Figure 2. Erd1 is required for Vps74-COPI dependent recycling of specific Golgi glycosyltransferases.

(A) Immunoblot analysis on yeast cell lysates from wild type, erd1$\mathrm{\Delta }$, and vps74$\mathrm{\Delta }$ mutant for glycosylation reporter, Gas1. (B) Growth of wild type, erd1$\mathrm{\Delta }$, and vps74$\mathrm{\Delta }$ mutant in the presence of indicated concentrations of Tunicamycin in YPD liquid cultures at 30° C after 24 hr. (C) Growth of serial dilutions of wild type, erd1$\mathrm{\Delta }$, vps74$\mathrm{\Delta }$, and erd1$\mathrm{\Delta }$vps74$\mathrm{\Delta }$ mutants on synthetic media at 26 °C and 40° C after 2 days. (D) Quantification of percent vacuolar fluorescence to total fluorescence of the indicated GFP tagged early and medial Golgi proteins in wild type, erd1$\mathrm{\Delta }$, and vps74$\mathrm{\Delta }$ mutant. (E) Live-cell fluorescence imaging of Erd1-GFP and vacuolar dye, CMAC in wild type and the vps74$\mathrm{\Delta }$ mutant. (F) Live-cell fluorescence imaging of mNeonGreen-Vps74 and medial Golgi marker, Gea2-3xMars in wild type and the erd1$\mathrm{\Delta }$ mutant. (G) Growth of serial dilutions of vps74$\mathrm{\Delta }$ mutant transformed with empty vector (EV) or plasmids overexpressing Vps74 and Erd1 on YPD with 50 µg/ml hygromycinB at 26 °C or synthetic media lacking uracil at 26 °C or 40° C after 2–3 days. (H) Growth of serial dilutions of wild type and erd1$\mathrm{\Delta }$ mutant transformed with the indicated Vps74 mutants at 26° C after 3 days. (I) Growth of serial dilutions of wild type, erd1$\mathrm{\Delta }$, sec21-1, and sec21-1 erd1$\mathrm{\Delta }$ mutants at 26 °C after 3 days. Scale bars: 2.5µ m.

Figure 2—figure supplement 1. Erd1 and Vps74 mutants exhibit similar glycosylation defects.

(A) Growth of serial dilutions of wild type, erd1$\mathrm{\Delta }$, and vps74$\mathrm{\Delta }$ mutants on YPD with or without 60 µg/ml hygromycinB at the indicated temperature after 2 days. (B) SAFE analysis of genetic interaction profiles of erd1$\mathrm{\Delta }$ and vps74$\mathrm{\Delta }$ mutant (cut-off (p < 3e-8)). (C) Immunoblot analysis on yeast cell lysates from wild type, mnn9$\mathrm{\Delta }$, och1$\mathrm{\Delta }$, ktr3$\mathrm{\Delta }$, kre2$\mathrm{\Delta }$, vps74$\mathrm{\Delta }$ and erd1$\mathrm{\Delta }$ mutants for glycosylation reporter, Gas1. (D) Growth of serial dilutions of wild type, kre2$\mathrm{\Delta }$, ktr3$\mathrm{\Delta }$, kre2$\mathrm{\Delta }$ktr3$\mathrm{\Delta }$, erd1$\mathrm{\Delta }$ and vps74$\mathrm{\Delta }$ mutants on YPD with or without 60 µg/ml hygromycinB at 26° C after 2 days. (E) Schematic depicting the known defects in the Vps74 mutants tested in Figure 2H. (F) Live-cell confocal fluorescence imaging of mNeonGreen-Vps74 along with early (Mnn9-mCherry), medial (Gea2-3xmMars), and late (Sec7-6xDsRed) Golgi markers. (G) Violin plots for the ratio of co-localized mNeonGreen-Vps74 with the Golgi markers in (F). Dashed lines indicate the median values, and dotted lines indicate the 95 CIs. Scale bars: 2.5µ m.

Figure 2—figure supplement 2. Pmr1 is required for glycosylation but not glycosyltransferase recycling.

(A) Immunoblot analysis on yeast cell lysates from wild type, pmr1$\mathrm{\Delta }$, vps74$\mathrm{\Delta }$ and erd1$\mathrm{\Delta }$ mutants for glycosylation reporter, Gas1. (B) Immunoblot analysis on indicated yeast cell lysates from untreated or cells grown in media supplemented with 50 mM phosphate buffer pH 6.2 for 4 hr at 30° C. (C) Immunoblot analysis on indicated yeast cell lysates from untreated or cells grown in media supplemented with 50 mM CaCl2, and 600 µM MnCl2 for 4 hr at 30 °C. (D) Live cell imaging of wild type or pmr1$\mathrm{\Delta }$ mutant expressing Kre2-GFP. Scale bars: 2.5µ m. (E) List of Golgi glycosyltransferases dependent on Erd1 and Vps74 for their recycling. The (F/L)-(L/I/V)-x-x-(R/K) motif known to bind Vps74 is highlighted in green.