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

Calvin Tiengwe,Peter J Bush,James D Bangs,Kent L Hill

doi:10.1371/journal.ppat.1006366

Calvin Tiengwe, Peter J Bush + Show 2 more

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https://doi.org/10.1371/journal.ppat.1006366

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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.

Highlights

Many eukaryotic secretory proteins such as surface antigens, adhesion proteins, and receptors are attached to the external leaflet of the plasma membrane by glycosylphosphatidylinositol (GPI) anchors [1, 2]
Protozoan parasites that cause African Sleeping Sickness, have two structurally related secretory proteins that are critical for their success as pathogens: variant surface glycoprotein (VSG), which is responsible for evasion of host immune
Responses, and transferrin receptor (TfR), which is responsible for acquisition of essential iron for nutritional purposes

Summary

Introduction

Many eukaryotic secretory proteins such as surface antigens, adhesion proteins, and receptors are attached to the external leaflet of the plasma membrane by glycosylphosphatidylinositol (GPI) anchors [1, 2]. GPI anchors function as vesicular transport signals for ER export, for post-Golgi sorting, and for subsequent delivery to the plasma membrane. In yeast, inhibition of GPI attachment leads to delayed ER exit of the major GPI-AP, Gas1p [3]; and GPI-APs exit the ER in cargo vesicles that are distinct from other secretory and plasma membrane proteins [4]. GPI-anchors serve as cell surface targeting signals by specific association with sterol/sphingolipid-rich detergent-insoluble membranes (a.k.a. lipid rafts) at the trans-Golgi network [5, 6]. Likewise at the plasma membrane, GPI-APs preferentially cluster in lipid raft microdomains [7]. Cell surface GPI-APs play critical roles in cell adhesion in fungi, inhibition of complement lysis in erythrocytes, and in defence against host immunity in parasitic protozoa like African trypanosomes [2, 8]

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