A reversibly aggregating fluorescent secretory cargo. (A) Strategy for generating and dissolving fluorescent aggregates. DsRed-Express2 tetramers (red) fused to a dimerizing variant of FKBP (gold) cross-link to form aggregates. Addition of the FKBP ligand SLF (blue) blocks dimerization, thereby dissolving the aggregates into soluble tetramers. (B) Dependence of aggregation on the dimerization of FKBP. A cargo construct containing the dimerizing FKBP^RD(C22V) variant aggregated in the ER, which was marked by Erg11-GFP. By contrast, a cargo construct containing the monomeric FKBP(C22V) variant did not form ER aggregates, and the cargo was sorted to the vacuole, which was marked by Vph1-GFP. Introduction of the vps10-104 mutation reduced the amount of the nonaggregating cargo in the vacuole. All images are projected confocal Z-stacks. Scale bar, 2 µm. (C) Quantification of the vacuolar fluorescence in B. For each cell, the green signal in the Vph1-GFP channel was used to create a mask for measuring the vacuolar fluorescence in the cargo channel. Data are average values from at least 110 cells for each strain. Error bars represent SEM.