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First published online 18 January 2005
doi: 10.1242/jcs.01645

Journal of Cell Science 118, 517-527 (2005)
Published by The Company of Biologists 2005
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Research Article

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

Carolin Stegmayer1, Angelika Kehlenbach2, Stella Tournaviti1, Sabine Wegehingel1, Christoph Zehe1, Paul Denny3, Deborah F. Smith3, Blanche Schwappach2 and Walter Nickel1,*

1 Heidelberg University Biochemistry Center, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany
2 Zentrum für Molekulare Biologie Heidelberg, 69120 Heidelberg, Germany
3 Wellcome Trust Laboratories for Molecular Parasitology, Department of Biological Sciences, Imperial College London, SW7 2AZ, UK

* Author for correspondence (e-mail: walter.nickel{at}urz.uni-heidelberg.de)

Accepted 11 November 2004

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.

Key words: Leishmania parasites, Surface coat, Hydrophilic acylated surface protein, Acylation, SH4 domain, Cell surface expression, Non-classical protein export, Fibroblast growth factor 2


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© The Company of Biologists Ltd 2005