RFT1 Protein Affects Glycosylphosphatidylinositol (GPI) Anchor Glycosylation

Abstract

The membrane protein RFT1 is essential for normal protein N-glycosylation, but its precise function is not known. RFT1 was originally proposed to translocate the glycolipid ManGlcNAc-PP-dolichol (needed to synthesize N-glycan precursors) across the endoplasmic reticulum membrane, but subsequent studies showed that it does not play a direct role in transport. In contrast to the situation in yeast, RFT1 is not essential for growth of the parasitic protozoan Trypanosoma brucei, enabling the study of its function in a null background. We now report that lack of T. brucei RFT1 (TbRFT1) not only affects protein N-glycosylation but also glycosylphosphatidylinositol (GPI) anchor side-chain modification. Analysis by immunoblotting, metabolic labeling, and mass spectrometry demonstrated that the major GPI-anchored proteins of T. brucei procyclic forms have truncated GPI anchor side chains in TbRFT1 null parasites when compared with wild-type cells, a defect that is corrected by expressing a tagged copy of TbRFT1 in the null background. In vivo and in vitro labeling experiments using radiolabeled GPI precursors showed that GPI underglycosylation was not the result of decreased formation of the GPI precursor lipid or defective galactosylation of GPI intermediates in the endoplasmic reticulum, but rather due to modifications that are expected to occur in the Golgi apparatus. Unexpectedly, immunofluorescence microscopy localized TbRFT1 to both the endoplasmic reticulum and the Golgi, consistent with the proposal that TbRFT1 plays a direct or indirect role in GPI anchor glycosylation in the Golgi apparatus. Our results implicate RFT1 in a wider range of glycosylation processes than previously appreciated.

Comparative multiplexed interactomics of SARS-CoV-2 and homologous coronavirus non-structural proteins identifies unique and shared host-cell dependencies

“…We observed shared interactors between OC43 and SARS-CoV-2, such as the N -glycosylation factor RFT1 39 , 40 and a sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA – ATP2A2) 41 . In addition, we identified shared interactors between OC43 and SARS-CoV-1, including a regulator of UPR-mediated apoptosis (WLS or GPR177) 42 , a member of the signal peptidase complex (SEC11A) 43 , and factors involved in cholesterol synthesis (IDI1, DHCR7) 44 – 47 .…”

Complexity of the eukaryotic dolichol-linked oligosaccharide scramblase suggested by activity correlation profiling mass spectrometry

“…Procedures were as previously described 17 . Briefly, 5x10 8 cells were cultured for 16 hours in the presence of 40 µCi of [ 3 H]ethanolamine.…”

“…The M5-DLO scramblase is critically important as N-glycosylation cannot occur without it (we therefore also refer to it as the N-glycosylation scramblase). The polytopic ER membrane protein Rft1 was proposed as the M5-DLO scramblase almost two decades ago 12,13 , but subsequent work showed that whereas Rft1 is clearly important for N-glycosylation, it appears to have no direct role in translocating M5-DLO across the ER membrane 6,[14][15][16][17] .…”

Complexity of the eukaryotic dolichol-linked oligosaccharide scramblase suggested by activity correlation profiling mass spectrometry

“…they are ATP-independent, and that they are mediated by ER membrane proteins (termed scramblases) with exquisite substrate specificity 8 – 10 , 16 , 17 . The ER membrane protein Rft1 was proposed as the M5-DLO scramblase almost two decades ago 18 , 19 , but subsequent work showed that whereas Rft1 is clearly important for N -glycosylation, it appears to have no direct role in translocating M5-DLO across the ER membrane 16 , 20 – 23 . Likewise, the mammalian protein Lec35/MPDU1 appeared to be involved in MPD scrambling, but likely in the role of a 'dolichol chaperone' that may assist in an as yet undefined way in the scrambling process 11 , 12 .…”