Fig 2: Confocal microscopy after tethering of autophagy adapter to target organelles.a Immunofluorescence staining of cell lines stably expressing ER hook and different GFP baits followed by imaging by confocal microscopy. LAMP1 staining (green), CALR staining (red), and merge including Hoechst counterstaining (blue) in the presence of biotin (40 µM), and after shifting to avidin (60 µM) for 12 h are shown. Lower panels show same experiment after addition of rapamycin (Rapa; 1 µM, 6 h). Scale bar equals 10 µm. b As in (a), but for cell lines expressing Golgi hook, and stained for B4GALT1 instead of CALR. c Quantification of colocalization by surface overlap coefficient for LAMP1 and CALR signal from images in (a). d As in (c), but for LAMP1 and B4GALT1 signal from images in (b). Bars indicate means ± standard deviation for at least three cells

Fig 3: Tethering autophagy adapters to subcellular structures does not trigger their removal.a Immunofluorescence staining for CALR in cell lines expressing ER–hook and SBP–GFP–LC3 (left) or p62-SBP–GFP (right) in the presence of biotin (40 µM), and after shifting to avidin (60 µM) for 12 h. Lower panels show same experiment but in the presence of 1 µM rapamycin (Rapa). Scale bar equals 10 µm. b As in (a), but for cell lines expressing Golgi hook, and staining for B4GALT1. c Quantification of relative fluorescence intensity (FI) of CALR immunofluorescence staining signal from images in (a). d As in (c), but of B4GALT1 immunofluorescence staining signal from images in (b). e Immunoblots of cell lines expressing ER hook and indicated SBP–GFP bait constructs in the presence of biotin (40 µM), after shifting to avidin (60 µM; 12 h), and after shifting to avidin + rapamycin (1 µM, 12 h). Primary antibodies used are indicated. f As in (e), but immunoblots of cell lines expressing Golgi hook. The two bands correspond to soluble and membrane-bound isoforms of B4GALT1. Immunoblots were quantified by densitometry and quantitative data are shown in (g) and (h). Bars indicate means ± standard deviation of at least three replicates (*p < 0.05 and **p < 0.01, two-tailed Student’s t test, compared to control cells)

Fig 4: The reverse RUSH system as a tool for tethering reporters to subcellular structures.a Scheme depicting the principle of the reverse RUSH system. In presence of biotin, streptavidin is bound by biotin preventing interaction with SBP. Upon addition of excess avidin, biotin is titrated out and SBP can bind to streptavidin. Using different fusion constructs of SBP and streptavidin, a protein of interest (here GFP) can be directed to different subcellular structures. b Immunofluorescence staining of cell lines stably expressing ER hook and different GFP baits. Streptavidin staining, GFP signal, CALR staining and merge in the presence of biotin (40 µM), and after shifting to avidin (60 µM) for 12 h are shown. Scale bar equals 10 µm. c As in (b), but for cell lines expressing Golgi hook, and stained for B4GALT1 instead of CALR. d Quantification of relative co-occupancy of CALR immunofluorescence signal with SBP–GFP–LC3 (left) or p62–SBP–GFP (right) from images shown in (b) at indicated time points after shifting to avidin, and at 12 h washout after 24 h incubation in presence of avidin (wash). e As in (d), but for images shown in (c) and staining for B4GALT1. Bars indicate means ± standard deviation of at least three replicates (*p < 0.05, **p < 0.01, and ***p < 0.001, two-tailed Student’s t test, compared to cells before avidin addition; #p < 0.05, two-tailed Student’s t test, compared to cells after avidin washout)