GPI valence and the fate of secretory membrane proteins in African trypanosomes

Abstract

Progression of GPI-anchored proteins in bloodstream African trypanosomes correlates with GPI-valence: homodimeric VSG (2 GPI) is a surface protein; heterodimeric transferrin receptor (1 GPI) localizes in the flagellar pocket; homodimeric GPI-minus VSG (0 GPI) is rapidly degraded in the lysosome. We test this relationship using three native secretory/endocytic proteins as monomeric GPI-plus and -minus reporters. GPI-minus procyclin trafficks to the lysosome and is degraded. GPI-plus procyclin trafficks to the flagellar pocket/cell surface and is released (∼50%) with an intact anchor, the remainder (∼50%) is degraded in the lysosome. GPI-plus BiPNHP, derived from the ER marker BiP, is released quantitatively (>80%), while GPI-plus p67HP, derived from the lysosomal marker p67, turns over by both release (∼15%) and lysosomal degradation (>50%). Turnover of endogenous transferrin receptor occurs primarily by lysosomal degradation (>90%). Thus shedding of monovalent GPI reporters correlates inversely with lysosomal targeting. We propose that mono-GPI reporters cycle through the flagellar pocket and endosome until they are disposed of by either shedding or lysosomal targeting. Partitioning between these fates may be a function of individual physical properties. Release is likely due to the exclusive use of C-14:0 myristate in the bloodstream stage GPI anchor. Up-regulation of transferrin receptor by culture in dog serum resulted in prominent cell surface localization, but not in elevated release. Surface receptor was non-functional for ligand binding suggesting that it may be bivalent homodimers of the GPI-anchored ESAG6 receptor subunit.

Streamlined Architecture and Glycosylphosphatidylinositol-dependent Trafficking in the Early Secretory Pathway of African Trypanosomes

“…Constructs were linearized with NotI and TbSec RNAi vectors were introduced separately into DM-BS cells and clonal cell lines selected for with phleomycin. Cloning of the BiPN:GPI reporter into pXS5 neo has been described previously (Schwartz et al, 2005). After replacement of neomycin resistance cassette with the puromycin cassette, the construct was linearized with XhoI, stably transfected into the TbSec23.1 and TbSec23.2 RNAi cell lines, and clonal transformants were selected with puromycin.…”

“…Soluble GPI-minus VSG is delayed in ER exit relative to native controls and is missorted to the lysosome during post-Golgi transport (Triggs and Bangs, 2003). Conversely, addition of a GPI anchor can rescue biosynthetic cargo destined for lysosomal delivery (Schwartz et al, 2005). Finally, the GPI anchor is critical for sorting and recycling of VSG, which is constantly taken up with cargo endocytosed for nutritional purposes (Grü nfelder et al, 2003).…”

Role of Protein Translocation Pathways across the Endoplasmic Reticulum in Trypanosoma brucei

“…To this end, an EP protein containing an HA tag and carrying multiple methionines to enable efficient labeling of the protein (EPMH) was used (Fig. 7, A and B) (54). Pulse-chase labeling and immunoprecipitation was performed with anti-HA antibodies.…”

“…7A demonstrate two major immunoprecipitated products, which were observed previously using the same construct; the first product was a ϳ40-kDa precursor that represents a protein that was modified by N-glycosylation and GPI anchoring and hence was able to translocate into the ER, and the second band represented fully modified GPI-anchored protein. The same experiment was performed using cells carrying the EPMH construct lacking the C-terminal domain with the GPI anchoring site (EPMH⌬GPI) (54). The level of immunoprecipitated EP was markedly reduced upon SEC71 silencing for both EPMH and EPMH⌬GPI, suggesting that it is not the ability to undergo GPI anchoring that causes the reduction in EP or its derivative upon SEC71 silencing.…”

Transferrin Binding Proteins as a Means to Obtain Iron in Parasitic Protozoa

“…Apparently, TFR is retained in the flagellar pocket by the single GPI anchor, while those that present two GPI anchors are targeted to the cell surface [Schwartz et al, 2005;Taylor and Kelly, 2010]. Then, GPI is essential for the correct formation of the VSG coat, for the expression of TbTFRs on the flagellar pocket, and to signal for clathrin-coated endocytosis [Allen et al, 2003].…”