Comparison of bulk flow-mediated versus signal-mediated ER export of fluorescent secretory cargo proteins. (A) Projected Z-stacks from Video 1 showing the dissolution of cargo aggregates. Cells containing ER-localized aggregates of the DsRed-Express2-FKBP^RD(C22V) cargo were mounted in a flow chamber and imaged while SLF-containing medium entered the chamber. The solubilized cargo colocalized with the ER marker Erg11-GFP. Scale bar, 2 µm. (B) Loss of ER-associated fluorescence for various cargo constructs after SLF addition. Strains expressed cargo variants with no export signal, the APVNTT hexapeptide, the APVNTA hexapeptide, or the APVNTT hexapeptide in an erv29Δ background. Growing cells were fixed at the indicated time points after SLF addition, then imaged by wide-field fluorescence microscopy. Scale bar, 2 µm. (C) Quantification of the loss of ER-associated fluorescence in B. The perinuclear cargo signal was measured as described in Materials and methods. Plotted is the average fluorescence signal per unit area. Data were from 27–54 cells per time point, and error bars represent SEM. (D) Immunoblot to assess N-linked glycosylation of the cargo. Growing cells containing ER-localized aggregates of either the APVNTT-DsRed-Express2-FKBP^RD(C22V) cargo or the APVNTA-DsRed-Express2-FKBP^RD(C22V) cargo were lysed with glass beads and centrifuged, and the proteins released were precipitated. Resuspended samples were treated or mock treated with endo H, followed by SDS-PAGE and immunoblotting for FKBP. Numbers represent molecular weights of reference markers.