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Molecular Analysis of the Unconventional Export Machinery of HASPB, a component of the surface coat of Leishmania parasites
Summary Leishmania HASPB is a lipoprotein that is exported to the extracellular space from both Leishmania parasites and mammalian cells via an unconventional secretory pathway (Denny et al., 2000; Stegmayer et al., 2005). 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 (Denny et al., 2000; Stegmayer et al., 2005). HASPB targeting to the plasma membrane depends on SH4 acylation, which 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. Furthermore, possible secretory mechanisms for HASPB are not yet clarified in molecular terms. In order to address this issue, a screening for clonal CHO mutants derived from somatic mutagenesis that are incapable of exporting HASPB, was performed. In such a CHO mutant cell line HASPB was myristoylated and palmitoylated and was 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 was largely reduced in this mutant despite a normal expression level of the HASPB reporter molecule compared to CHO wild-type cells. These findings indicate that the subcellular site of HASPB membrane translocation is the plasma membrane as the reporter molecule accumulates in this location when export is blocked. Hence, based on these results a two-step process of HASPB cell surface biogenesis can be defined, in which SH4 acylation of HASPB mediates intracellular targeting to the plasma membrane. In a second step, the plasma membrane-resident machinery that is apparently disrupted in the CHO mutant cell line, mediates membrane translocation of HASPB. Intriguingly, the angiogenic growth factor FGF-2 and Galectin-1, a lectin of the extracellular matrix, proteins secreted by unconventional means, were 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. However, as revealed from the mutagenesis analysis the identified chemokine orphan receptor 1 was not the reason for the perturbed HASPB membrane localization in CHO K3 cells since its expression did not seem to be affected in CHO K3 cells. In the second chapter of this thesis, the SH4 protein HASPB, besides being localized to the cell surface, was found to be associated with extracellular vesicles. HASPB induces curvature of the plasma membrane resulting in the formation of highly-dynamic non-apoptotic plasma membrane blebs (Tournaviti et al., 2006 submitted). Based on these observations cell culture supernatants from HASPB expressing CHO cells were analyzed. As revealed by flotation analysis the detected sedimentable material contained a vesicle-associated HASPB population. Importantly, other SH4 proteins such as Src, Fyn, Yes and Lck were not detectable in extracellular vesicles. Since these proteins were able to induce the formation of plasma membrane blebs, the extracellular HASPB vesicle population was shown not to be a consequence of plasma membrane blebbing, a process promoting the shedding of plasma membrane-derived vesicles that are released into the extracellular space. This observation was confirmed by results obtained from HeLa cells that were able to produce HASPB-containing vesicles. Importantly, the formation of plasma membrane blebs was largely reduced in this cell line. As judged by protease protection experiments HASPB was shown to be located on the inner leaflet of the vesicle membrane. Colocalization analysis with different exosomes markers further confirmed that HASPB might be exported via vesicles being released by the MVB sorting machinery. Furthermore employing linear sucrose gradients and electron microscopy, these vesicles corresponded to the reported density of 1.15 g/ml for exosomes (Heijnen et al., 1999) as well as to the exosomal size with diameters <100 nm (Stoorvogel et al., 2002). Together, these findings strongly suggest that dually acylated HASPB localized on the inner leaflet of the plasma membrane is delivered into vesicles that are released by the MVB sorting machinery into the extracellular space.
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570 Biowissenschaften, Biologie
570 Life sciences