01645 |
Keywords | Leishmania parasites, Surface coat, Hydrophilic acylated surface protein, Acylation, SH4 domain, Cell surface expression, Non-classical protein export, Fibroblast growth factor, 2fibroblast-growth-factor, Secretory signal sequence, Gene B-protein, Factor- |
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