FIGURE 7.

Inducible tools to study surface proteins. (A) Schematic diagram showing repression of the CUP1 promoter in the presence of bathocuproine disculfonic acid (BCDS) (left), and the copper induced expression of Fur4‐GFP, which localizes to the PM and vacuole (middle) and Fur4^∆N‐GFP that localizes exclusively to the surface. (B–D) Wild‐type cells transformed with CUP1‐Fur4‐GFP (B) and CUP1‐Fur4^∆N‐GFP plasmids were grown under indicated conditions prior to fluorescence microscopy. A mixture of these cells, with vacuoles of Fur4‐GFP expressing cells first labelled with FM4‐64, were also imaged simultaneously (D). (E) Indicated cells expressing Fur4^∆N‐GFP were grown to log‐phase followed by confocal microscopy. (F) Cells co‐expressing Tna1‐GFP and Sec63‐RFP (upper) or Fur4^∆N‐GFP and Fur4‐mCherry (lower) were imaged by Airyscan microscopy. An example of Tna1‐GFP localized to the cortical ER (upper) and Fur4^∆N‐GFP localized to eisosomes is included on right panels. White arrows indicate regions of the cortical ER that are not closely associated with the PM, yellow arrows indicate eisosomes. (G) The fluorescence signal of perinuclear ER was compared with peripheral signal (either cortical ER or PM) for Sec63‐RFP, Tna1‐GFP and Fur4^∆N‐GFP. (H) Wild‐type cells expressing Fur4^∆N‐GFP + Sec63‐RFP were grown to mid‐log phase and then imaged by Airyscan microcopy. (I) A zoomed in region of the periphery from (H) indicating Fur4^∆N‐GFP localizing exclusively to the PM (upper) and membrane contact sites with the ER (lower)