Figure 5.

The interactions of mutant VPS33A and autophagic SNARE complex were increased. (A) MYC-VPS33A^WT or VPS33A^D251E and FLAG-STX17 were cotransfected in HEK293T cells. Cells were treated with DMSO or rapamycin (100 nM) for 4 h before being harvested, immunoprecipitated with FLAG beads, and followed by anti-MYC immunoblotting. FLAG-STX17 precipitated more mutant MYC-VPS33A with or without rapamycin treatment as shown in the blots. (B) MYC-VPS33A ^WT or VPS33A^D251E and FLAG-SNAP29 or VAMP8 were cotransfected in HEK293T cells. After 24 h, the cells were harvested for immunoprecipitation analyses. FLAG-SNAP29 or VAMP8 precipitated more mutant MYC-VPS33A as shown in the blots. (C) COS7 cells were cotransfected with MYC-VPS33A (WT or D251E) and FLAG-STX17 for 24 h. Cells were cultured in DMEM without amino acids for 2 h before fixation. Then cells were fixed, made permeable in 0.1% saponin, stained with anti-MYC and anti-FLAG antibodies, and finally examined under a confocal microscope. Scale bars: 10 µm. Quantification of colocalization represented by Pearson correlation coefficient showed that STX17 colocalized more with the mutant VPS33A than the wild-type. /P< 0.05 (P = 0.0419), n = 20 cells. (D) FLAG alone, FLAG-VPS33A^WT or FLAG-VPS33A^D251E were expressed in HEK293T cells, and immunoprecipitated with FLAG beads. Then the FLAG beads were incubated with bacterially purified GST-STX17. Samples were immunoblotted with anti-GST antibody. FLAG-D251E binds more GST-STX17 as shown in the blots. (E) STX17 pulled down almost equal amounts of endogenous VAMP8 and SNAP29 in both wide-type and bf MEFs. IB, immunoblotting; IP, immunoprecipitation.