Fig. 1: High copy suppressors of rft1∆.
From: Rft1 catalyzes lipid-linked oligosaccharide translocation across the ER membrane
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.
