Controlling transferrin receptor trafficking with GPI-valence in bloodstream stage African trypanosomes

Abstract

Bloodstream-form African trypanosomes encode two structurally related glycosylphosphatidylinositol (GPI)-anchored proteins that are critical virulence factors, variant surface glycoprotein (VSG) for antigenic variation and transferrin receptor (TfR) for iron acquisition. Both are transcribed from the active telomeric expression site. VSG is a GPI2 homodimer; TfR is a GPI1 heterodimer of GPI-anchored ESAG6 and ESAG7. GPI-valence correlates with secretory progression and fate in bloodstream trypanosomes: VSG (GPI2) is a surface protein; truncated VSG (GPI0) is degraded in the lysosome; and native TfR (GPI1) localizes in the flagellar pocket. Tf:Fe starvation results in up-regulation and redistribution of TfR to the plasma membrane suggesting a saturable mechanism for flagellar pocket retention. However, because such surface TfR is non-functional for ligand binding we proposed that it represents GPI2 ESAG6 homodimers that are unable to bind transferrin—thereby mimicking native VSG. We now exploit a novel RNAi system for simultaneous lethal silencing of all native TfR subunits and exclusive in-situ expression of RNAi-resistant TfR variants with valences of GPI0–2. Our results conform to the valence model: GPI0 ESAG7 homodimers traffick to the lysosome and GPI2 ESAG6 homodimers to the cell surface. However, when expressed alone ESAG6 is up-regulated ~7-fold, leaving the issue of saturable retention in the flagellar pocket in question. Therefore, we created an RNAi-resistant GPI2 TfR heterodimer by fusing the C-terminal domain of ESAG6 to ESAG7. Co-expression with ESAG6 generates a functional heterodimeric GPI2 TfR that restores Tf uptake and cell viability, and localizes to the cell surface, without overexpression. These results resolve the longstanding issue of TfR trafficking under over-expression and confirm GPI valence as a critical determinant of intracellular sorting in trypanosomes.

The Trypanosomal Transferrin Receptor of Trypanosoma Brucei—A Review

“…The TfR has also been modelled to spread out from the surrounding VSG molecules, which previously was presumed to allow adequate contact and capture of Tf [74]. However, this hypothesis has recently been contradicted by evidence that the TfR outside of the FP is not functional, as it is composed of an ESAG6 homodimer, rather than an ESAG6/7 heterodimer [110]. The GPI valence in trypanosomes has been shown to be a critical determinant of intracellular sorting, with molecular complexes with two GPIs (GPI 2 ) being trafficked out of the FP, one GPI (GPI 2 ) being retained within the FP, and non-GPI-anchored complexes being degraded in the lysosome [110].…”

“…However, this hypothesis has recently been contradicted by evidence that the TfR outside of the FP is not functional, as it is composed of an ESAG6 homodimer, rather than an ESAG6/7 heterodimer [110]. The GPI valence in trypanosomes has been shown to be a critical determinant of intracellular sorting, with molecular complexes with two GPIs (GPI 2 ) being trafficked out of the FP, one GPI (GPI 2 ) being retained within the FP, and non-GPI-anchored complexes being degraded in the lysosome [110]. Given that ESAG6 is the GPI-anchored partner in the complex, when over-expressed as homodimer during iron starvation, its GPI valence allows its escape from the FP [110].…”

The crystal structure and localization ofTrypanosoma bruceiinvariant surface glycoproteins suggest a more permissive VSG coat in the tsetse-transmitted metacyclic stage

“…This restricted localization has been proposed to be due partly to the presence of only one GPI anchor molecule, differing from VSGs which form homodimers (i.e. two GPI anchors) that allow their homogeneous surface expression 73 . In contrast, our crystallographic and in vivo labelling evidence show that MISP appear to be displayed on the entire cell surface.…”

Evolution of protein trafficking in kinetoplastid parasites: Complexity and pathogenesis

“…A second GPI‐anchored surface protein, the transferrin receptor, is a heterodimer with only one subunit possessing a GPI‐anchor and is retained within the flagellar pocket. This has been proposed as a general model for localization of specific proteins using “GPI‐valance.” Tests of this model, including the production of a dual GPI‐anchored transferrin receptor, lend support, but neither a molecular mechanism nor an exhaustive exploration of the surface protein repertoire has been described. Interestingly, the localization of VSG can be significantly altered by changes to the size of the VSG ectodomain alone, which may suggest that membrane anchoring has a lesser role compared with the architecture of the protein itself …”