Figure 4.

Protein enrichment at vertices of tethered vacuoles. Reaction inhibitors, including 50 ng/μl excess Sec17p, 60 ng/μl anti-Vam3 Fab, 600 ng/μl recombinant Gyp1p, 500 μM jasplakinolide, 15 μM PX domain, 500 μM latrunculin B, and 5 μM full-length Vam7p were added to docking assays (see Materials and methods) with GFP-tagged proteins as indicated. After incubation, vacuoles were observed by fluorescence microscopy and images were acquired in GFP and rhodamine channels. Vacuole clusters from random fields were analyzed and ratio values of GFP:FM4–64 were generated from outside edge membranes (black) and vertex membranes (red). An average of 200 normalized ratio values for each treatment were plotted against their percentile (cumulative distribution plot).