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. Author manuscript; available in PMC: 2016 Dec 1.
Published in final edited form as: Traffic. 2015 Nov 16;16(12):1318–1329. doi: 10.1111/tra.12331

Figure 5. Silencing of LUCID reporter correlates with delivery to luminal vesicles, but not with vacuolar proteolysis.

Figure 5

(A–B) LUCID assay of Sna3-FLuc (A) or FLuc-GFP-CPS (B) in cells with no luminal cargo sorting (vps4Δ) or with luminal cargo sorting but with reduced vacuole protease activity (pep4Δ prb1Δ). Bars each represent the mean of 4 biological replicates. (C) Fluorescence microscopy of GFP signal from FLuc-GFP-CPS. Correctly sorted MVB cargo is indicated by GFP signal from the vacuole lumen, whereas missorted cargo instead appears at the vacuole limiting membrane and adjacent puncta called class E compartments. (D) Western blot analysis of yeast cell lysates. Delivery of FLuc-GFP-CPS to the MVB/vacuole lumen results in proteolytic processing in wild type cells, indicated by soluble GFP that resists degradation in the vacuole. Mutants show no soluble GFP, but instead accumulate full-length and high MW partial degradation products.