Subcellular localization of GFP-Ecm33.

(A) GFP-tagged Ecm33 was functionally similar to the non-tagged protein. Cells transformed with the multicopy vector harboring gene encoding GFP-Ecm33 or the empty vector was streaked onto each plate containing YPD or YPD plus 0.12 M MgCl₂, and then incubated for 4 days at 30°C. (B) GFP-Ecm33 localized to the cell surface or medial regions in wild-type cells. Wild-type cells expressing chromosome-borne GFP-Ecm33 were cultured in EMM medium at 27°C, and were examined by fluorescence microscopy; Sterol localization detected by filipin staining (see Materials and Methods) in wild-type cells. Bar: 10 µm. (C) GFP-Ecm33 localized to the structure surrounding the nucleus in wild-type cells when cells were treated with BE49385A. Wild-type cells expressing chromosome-borne GFP-Ecm33 were cultured in EMM medium and incubated at 27°C, and were examined by fluorescence microscopy at 0 hour, 1 hour, 2 hour, and 4 hour after 1 µg/ml BE49385A was added to the medium. Bar: 10 µm. (D) GFP-Ecm33 primarily localized to the structure surrounding the nucleus and to the cell surface in its8-1 mutant, while the localization of GFP-Ecm33 was normal in Δcis4 mutant. The its8-1 mutant and Δcis4 mutant expressing chromosome-borne GFP-Ecm33 were cultured in EMM medium and incubated at 27°C, and were examined by fluorescence microscopy. Bar: 10 µm. (E) In the zinc-deficient medium GFP-Ecm33 primarily localized to the intracellular dots and to the ER in Δcis4 cells. The wild-type cells and Δcis4 mutant expressing chromosome-borne GFP-Ecm33 were cultured in the zinc deficient EMM medium at 27°C for 48 hours, and were examined by fluorescence microscopy. Bar: 10 µm.