In the presence of IC261, cargo is exported more rapidly but remains at peripheral pre-Golgi sites
a, The hrr25 mutant was grown in galactose (lanes 1–3), or glucose (lanes 4–6) supplemented media for 10 h. Total (T) lysates were centrifuged at 150,000 g and the supernatant (S) and pellet (P) fractions were analyzed. The starred band is a degradation product of HA-Hrr25p. b, NRK cells that accumulated VSV-G-GFP in the ER at 40°C were shifted to 15°C for 30 min in the presence of DMSO (top) or 100 μM IC261 (bottom) to allow cargo transit from the ER to the ER/Golgi interface. Left, VSV-G-GFP fluorescence; middle, rbet1; right, gpp130. Insert in left corner is an expansion of the dotted box. Arrows, peripheral cargo-containing punctate structures that colocalize with rbet1 but not gpp130. c, Quantitation, from Fig. 5b, of VSV-G-GFP fluorescence in pre-Golgi structures divided by VSV-G-GFP remaining in the ER (see METHODS for calculation). S.E.M., N=20 cells/bar, P<0.0001 Student’s t test. d, Same as b only cells were incubated at 15°C for 60 min. Left, VSV-G-GFP fluorescence; middle, gpp130; right, merge of VSV-G-GFP (green) and gpp130 (red). Arrowheads, cargo that concentrated in the Golgi area. e, Quantitation, from Fig. 5d, of VSV-G-GFP fluorescence in punctate structures overlapping the Golgi divided by total fluorescence in punctate structures (see METHODS for calculation). S.E.M., N=20 cells/bar, P<0.0001 Student’s t test. The nuclei are stained with DAPI and the scale bars are 10 microns.