The YPX3L motif of PAR1 is essential for MVB/lysosomal sorting. (A) HeLa cells expressing FLAG-PAR1 WT or Y206A mutant were incubated with 100 µM SFLLRN at 37°C, as indicated. PAR1 degradation was analyzed as described in Fig. 2 B. IB, immunoblotting. The data (mean ± SD) are expressed as a fraction of untreated control (Ctrl) and were analyzed by a two-way analysis of variance (*, P < 0.05; ***, P < 0.001; n = 3). (B) HeLa cells expressing FLAG-PAR1 WT or Y206A were untreated (control) or treated with 100 µM SFLLRN (SF) for 15 min at 37°C. Membranes were isolated and treated with proteinase K or proteinase K with 0.1% Triton X-100, and PAR1 or EEA1 was detected by immunoblotting. The data (mean ± SD) are expressed as the fraction of PAR1 remaining after proteinase K treatment compared with untreated control (−) from either the control or SFLLRN (+SF)-treated samples and were analyzed by Student’s t test (*, P < 0.05; n = 3). (C and D) HeLa cells expressing either FLAG-PAR1 WT or Y206A mutant were prelabeled with anti-FLAG antibody at 4°C and incubated with 100 µM SFLLRN at 37°C, as indicated. Cells were immunostained for either EEA1 or LAMP1 and analyzed, as described in Fig. 1 A. Insets show magnifications of boxed areas. Bars, 10 µm. The data (mean ± SD) represent Pearson’s correlation coefficients that were calculated, as described in Fig. 1 A, and were analyzed by Student’s t test (**, P < 0.01; ***, P < 0.001; n = 6).
