Chaperone Requirements for Biosynthesis of the Trypanosome Variant Surface Glycoprotein

BACKGROUND: Trypanosoma brucei does not respond transcriptionally to several endoplasmic reticulum (ER) stress conditions, including tunicamycin or dithiothreitol, indicating the absence of a conventional unfolded protein response. This suggests divergent mechanisms for quality control (QC) of ER protein folding and export may be present in trypanosomes. As the variant surface glycoprotein (VSG) represents ∼90% of trypanosome plasma membrane protein, it is possible that VSG has evolved to fold efficiently to minimize ER folding burden. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate the presence of a QC system by pharmacological inhibition of the trypanosome 26S proteasome. This indicates active proteasome-mediated VSG turnover as ∼2.5 fold more VSG is recovered from cell lysates following MG132 inhibition. An in silico scan of the trypanosome genome identified 28 open reading frames likely to encode polypeptides participating in ER nascent chain maturation. By RNA interference we monitored the importance of these gene products to proliferation, VSG abundance and cell morphology. 68% of the cohort were required for normal proliferation, and depletion of most of these factors resulted in increased VSG abundance, suggesting involvement in ERQC and degradation. CONCLUSIONS/SIGNIFICANCE: The retention of genes for, and the involvement of many gene products in, VSG folding indicates a substantial complexity within the pathways required to perform this role. Counterintuitively, for a super-abundant antigen VSG is apparently made in excess. The biosynthetic excess VSG appears to be turned over efficiently by the proteasome, implying that considerable VSG is rejected by the trypanosome ERQC mechanism. Accordingly, the VSG polypeptide is not well optimized for folding, as only ∼30% attains the native state. Finally as much of the core ERQC system is functionally conserved in trypanosomes, the pathway has an ancient evolutionary origin, and was present in the last common eukaryotic ancestor.