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

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 GPI 2 homodimer; TfR is a GPI 1 heterodimer of GPI-anchored ESAG6 and ESAG7. GPI-valence correlates with secretory progression and fate in bloodstream trypanosomes: VSG (GPI 2) is a surface protein; truncated VSG (GPI(0)) is degraded in the lysosome; and native TfR (GPI 1) 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 GPI 2 ESAG6 homodimers that are unable to bind transferrin D 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 GPI(0-2). Our results conform to the valence model: GPI(0) ESAG7 homodimers traffick to the lysosome and GPI(2) ESAG6 homodimers to the cell surface. However, when expressed alone ESAG6 is up-regulated similar to 7-fold, leaving the issue of saturable retention in the flagellar pocket in question. Therefore, we created an RNAi-resistant GPI(2) TfR heterodimer by fusing the C-terminal domain of ESAG6 to ESAG7. Co-expression with ESAG6 generates a functional heterodimeric GPI(2) 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.