Article
7.5 μM Atg1 complex (Atg1(D211A), Atg13–SNAP, SNAP–Atg17–Atg29–
Atg31) were formed by dilution of proteins from a stock solution into
buffer (final concentrations: 50 mM Bis-Tris-HCl, pH 5.5, 400 mM NaCl)
at 25 °C. In Fig. 2e, 4.8 μM Atg1 complexes (SNAP–Atg1(D211A), Atg13–
SNAP, SNAP–Atg17–Atg29–Atg31) were formed by dilution of proteins
from a stock solution into buffer (final concentrations: 50 mM MES,
pH 6.0, 300 mM NaCl) with subsequent incubation for 30 min at 25 °C. In
Fig. 2g and Extended Data Fig. 2h, liquid droplets of 6 μM Atg13–Atg17–
Atg29–Atg31 containing the indicated mutations were formed by dilu-
tion of proteins from a stock solution into buffer (final concentrations:
50 mM HEPES, pH 7.0, 250 mM NaCl) with subsequent incubation at
25 °C for the indicated time. In Extended Data Fig. 2d, liquid droplets
of 6 μM Atg13–Atg17–Atg29–Atg31 were formed by dilution of proteins
from a stock solution into buffer (final concentrations: 50 mM HEPES,
pH 7.0, 250 mM NaCl) with subsequent incubation for 10 min at 25 °C.
Next, 1,6-hexanediol or buffer was added at a final concentration of 5%
to liquid droplets at 25 °C. In Extended Data Fig. 2i, liquid droplets of the
indicated concentration of Atg13–Atg17–Atg29–Atg31 were formed by
dilution of proteins from a stock solution into buffer (final concentra-
tions: 50 mM MES, pH 6.0, 400 mM NaCl) with subsequent incubation
for 3 min at 25 °C. Phase separation was scored ‘droplet’ or ‘no droplet’,
depending on the presence or absence of protein droplets. In Fig. 3b,
liquid droplets of 2.7 μM Atg13–Atg17–Atg29–Atg31 containing either
phosphorylated Atg13 or non-phosphorylated Atg13 were formed by
dilution of proteins from a stock solution into buffer (final concentra-
tions: 50 mM HEPES, pH 7.0, 250 mM NaCl) with subsequent incubation
at 25 °C for the indicated times.
These samples were mixed in a microtube and imaged on a glass-
bottom dish (Mattek) coated with 3% bovine serum albumin (BSA)
(Wako). A FV3000RS was used for fluorescence imaging. 488-nm, 561-
nm, and 640-nm lasers were used for excitation of Atg1 labelled with
SNAP-Surface Alexa Fluor 488, Atg13 labelled with SNAP-Surface 549,
and Atg17 labelled with SNAP-Surface Alexa Fluor 647, respectively, and
fluorescence was recorded in linear sequential mode using a galvano
scanner. Quantitative analysis was carried out with Fiji.
In vitro pull-down assay
Purified proteins were incubated with GST-accept beads (Nacalai
Tesque) at 4 °C for 30 min. After the beads were washed three times
with PBS, proteins were eluted by 10 mM glutathione in 50 mM Tris-HCl
(pH 8.0). The samples were separated by SDS–PAGE. Protein bands
were detected by One Step CBB (BIO CRAFT).
Phosphorylation of Atg13 by TORC1
Purification of TORC1 from yeast was performed as previously
reported^17. In the final step of purification, TORC1 was eluted with elu-
tion buffer (250 ng μl−1 Flag peptide (Sigma, F3290), 100 mM NaCl, 31
mM Tris-HCl, pH 7.5). Atg13 (3 μM) was phosphorylated by TORC1 in
reaction buffer (1 mM ATP, 1 mM MgCl 2 , 1× protease inhibitor cocktail
(Nacalai, 03969-21), 1 mM PMSF, 100 mM NaCl, 50 mM Tris-HCl, pH 7.5)
for 17 h at 20 °C. After the reaction, NaCl was added to a final concentra-
tion of 500 mM. Dilution with buffer (20 mM HEPES, pH 7.0, 500 mM
NaCl) and concentration were repeated for phosphorylated Atg13 to
exchange the buffer. Samples were separated by SDS–PAGE and Zn2+-
Phos-tag SDS–PAGE (Wako). Zn2+-Phos-tag SDS–PAGE was performed
using 20 μM Phos-tag solution. Protein bands were detected by One
Step CBB. For western blotting, protein bands were detected by C-DiGit
Blot Scanner (LI-COR Biotechnology).
Phosphorylation of the Atg1 complex
For Extended Data Fig. 3f, liquid droplets of 3.3 μM Atg1 complex con-
taining either wild-type Atg1 or Atg1(D211A) were formed by dilution
of the protein from a stock solution into buffer (final concentration;
50 mM HEPES, pH 7.0, 250 mM NaCl) and then incubated for 10 min
at 25 °C. Next, one-tenth volume of ATP solution (10 mM ATP, 10 mM
MgCl 2 , 10 mM HEPES, pH 7.0, 250 mM NaCl) was added to liquid droplets
at 25 °C. Samples were then collected at the indicated time points. The
samples were separated by SDS–PAGE and detected by One Step CBB.
Fluorescence of the same samples was imaged using FV3000RS on 3%
BSA coated glass-bottom dishes. Turbidity was measured by sample
optical density at 350 nm using a NanoDrop 2000 (Thermo Scientific).Autophosphorylation assays
For confirming the specificity of anti-T226-P antibodies (Extended
Data Fig. 3b), ATP (–) samples were prepared by incubating the Atg1
complex containing either wild-type or T226A of SNAP–Atg1–Flag–
His 6 in 50 mM MES, pH 6.0, 150 mM NaCl for 30 min at 25 °C. ATP (+)
samples were prepared by incubating the same protein complex in 50
mM MES, pH 6.0, 150 mM NaCl for 30 min at 25 °C followed by incuba-
tion with 1 mM ATP-Mg in the same buffer solution for 30 min at 25 °C.
The samples were separated by SDS–PAGE and subjected to western
blotting using anti-T226-P and anti-Flag antibodies. For Fig. 3c, liquid
droplets of 0.2 μM Atg1 complex containing either wild-type Atg13 or
Atg13(F430A) were formed by incubation in 50 mM MES, pH 6.0, 300
mM NaCl for 30 min at 25 °C. A sample comprising Atg1 alone was also
prepared in the same buffer. Next, the ATP-Mg solution was added to
samples at a final concentration of 1 mM at 25 °C. Samples were then
collected at indicated time points. Samples were separated by SDS–
PAGE and subjected to western blotting. Protein bands were detected
by C-DiGit Blot Scanner (LI-COR Biotechnology). Quantitative analyses
were carried out using Fiji.Dephosphorylation of the Atg1 complex
For Fig. 3d, e, liquid droplets of 2 μM Atg1 complex were formed by dilu-
tion of the protein from a stock solution into buffer (final concentration;
50 mM HEPES, pH 7.0, 250 mM NaCl) with subsequent incubation for 10
min at 30 °C. Next, the solution containing Atg1-complex droplets was
supplemented with one-tenth volume of the ATP solution (10 mM ATP,
10 mM MgCl 2 , 10 mM HEPES, pH 7.0 and 250 mM NaCl) and incubated
for 20 min at 30 °C. Finally, the solution was supplemented with Ptc2
and MnCl 2 to final concentrations of 1 μM and 3 mM, respectively, and
incubated for 40 min at 30 °C. Samples were collected at the indicated
time points for further analysis.Antibodies
Polyclonal antibodies against S. cerevisiae Atg1 with phosphorylation
at Thr226 (anti-T226-P antibody) were raised according to a previ-
ous report^18. Antibodies were raised in rabbits against a phosphoryl-
ated peptide, FLPNTSLAE[pThr]LCGSPLY, which corresponds to the
sequence of the activation loop of Atg1. ELISA was performed using
the peptides with and without phosphorylation at Thr226 to confirm
the specificity of antibodies against the phosphorylated peptide. The
peptide synthesis, antibody generation, ELISA, and purification were
performed by GenScript. Polyclonal antibodies against S. cerevisiae
Atg13 with phosphorylation at Ser428 and Ser429 (anti-S428/9-P anti-
body) were generated as described in a previous study^16. Anti-Flag M2
antibody was purchased from Sigma (F3165). Anti-HA antibody was
purchased from MBL (M180-3S). Anti-mouse IgG (Fab specific)–per-
oxidase antibody produced in goat was purchased from Sigma (A9917).
Anti-rabbit IgG (whole molecule)–peroxidase antibody produced in
goat was purchased from SIGMA (A6154).Sample preparation for HS-AFM observation
For HS-AFM imaging, coverslips (24 × 32 mm, 0.13–0.17 mm thick) (Mat-
sunami Glass) were used as a solid support. Coverslips were immersed
in 5 M KOH solution for 1 h, followed by three washes in Milli-Q water.
Cleaned coverslips were subsequently sonicated in Milli-Q water
for 20 min and stored in ethanol at 4 °C until use. Ethanol was com-
pletely eliminated before each experiment. Atg13–SNAP (1 μM) and
SNAP–Atg17–Atg29–Atg31 (1 μM) were mixed in 20 μl of observation