Figure 7

MGRN1-mediated monoubiquitination of TSG101 rescues lysosomal fusion events and restores degradation of cargo from autophagy and heterophagy pathways. (a) Experimental logic to establish that MGRN1 interacts with and ubiquitinates TSG101 to mediate vesicular fusion with lysosomes. (i) Monoubiquitination of TSG101 is essential for this. (ii) and (iii) Perturabation of the interacting regions of either MGRN1 (PSAP motif) or TSG101 (UEV domain) compromises vesicle fusions with lysosomes. (b) HeLa cells co-transfected with MGRN1 or MGRN1ΔR, HA-TSG101 along with K0 Ub or ΔG75/76 Ub were lysed and immunoblotted to analyze the levels of endogenous LC3 II in the presence or absence of 300 nM bafilomycin A1. Note that K0 Ub, a lysine-less ubiquitin mutant promotes only monoubiquitination; ΔG75/76 Ub cannot be conjugated to substrates, but binds noncovalently to ubiquitin interacting domains and acts as a competitive inhibitor of Ub binding. GAPDH was used as loading control. Efficiencies of all transfections were checked. The blots are representative of at least three experiments. Note that overexpression of TSG101, without its monoubiquitination cannot rescue autophagosomal degradation. indicates HA-TSG101; endogenous TSG101. (c) Quantification of data from (b) denotes fold change in endogenous LC3 II level when normalized against GAPDH from three independent experiments. **P≤0.05, n.s., not significant (P=0.3 and 0.6 in the presence of MGRN1 and MGRN1ΔR, respectively) using Student’s t-test. Error bars, ±S.E.M. (d) HeLa cells co-transfected with MGRN1or MGRN1 (SRAP) along with HA-TSG101 or HA-TSG101ΔUEV and K0 Ub, as indicated, were lysed and immunoblotted to analyze the levels of endogenous LC3 II in the presence or absence of 300 nM bafilomycin A1. GAPDH was used as loading control. Efficiencies of all transfections were checked. Note that the lack of interaction between MGRN1 and TSG101 (when either MGRN1 (SRAP) or HA-TSG101ΔUEV was used) disrupts autophagosomal degradation. indicates HA-TSG101; indicates endogeneous TSG101, indicates HA-TSG101ΔUEV. (e) Graphical representation of LC3 II fold change as normalized against loading control. **P≤0.05, n.s., not significant (P=0.22 and 0.77 in the presence of MGRN1 (SRAP) and HA-TSG101ΔUEV, respectively) using Student’s t-test. Error bars, ±S.E.M. (f) SHSY5Y cells co-transfected with MGRN1 or MGRN1ΔR, HA-TSG101 or HA-TSG101ΔUEV along with K0 Ub or ΔG75/76 Ub were lysed and immunoblotted to analyze the levels of endogenous LC3 II in the presence or absence of 60 nM bafilomycin A1. GAPDH was used as loading control. Efficiencies of all transfections were checked. The blots are representative of at least three experiments. Note that overexpression of TSG101, without its monoubiquitination cannot rescue autophagosomal degradation. indicates HA-TSG101; indicates HA-TSG101ΔUEV. (g) Graphical representation of LC3 II fold change as normalized against loading control. **P≤0.05, n.s., not significant (P=0.74, 0.45 and 0.92 in the presence of ΔG75/76 Ub,MGRN1 (SRAP) and HA-TSG101ΔUEV, respectively) using Student’s t-test. Error bars, ±S.E.M. (h) HeLa cells treated with the indicated siRNAs, transiently co-transfected with HA-TSG101 along with K0 Ub or ΔG75/76 Ub was subjected to Alexa-Fluor 488 EGF uptake. Cells were washed, fixed at indicated time points and imaged. Note that overexpression of TSG101 and its monoubiquitination are required together to restore endo-lysosomal pathway. Scale bar, 5 μm