Copper-dependent internalization of the Ctr4-Ctr5 complex.

A, Cells harboring a ctr4Δ ctr5Δ double deletion were co-transformed with the ctr4+-VC and ctr5+-VN alleles. Co-transformed cells were grown in EMM containing 160 nM of copper (low Cu) to an A600 of 0.5 (T0), and then were left untreated (low Cu), or were treated with BCS (100 µM), CuSO4 (1, 25, and 100 µM) (Cu) or FeCl3 (100 µM) (Fe). After being incubated for 3 h, the cells were visualized by fluorescence microscopy (BiFC). The cells were also examined by Nomarski microscopy for cell morphology. B, ctr4Δ ctr5Δ cells co-expressing the Ctr4-GFP and Ctr5-MYC12 fusion proteins were grown to mid-logarithmic phase in EMM copper-poor (160 nM) media (low Cu). Cells were then incubated in the absence (low Cu) or the presence of CuSO4 (100 µM) or BCS (100 µM). After a 3 h treatment, the full-length Ctr4-GFP protein was viewed by direct fluorescence microscopy (GFP). The corresponding Nomarski images are also shown for each GFP panel.