Figure 5. NAC mutants have mild... | Rockefeller University Press

Figure 5.

$Figure 5. NAC mutants have mild SRP-dependent translocation defects. (A) WT (BY4741), Δegd1, Δegd2, Δbtt1, and rpl25GFP strains (control) were transformed with a PHO8-URA3 reporter plasmid (pMP234) and streaked onto SD–leu (selects for plasmid) or SD–leu –ura media (selects for translocation defect) and grown for 7 d at 16°C. (B) Translocation assay was performed as in A, but with CPY-URA3. (C) WT (W303), sec65-1 (CSY128), Δegd2, and sec65-1Δegd2 (YNY1) strains were grown to mid-log phase and serial dilutions spotted onto YPD media and grown at 30°C for 3 d. (D) PHO8-URA3 reporter assay was repeated as in A, but with strains as in C. (E) Total and ribosome fractions from cycloheximide-treated cells for strains as in C were analyzed by Western blot for Sec65, Rpl17, Egd1, and Egd2. (F) Dap2 translocation in WT (W303), sec65-1 (CSY128) cells, and sec65-1 cells overexpressing NAC (pMP304) or Egd2 (pMP302) was analyzed by pulse labeling. Nontranslocated (Dap2) and glycosylated (translocated) forms (g-Dap2) are indicated. (G) Ribosome pellets, prepared from WT and sec65-1 strains overexpressing Egd1, Egd2, or NAC were treated with DSS and analyzed by blotting for Egd2.$

NAC mutants have mild SRP-dependent translocation defects. (A) WT (BY4741), Δegd1, Δegd2, Δbtt1, and rpl25GFP strains (control) were transformed with a PHO8-URA3 reporter plasmid (pMP234) and streaked onto SD–leu (selects for plasmid) or SD–leu –ura media (selects for translocation defect) and grown for 7 d at 16°C. (B) Translocation assay was performed as in A, but with CPY-URA3. (C) WT (W303), sec65-1 (CSY128), Δegd2, and sec65-1Δegd2 (YNY1) strains were grown to mid-log phase and serial dilutions spotted onto YPD media and grown at 30°C for 3 d. (D) PHO8-URA3 reporter assay was repeated as in A, but with strains as in C. (E) Total and ribosome fractions from cycloheximide-treated cells for strains as in C were analyzed by Western blot for Sec65, Rpl17, Egd1, and Egd2. (F) Dap2 translocation in WT (W303), sec65-1 (CSY128) cells, and sec65-1 cells overexpressing NAC (pMP304) or Egd2 (pMP302) was analyzed by pulse labeling. Nontranslocated (Dap2) and glycosylated (translocated) forms (g-Dap2) are indicated. (G) Ribosome pellets, prepared from WT and sec65-1 strains overexpressing Egd1, Egd2, or NAC were treated with DSS and analyzed by blotting for Egd2.

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