A significant fraction of the Dsc complex localizes to the vacuole membrane. (A) Left: Schematics illustrating the position where GFP (or Phluorin) is inserted. Tul1 contains an N-terminal luminal domain with multiple glycosylation sites, seven transmembrane helices, and a C-terminal RING domain. Right: Localization of Tul1-GFP, Tul1-pH-Ring, and Cot1-Mars 6 h after Zn2+ withdrawal from the YNB media. Both Tul1-GFP and Tul1-pH-Ring were overexpressed under the tet-O7 promoter, and Cot1-Mars was chromosomally tagged. White arrows highlight the punctae outside the vacuole. (B) Cot1-Mars was constitutively internalized into the vacuole lumen in cells overexpressing Tul1-pH-Ring, whereas Cot1-Mars stayed on the vacuole membrane in cells transformed with an empty vector (yellow arrows). Tul1-pH-Ring was overexpressed under the tet-O7 promoter, and Cot1-Mars was chromosomally tagged. (C) Localization of chromosomally tagged Ubx3-GFP and Cot1-Mars before and 6 h after removing Zn2+ from the YNB media. White lines highlight the position for line-scanning analysis, which was shown on the right. Bar, 1 µm.
