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Abstract
The critical virulence factor of bloodstream-form Trypanosoma brucei is the glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG). Endoplasmic reticulum (ER) exit of VSG is GPI dependent and relies on a discrete subset of COPII machinery (TbSec23.2/TbSec24.1). In other systems, p24 transmembrane adaptor proteins selectively recruit GPI-anchored cargo into nascent COPII vesicles. Trypanosomes have eight putative p24s (TbERP1 to TbERP8) that are constitutively expressed at the mRNA level. However, only four TbERP proteins (TbERP1, -2, -3, and -8) are detectable in bloodstream-form parasites. All four colocalize to ER exit sites, are required for efficient GPI-dependent ER exit, and are interdependent for steady-state stability. These results suggest shared function as an oligomeric ER GPI-cargo receptor. This cohort also mediates rapid forward trafficking of the soluble lysosomal hydrolase TbCatL. Procyclic insect-stage trypanosomes have a distinct surface protein, procyclin, bearing a different GPI anchor structure. A separate cohort of TbERP proteins (TbERP1, -2, -4, and -8) are expressed in procyclic parasites and also function in GPI-dependent ER exit. Collectively, these results suggest developmentally regulated TbERP cohorts, likely in obligate assemblies, that may recognize stage-specific GPI anchors to facilitate GPI-cargo trafficking throughout the parasite life cycle. IMPORTANCE African trypanosomes are protozoan parasites that cause African sleeping sickness. Critical to the success of the parasite is the variant surface glycoprotein (VSG), which covers the parasite cell surface and which is essential for evasion of the host immune system. VSG is membrane bound by a glycolipid (GPI) anchor that is attached in the earliest compartment of the secretory pathway, the endoplasmic reticulum (ER). We have previously shown that the anchor acts as a positive forward trafficking signal for ER exit, implying a cognate receptor mechanism for GPI recognition and loading in coated cargo vesicles leaving the ER. Here, we characterize a family of small transmembrane proteins that act at adaptors for this process. This work adds to our understanding of general GPI function in eukaryotic cells and specifically in the synthesis and transport of the critical virulence factor of pathogenic African trypanosomes.
Highlights
The critical virulence factor of bloodstream-form Trypanosoma brucei is the glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG)
The deduced TbERP proteins range in size from 203 to 253 amino acids in length and exhibit the classic conserved architecture of the p24 family (N to C): signal peptide, luminal GOLD domain, a heptad repeat region, a single transmembrane domain, and a short cytoplasmic tail (10 to 15 amino acids) with predicted COPI and coat protein II (COPII) interaction motifs [K(X)KXX and dihydrophobic, respectively] [24]
TbERP1, TbERP2, TbERP3, and TbERP8 localize to endoplasmic reticulum (ER) exit sites in BSF cells
Summary
The critical virulence factor of bloodstream-form Trypanosoma brucei is the glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG). A separate cohort of TbERP proteins (TbERP1, -2, -4, and -8) are expressed in procyclic parasites and function in GPI-dependent ER exit These results suggest developmentally regulated TbERP cohorts, likely in obligate assemblies, that may recognize stage-specific GPI anchors to facilitate GPI-cargo trafficking throughout the parasite life cycle. Robust GPI-anchored surface coats and the capacity to change them in response to environmental conditions are critical to trypanosome transmission and pathogenesis Despite this crucial role of GPI-cargo proteins throughout the parasite life cycle, the endogenous machinery involved in the manufacturing and transport of these critical virulence factors remains poorly characterized. In PCF cells, GPI anchors on secretory reporter molecules, including ectopically expressed VSG, accelerate transport kinetics [11, 12] Combined, these data indicate that the GPI anchor alone is a forward trafficking signal in the early secretory pathway of trypanosomes
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