Figure 10. Distinct localization patterns of tagged Vps10 and Kex2.
(A) Vps10-GFP localizes to both the Golgi and prevacuolar endosomes whereas Kex2-GFP is largely restricted to the Golgi. Late Golgi compartments were tagged with Sec7-mCherry, or else prevacuolar endosome compartments were tagged with Vps8-mCherry. The localizations of the Vps10-GFP and Kex2-GFP proteins were then examined with reference to these two compartments. As a control, Vps8-GFP was expressed together with Sec7-mCherry to confirm that the two compartments were separate. Representative projected confocal images are shown. Scale bar, 2 μm. (B) Quantitation of the data from (A). To analyze an image, a mask was created from the punctate compartment signal, and the percentage of the protein signal visible through the mask was then measured (Levi et al., 2010). For each strain, 40 images with ~2–4 cells per image were quantified. Bars represent mean percentage values with s.e.m. Based on the analysis of Vps8 and Sec, the background signal due to chance overlap in this assay was approximately 8–16%.
Figure 10—figure supplement 1. Example of an unusual cell with some Kex2-GFP visible in prevacuolar endosomes.
