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. Author manuscript; available in PMC: 2023 Dec 7.
Published in final edited form as: Nat Cell Biol. 2016 Jun 13;18(7):765–776. doi: 10.1038/ncb3372

Figure 6.

Figure 6

USP19 recruits misfolded proteins to the ER and ER-associated late endosomes. (a) USP19 preferentially recruits GFP1–10 to membranes. The cytosol and membrane fractions from HEK293T cells co-transfected with the indicated constructs were analysed by immunoblotting. (b) Knockout of USP19 reduces membrane-associated GFP1–10. Control and USP19 CRISPR (KO) cells were transfected with GFP1–10. After fractionation, GFP1–10 in the membrane and cytosol fractions was analysed by immunoblotting. The graph shows the level of membrane-associated GFP1–10 relative to cytosolic GFP1–10 (mean ± s.e.m., n = 3 independent experiments, ∗∗P < 0.01). (c) USP19 recruits GFP1–10 to the ER membrane. COS7 cells co-transfected with GFP1–10 and FLAG-USP19 were permeabilized, stained with GFP and FLAG antibodies and analysed by confocal microscopy. Bottom panels show the outline of GFP1–10 and USP19 signal in the outlined area. Scale bar, 5 μm. (d,e) A photobleaching-based assay reveals vesicles containing MAPS cargos. (d) A schematic illustration of the photobleaching experiment shown in e. The marked area in d and e was photobleached with a 568nm laser to remove cytosolic and ER-associated mCh–GFP1–10 background. Arrows indicate a few examples of vesicles revealed after photobleaching. Scale bar, 5 μm. (f) MAPS vesicles were labelled with Rab9. Shown are two frames from a live-cell imaging experiment using cells transfected with mCe–USP19, mCi–Rab9, and mCh–GFP1–10 after photobleaching. Panels 4–6 are enlarged views of the indicated area in panel 1 after 20s. (g) Structure-illuminated microscopy analysis of MAPS vesicles. Cells transfected with mCe–USP19, Ubl4A–Venus and mCh–Rab9 were permeabilized, fixed and imaged. Note that Ubl4A–Venus is detected on the membranes of intraluminal vesicles, but not in the lumen of these vesicles. (h) Transmission electron microscopy analyses of MAPS vesicles. COS7 cells expressing FLAG-tagged GFP1–10 were permeabilized, fixed, and stained with anti-FLAG antibodies and immunogold-labelled secondary antibodies. Blue arrows show examples of luminal GFP1–10 signals. Arrowheads show PM-associated GFP1–10. The inset shows an example of GFP1–10 association with the limiting membrane on the luminal side (yellow arrows). Statistics source data for b can be found in Supplementary Table 2. Unprocessed original scans of blots are shown in Supplementary Fig. 8.