FIGURE 3:

Npl4, but not Cdc48, colocalizes with Ddi1 to the class E compartment in an MVB mutant. (A) Localization of Cdc48 in WT cells. WT (W303) cells expressing Cdc48-GFP from a single-copy plasmid were transformed with plasmids expressing either Snx4-GFP, RFP-Vps27, or Vps10-RFP, as indicated. Alternatively, WT cells expressing RFP-Snf7 from the genome were transformed with the same plasmid expressing Cdc48-GFP. Cells were grown to mid–log phase at 26°C and examined by confocal microscopy. White arrows indicate colocalization. Merge indicates merger of the GFP and RFP windows. Light indicates the DIC window. Bar = 1 μm. (B) Cdc48 does not colocalize with the LE/MVB in MVB sorting mutants. vps23Δ, ddi1Δ, and doa1Δ cells expressing Cdc48-GFP from a single-copy plasmid were transformed with a 2μm plasmid expressing RFP-Vps27. Cells were grown and visualized as in A. (C) Cdc48 does not colocalize with Ddi1 in an MVB sorting mutant. vps27Δ cells expressing Cdc48-GFP from a single-copy plasmid were transformed with a 2μm plasmid expressing RFP-Ddi1. Cells were grown and visualized as in A. (D) Npl4 and Ddi1 colocalize to the nucleus and perivacuolar structures in an MVB mutant. WT (BY4741) and vps23Δ cells expressing GFP-Npl4 from a 2μm plasmid were transformed with plasmids expressing RFP-Ddi1 or RFP-Vps27, as indicated. Cells were grown and visualized as in A. White arrowheads designate colocalization at the class E compartment.