Figure 2.
IQGAP1 is necessary for degradative autophagy and bridges ATG9A with CHMP2A. (A) Schematic, TMRHT release assay. TMRHT-LC3B (HaloTag-LC3B) is processed by lysosomal hydrolases releasing the TMRHT fragment from a fusion with LC3B. Released (HaloTag stabilized by TMR) is detectable by in-gel fluorescence and immunoblotting. Top, open phagophores do not yield the TMRHT fragment. Bottom, closed autophagosomes fuse with lysosomes and the TMRHT fragment is released. (B)TMRHT release in IQGAP1 knockdown or control (siScr) cells stably expressing HT-LC3B. TMR+, cells incubated with TMR for 30 min. Cells were starved in EBSS for 90 min, lysed, and processed for in-gel fluorescence and immunoblotting. (i) In-gel fluorescence detection of released TMRHT. (ii) Immunoblot detection of released TMRHT. (iii) quantification of released TMRHT in immunoblots. (C) ESCRT protein subcomplexes with components present in or absent from LC-MS/MS after proximity biotinylation with APEX2-ATG9A. Cells (FLAG-APEX2-ATG9A Flp-In T-REx HEK293[TetON]) were incubated in EBSS (90 min) or treated with CCCP in full medium for 6 h. Black, proteins detected in all conditions; blue, detected only in EBSS; purple, detected only in CCCP; red, not detected in any samples. Note ESCRTs absent from proteomic dataset (red color). (D) Co-IP analysis of GFP-ATG9A with endogenous CHMP2A in Huh7 cells, control (siScr) or knocked down for IQGAP1 by siRNA. Cells were treated with protonophore CCCP for 6 h as a means to collapse organellar proton gradients. (i) Immunoblot, IQGAP1 knockdown in Huh7 cells. (ii) Western blot, Co-IP analysis of GFP-ATG9A (GFP pulldown), and endogenous CHMP2A in control and IQGAP1 depleted cells. (iii) Quantification of Co-IP analyses (CHMP2A band intensity was ratioed to the intensity of the upper band in GFP-ATG9A blots). Data, means ± SD, n = 3 ANOVA. (E) Summary of findings in Fig. 2. Source data are available for this figure: SourceData F2.

IQGAP1 is necessary for degradative autophagy and bridges ATG9A with CHMP2A. (A) Schematic, TMRHT release assay. TMRHT-LC3B (HaloTag-LC3B) is processed by lysosomal hydrolases releasing the TMRHT fragment from a fusion with LC3B. Released (HaloTag stabilized by TMR) is detectable by in-gel fluorescence and immunoblotting. Top, open phagophores do not yield the TMRHT fragment. Bottom, closed autophagosomes fuse with lysosomes and the TMRHT fragment is released. (B)TMRHT release in IQGAP1 knockdown or control (siScr) cells stably expressing HT-LC3B. TMR+, cells incubated with TMR for 30 min. Cells were starved in EBSS for 90 min, lysed, and processed for in-gel fluorescence and immunoblotting. (i) In-gel fluorescence detection of released TMRHT. (ii) Immunoblot detection of released TMRHT. (iii) quantification of released TMRHT in immunoblots. (C) ESCRT protein subcomplexes with components present in or absent from LC-MS/MS after proximity biotinylation with APEX2-ATG9A. Cells (FLAG-APEX2-ATG9A Flp-In T-REx HEK293[TetON]) were incubated in EBSS (90 min) or treated with CCCP in full medium for 6 h. Black, proteins detected in all conditions; blue, detected only in EBSS; purple, detected only in CCCP; red, not detected in any samples. Note ESCRTs absent from proteomic dataset (red color). (D) Co-IP analysis of GFP-ATG9A with endogenous CHMP2A in Huh7 cells, control (siScr) or knocked down for IQGAP1 by siRNA. Cells were treated with protonophore CCCP for 6 h as a means to collapse organellar proton gradients. (i) Immunoblot, IQGAP1 knockdown in Huh7 cells. (ii) Western blot, Co-IP analysis of GFP-ATG9A (GFP pulldown), and endogenous CHMP2A in control and IQGAP1 depleted cells. (iii) Quantification of Co-IP analyses (CHMP2A band intensity was ratioed to the intensity of the upper band in GFP-ATG9A blots). Data, means ± SD, n = 3 ANOVA. (E) Summary of findings in Fig. 2. Source data are available for this figure: SourceData F2.

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