Rft1 catalyzes lipid-linked oligosaccharide translocation across the ER membrane

Abstract

The eukaryotic asparagine (N)-linked glycan is pre-assembled as a fourteen-sugar oligosaccharide on a lipid carrier in the endoplasmic reticulum (ER). Seven sugars are first added to dolichol pyrophosphate (PP-Dol) on the cytoplasmic face of the ER, generating Man5GlcNAc2-PP-Dol (M5GN2-PP-Dol). M5GN2-PP-Dol is then flipped across the bilayer into the lumen by an ER translocator. Genetic studies identified Rft1 as the M5GN2-PP-Dol flippase in vivo but are at odds with biochemical data suggesting Rft1 is dispensable for flipping in vitro. Thus, the question of whether Rft1 plays a direct or an indirect role during M5GN2-PP-Dol translocation has been controversial for over two decades. We describe a completely reconstituted in vitro assay for M5GN2-PP-Dol translocation and demonstrate that purified Rft1 catalyzes the translocation of M5GN2-PP-Dol across the lipid bilayer. These data, combined with in vitro results demonstrating substrate selectivity and rft1∆ phenotypes, confirm the molecular identity of Rft1 as the M5GN2-PP-Dol ER flippase.

Fig. 1. High copy suppressors of rft1∆.

a Schematic of ER lipid-linked oligosaccharide biosynthesis of Man5GlcNAc2 (M5GN2)-PP-Dol in vivo, with the luminal translocation of M5GN2-PP-Dol in magenta. b Complementation of the rft1Δ growth defect. YEp351-GAPII plasmids, containing ScRFT1, HsRFT1, TbRFT1, HhRFT1, HvRFT1, HhAGL23, HsCLPTM1L, TaMURJ, HhAGL22 or empty vector, were introduced in W303a-rft1Δ mutants containing pRS316-ScRFT1. Serial dilutions of each strain were plated on SD-Leu containing 5-FOA and incubated for 2 days at 30 °C. c Western blot analysis of CPY. left: Lysates were prepared from the glucose (Glc)-repressible GALpr-RFT1 strain grown in galactose (Gal)- or Glc for 12 h at 30 °C and analyzed by SDS-PAGE followed by western blotting with anti-CPY antibodies; right: CPY in lysates from rft1∆ harboring high-copy Group I or Group II suppressors and was analyzed by Western blotting. Fully glycosylated (mCPY) and hypoglycosylated (lacking up to four oligosaccharides) CPY are indicated. Images are representative of two independent experiments. d UPLC chromatograms of oligosaccharides released from the LLO intermediates in different strains corresponding to those in b and in GALpr-RFT1 cells at 12 h after shift to glucose-containing medium. Major peaks of GlcNAc2 and Man5GlcNAc2 are marked. Man and GlcNAc are indicated by circles (green) and squares (blue), respectively.

Fig. 2. Rft1 or HhAgl23 catalyze the translocation of M5GN2-PP-Pyh and M5GN2-PP-Dol across a lipid bilayer.

a Schematic of α1-2 mannosidase-based translocation assay by UPLC-MS analysis. Lipid-linked Man5GlcNAc2 (M5GN2) in the outer leaflet of liposomes is digested to Man3GlcNAc2 (M3GN2) by α1-2 mannosidase. In the absence of a flippase, there is a 1:1 distribution of M3GN2:M5GN2. Flippase activity is measured as an increase in this ratio. b Diagram of the phytanyl- and dolichyl- pyrophosphate lipid carriers used in this study. c SDS-PAGE analysis of purified FLAG-ScRft1, HsRft1-FLAG, HhAgl22 and HhAgl23 tagged with a cellulose binding domain (CBD). Proteins were detected by Coomassie blue-staining. Images are representative of two independent experiments. d Time course of translocation (i.e., M5GN2-PP-Phy hydrolysis by α1-2 mannosidase) in proteoliposomes using protein to phospholipid ratio (PPR) of ∼2 mg/mmol. e Translocation rate as a function of different PPRs. Mannosidase-mediated hydrolysis M5GN2-PP-Phy in ScRft1-proteoliposomes was for 2 h at different PPRs. f Translocation of M5GN2-PP-Dol. Mannosidase-mediated hydrolysis by α1-2 mannosidase for 2 h in Rft1-proteoliposomes using PPR of ∼2 mg/mmol. Data in d, f are mean ± SEM of three independent biological repeats and P values are from t test (unpaired and two-tailed).

Fig. 3. Rft1 is required to translocate M5GNA2-PP-Phy across lipid bilayer.

a Overexpression of HRD3 rescues the growth defect of yeast rft1Δ cells. YEp351-GAPII plasmids, containing ScRFT1, HRD3 or empty vector, were introduced in W303a-rft1Δ mutants containing pRS316-ScRFT1. Serial dilutions of each strain were plated on SD-Leu containing 5-FOA and incubated for 2 days at 30 °C (upper); growth curves of W303a- rft1Δ+ScRFT1 and W303a- rft1Δ+HRD3 cultured in YPAD at 30 °C (below). b Overexpression of HRD3 fails to rescue the rft1∆-dependent hypo glycosylation of CPY. The positions of fully glycosylated (mCPY) and hypoglycosylated CPY (lacking up to four oligosaccharides) are indicated in a western blot of CPY from rft1Δ + ScRFT1 or + HRD3 strains. Images are representative of two independent experiments. c UPLC chromatograms of glycan chains released from the lipid-linked oligosaccharide intermediates from wild type or rft1Δ+HRD3 cells. d M5GN2-PP-Phy hydrolysis by α1-2 mannosidase for 2 h in proteoliposomes reconstituted with membrane proteins extracted from WT (gray) and rft1Δ+HRD3 cells (pink) using PPR of ∼20 mg/mmol. Protein-free liposomes (green) were used as a negative control. Data in a (below) and d are mean ± SEM of three independent biological repeats and P values are from t test (unpaired and two-tailed).

Fig. 4. Rft1 fails to translocate M3GNA2-PP-Phy across the lipid bilayer.

a Schematic of α1-2,3 mannosidase-based translocation assay by UPLC-MS analysis. M3GN2-PP-Phy in the outer leaflet of liposomes is digested to M2GN2-PP-Phy by α1-2,3 mannosidase. In the absence of a flippase, there is a 1:1 distribution of M3GN2:M2GN2. Flippase activity is measured as an increase in this ratio. b Translocation of M3GN2-PP-Phy. Mannosidase-mediated hydrolysis by α1-2,3 mannosidase in proteoliposomes using PPR of ∼2 mg/mmol. Triton X-100 was added after 4 h incubation. Data in b are mean ± SEM of three independent biological repeats.