Figure 1

A Co‐localization of endogenous TBC1D5 and VPS35 with mCherry‐ATG9A, in steady state and autophagy induced by mTOR inhibitor KU0063794 (6 h).

B, C Quantification of co‐localization experiments presented in (A).

Co‐immunoprecipitation

of HA‐Flag‐TBC1D5 with myc‐VPS29, transiently overexpressed in 293T cells, DMSO or KU0063794 (6 h) treated.

E, F Co‐immunoprecipitation of endogenous TBC1D5 and ATG9A with myc‐VPS29

(E) transiently overexpressed in 293T cells or with HA‐Flag‐VPS35 (F) transiently overexpressed in 293T cells, treated with DMSO or KU0063794 (6 h).

G Co‐immunoprecipitation of endogenous VPS29 and ATG9A with HA‐Flag‐TBC1D5

stably expressed in 293T cells, treated with DMSO or KU0063794 (6 h).

H Co‐immunoprecipitation of endogenous ULK1 and pATG13 (Ser318) with HA‐Flag‐TBC1D5, stably expressed in T‐REx 293T cells, induced by doxycycline (50 ng/ml) for 20 h prior to DMSO or KU0063794 (6 h) treatment.

I Magnified still images extracted from Supplementary Movie S2. mCherry‐TBC1D5 co‐localizes with GFP‐ATG9 upon autophagy induction by mTOR inhibitor, KU0063794.

H Co-immunoprecipitation of endogenous

ULK1

and pATG13 (Ser318) with HA-Flag-TBC1D5, stably expressed in T-REx 293T cells, induced by doxycycline (50 ng/ml) for 20 h prior to DMSO or KU0063794 (6 h) treatment.

Figure 2

A Lysates of U2OS cells stably expressing control shRNA or shRNA targeting VPS29 or TBC1D5. Cells were treated with DMSO or KU0063794 (6 h), lysed in RIPA buffer (1% SDS) and lysates were subjected to SDS-PAGE.

B U2OS cells stably expressing mCherry‐ATG9 were depleted of VPS29 and TBC1D5 using shRNAs, treated with DMSO or KU0063794 (6 h), fixed with 2% PFA, and immunostained with anti‐LAMP1 antibody.

C Quantification of ATG9 vesicular structures in cells from (B) under non‐stimulated conditions.

D Quantification of ATG9 and LAMP1 co‐localization in cells presented in (B).

E Quantification of ATG9 and LAMP1 co−localization in cells stably depleted for TBC1D5 (shRNA#1) or VPS29 (shRNA#2), transiently transfected with myc−TBC1D5 plasmid resistant to

TBC1D5

shRNA#1. Cells were treated with KU0063794 (6 h), 20 h post−transfection, fixed, and

immunostained

with anti−LAMP1 and anti−myc antibodies.

F Control shRNA and U2OS cell lysates depleted for TBC1D5 (shRNA#1), transfected with empty plasmid or with shRNA‐resistant myc‐TBC1D5.

G Immunofluorescence of TBC1D5 (shRNA#1) cells transiently transfected with shRNA‐resistant myc‐TBC1D5, treated with KU0063794 (6 h). 20 h post‐transfection cells were treated, subsequently fixed, and immunostained with anti‐myc and anti‐LAMP1 antibodies.

Figure 3

A HA‐Flag‐TBC1D5 expression was induced in T‐REx HeLa cells for 20 h, lysates were subjected to GSTpull‐down, −AP2M1. Co‐precipitatedTBC1D5 was detected with anti−Flag antibody.

B Control cells and TBC1D5 (shRNA#1) U2OS cell lysates subjected to SDS-PAGE, subsequently blotted with endogenous antibodies for AP2 subunits AP2A1 and AP2M1.

C Immunofluorescent staining of AP2A1 in U2OS shRNA control, or shRNA#1 TBC1D5 cells treated with KU0063974 or DMSO (6 h).

D U2OS cells stably expressing HA‐Flag‐ATG9A were fixed and stained for endogenous AP2A1 and TBC1D5. Regions of co‐localization are indicated with arrows.

E U2OS cells stably expressing HA‐Flag‐ATG9A were starved in EBSS (4 h) or treated with KU0063794 (6 h), subsequently fixed and stained with anti‐AP2A1 antibody.

F U2OS cells stably expressing mCherry‐ATG9A were treated with KU0063794 (6 h), lysed in co‐immunoprecipitation buffer. Lysates from DMSO‐ or KU0063794‐treated cells were split and equal volumes were incubated with RFP‐Trap beads or GFP‐Trap beads, or agarose as a negative control, overnight at 4°C. Precipitated proteins were analyzed by SDS-PAGE.

Figure 4

A U2OS cells stably expressing HA‐Flag‐ATG9A transfected with siRNA targeting AP2A1 (40 nM), or siRNA AllStar control (40 nM) were treated with KU0063794 or DMSO (6 h) 4 days post‐transfection, fixed, and stained with anti‐HA antibody.

B Co‐localization of HA‐Flag‐ATG9A with TGN46.

C Lysates of U2OS cells transfected with siRNA targeting AP2A1, or siRNA control AllStars analyzed by SDS-PAGE.

D U2OS cells stably expressing mCherry−ATG9 were transiently transfected with GFP−Dynamin 2 or GFP−

Dynamin 2

(K44A), 20 h post−transfection cells were fixed and with anti−TBC1D5 antibody.

ATG9A

and Dynamin 2co−localization is indicated by arrows.

Figure 5

A U2OS cells stably expressing HA−Flag−ATG9A treated with DMSO or Dynasore (100 μM) 15 min, fixed and

immunostained

with anti−HA and anti−TBC1D5 antibodies.

B Quantification of ATG9A

and AP2A1co‐localization from experiment in (A), pictures from 3 independent experiments were analyzed and co‐localization quantified, statistics is calculated as described in Supplementary Materials and Methods.

C RGB‐line profile across the vesicle indicated with arrow in (B); red-AP2A1; green-ATG9A; blue-TBC1D5.

D Quantification of ATG9A and AP2 co‐localization. Cells were starved in EBSS media (45 min) or pretreated with Dynasore (15 min) and subsequently starved for 45 min in combination with Dynasore. Control cells were treated with DMSO.

E Quantification of LC3 puncta in cells stably expressing HA‐Flag‐ATG9A, treated and presented as in (D). 100 cells were quantified in 3 independent experiments.

F T‐REx HeLa cells were transfected with siRNA Control oligo or siRNA oligo#1 targeting (40 nM). 72 h post−transfection, expression of HA‐Flag−TBC1D5 was induced with doxycycline (50 ng/ml), and 96 h post−transfection cells were treated with Dynasore for 15 min, or pretreated with Dynasore and subsequently starved in combination with Dynasore for additional 45 min. Cells were lysed in co‐immunoprecipitation buffer, and lysates were incubated with Myc antibody or M2 antibody coupled with agarose overnight at 4°C. Beads were washed 3 times with incubation buffer and subjected to SDS‐PAGE.