Trypanosoma brucei: trypanosome-specific endoplasmic reticulum proteins involved in variant surface glycoprotein expression.
In Trypanosoma brucei the GPI-anchored variant surface glycoprotein (VSG) represents approximately 90% of cell surface protein and a major proportion of endoplasmic reticulum (ER) biosynthetic output. We identified four trypanosomatid-specific genes encoding candidate ER-resident proteins; all were required for normal proliferation. For Tb11.01.2640 and Tb11.01.8120, an increase in VSG abundance was found on silencing, while the protein products localized to the ER; we designated these ERAP32 and ERAP18 for ER-associated protein of 32kDa and 18kDa. Silencing ERAP32 or ERAP18 did not alter expression levels of ISG65 or ISG75, the major surface trans-membrane domain proteins. Surface biotinylation or immunoflorescence did not identify intracellular VSG accumulation, while FACS and fluorescence microscopy indicated that the cells were not increased in size, arguing for increased VSG density on the cell surface. Therefore, ERAP32 and ERAP18 are trypanosome-specific ER-localized proteins with a major role in VSG protein export and, contrary to current paradigms, VSG is not saturated on the cell surface.
Wang
Ya-Nan
YN
Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, UK.
Wang
Ming
M
Field
Mark C
MC
D019921:Biotinylation; D015153:Blotting, Western; D015536:Down-Regulation; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D018503:Genome, Protozoan; D008854:Microscopy, Electron; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000096:biosynthesis* / Q000235:genetics
D015800:Protozoan Proteins; D014643:Variant Surface Glycoproteins, Trypanosoma