Article
Methods
No statistical methods were used to predetermine sample size. The
experiments were not randomized. The investigators were not blinded
to allocation during experiments and outcome assessment.
Yeast strains and media
S. cerevisiae strains and plasmids used in this study are listed in Sup-
plementary Tables 1 and 2, respectively. Standard protocols were used
for yeast manipulation^30. Cells were cultured at 30 °C in nutrient-rich
SD + CA medium (0.17% yeast nitrogen base without amino acids and
ammonium sulfate, 0.5% ammonium sulfate, 0.5% casamino acids, and
2% glucose) supplemented with appropriate nutrients. Autophagy was
induced by transferring cells to nitrogen-starvation SD(−N) medium
(0.17% yeast nitrogen base without amino acids and ammonium sulfate,
and 2% glucose) or by treating cells with 0.5 μg ml−1 rapamycin (Sigma-
Aldrich). Treatment of yeast cells with 1,6-hexanediol was performed
by adding 1,6-hexanediol and digitonin at a final concentration of 10%
and 10 μg ml−1, respectively, to the medium^14. For replenishing nutri-
ents to starved cells, an equal volume of 2× SD + CA medium (doubled
concentration of each constituent) was added to culture media.
For galactose induction of GFP–Atg13 protein using the GAL1 pro-
moter, cells were cultured at 30 °C in nutrient-rich SD + CA medium
(0.17% yeast nitrogen base without amino acids and ammonium sulfate,
0.5% ammonium sulfate, 0.5% casamino acids, 2% raffinose and 0.1%
glucose) supplemented with appropriate nutrients. The following day,
the cell culture (OD 600 = 1.0–2.0) was supplemented with galactose to a
final concentration of 2%, and cultured for an additional 6 h. Autophagy
was induced by treating cells with 0.5 μg ml−1 rapamycin for 10 min.
Construction of expression plasmids
The pRS316-based low-copy plasmid for expression of GFP–Atg1 and
GFP–Atg13 in yeasts under the control of the AT G 1 and GPD promoters,
respectively, were constructed as described previously^16. The pRS426-
based multi-copy plasmid for expression of GFP–Atg13 in yeasts under
control of the GPD promoter and GAL1 promoter, respectively, were also
constructed similarly. To construct coexpression plasmids encoding
SNAP-tagged Atg17, Atg29 and hexahistidine (His 6 )-tagged Atg31, genes
were amplified by PCR and cloned into the pET28a (+) vector (Novagen)
for SNAP-tagged Atg17 and the pACYCDuet-1 vector (Novagen) for
Atg29 and His 6 -tagged Atg31. To construct expression plasmids encod-
ing N-terminal His 6 -tagged and C-terminal SNAP–Twin-Strep-tagged
Atg13, genes were amplified by PCR and cloned into the pET11a vector.
To construct expression plasmids encoding N-terminal glutathione-
S-transferase (GST)-tagged and C-terminal SNAP-tagged Vac8, genes
were amplified by PCR and cloned into the pGEX6p-1 vector. To con-
struct expression plasmids encoding N-terminal GST-tagged Ptc2,
genes were amplified by PCR and cloned into the pGEX6p-1 vector. To
construct expression plasmids encoding the N-terminal SNAP-tagged
Atg1 with a HRV3C protease site followed by Flag and His 6 tags, the
SNAP-tag gene was amplified by PCR and cloned into the pFastBac
Dual-based Atg1 expression vector^6. The NEBuilder HiFi DNA Assembly
Cloning Kit (New England Biolabs) was used for cloning. Mutations
to generate the indicated amino acid substitutions were introduced
by PCR-mediated site-directed mutagenesis. All constructs were
sequenced to confirm accuracy of cloning.
Protein Expression and Purification
E. coli strain BL21(DE3) cells were used for expression of all recombi-
nant proteins except Atg1. His 6 -tagged Atg31 was coexpressed with
SNAP–Atg17 and Atg29. After cell lysis, the SNAP–Atg17–Atg29–Atg31
complex was purified by affinity chromatography using a Ni-NTA col-
umn (Qiagen). After affinity chromatography, the protein complex
was purified on a HiLoad 26/60 Superdex 200 PG column (GE Health-
care) eluted with 20 mM Tris-HCl pH 8.0 and 150 mM NaCl. N-terminal
His 6 -tagged and C-terminal SNAP–Twin-Strep-tagged Atg13 was first
purified with a Ni-NTA column and then purified using a Strep-TactinXT
resin column (IBA Lifesciences). Finally, the proteins were purified on a
HiLoad 26/60 Superdex 200 PG column eluted with 20 mM HEPES pH
7.0 and 500 mM NaCl. GST–Vac8–SNAP was first purified using a glu-
tathione–Sepharose 4B (GS4B) column (GE Healthcare). After affinity
chromatography, GST was excised using human rhinovirus 3C protease.
Vac8–SNAP was again applied to a GS4B column in order to remove
the excised GST. Finally, the protein was purified on a HiLoad 26/60
Superdex 200 PG column eluted with 20 mM Tris-HCl, pH 8.0 and 150
mM NaCl. GST-Ptc2 was first purified using a glutathione–Sepharose
4B (GS4B) column (GE Healthcare). After affinity chromatography, GST
was excised using human rhinovirus 3C protease. Ptc2 was again applied
to a GS4B column in order to remove the excised GST. Recombinant Atg1
was expressed using the baculovirus expression system (Invitrogen)
and then purified as described previously^6. SNAP tag of Atg1, Atg13, and
Atg17 was labelled with SNAP-Surface Alexa Fluor 488, SNAP-Surface
549, and SNAP-Surface Alexa Fluor 647 (all from New England Biolabs),
respectively, according to the manufacturer’s protocol, except for the
samples used for AFM experiments, where the SNAP tag of Atg17 was
labelled with SNAP-Surface Alexa Fluor 488. For GUV experiments,
proteins were dialysed against 20 mM HEPES pH 7.0 and 500 mM NaCl
using dialysis tubes, 8 kDa cut-off (GE Healthcare).FRAP measurements and analysis
For FRAP experiments assessing the PAS, cells treated with rapamycin
were imaged on concanavalin A coated glass-bottom dishes (Mattek)
to immobilize cells. For FRAP experiments of Atg1-complex drop-
lets attached to a giant liposome, multilamellar liposomes instead
of GUVs were used in order to reduce the movement of droplets on
the liposome. During FRAP experiments, Atg1 sample was continu-
ously added in the vicinity of the liposome using a micropipette. FRAP
experiments were carried out with a FV3000RS confocal laser scanning
microscope (Olympus) equipped with an UPLSAPO60XO, NA 1.42 Oil
objective (Olympus). For imaging of GFP and SNAP-Surface Alexa Fluor
488 fluorescence, excitation was performed using a 488-nm laser and
fluorescence was recorded in a linear sequential mode using a galvano
scanner to capture one z-stack for the PAS and 6.9-μm z-stacks with
1.4-μm spacing for the liposome-tethered droplets. Photobleaching
was performed using 405-nm and 488-nm laser pulses (1 repeat, 10%
intensity, dwell time 5–50 ms) for the PAS and 488-nm laser pulses (1
repeat, 10% intensity, dwell time 45 ms) for the liposome-tethered
droplets. Image analysis was carried out with FIJI v.1.52e^31 or FV31S-SW
v.2.1.1.98 (Olympus). For kinetic analysis, relative fluorescence intensity
was plotted against time by setting the intensity before quenching as
1.0 and the minimum intensity after quenching as 0.0, and fitted to
an exponential recovery curve: F = A 0 (1 − exp(koff(t)) in which A 0 is the
maximum recovery at t = infinity and t is time in seconds. This equation
was used to determine koff. The t1/2 value was calculated as ln(2)/koff.Fluorescence correlation spectroscopy
The FCS data shown in Fig. 1 are taken with a TCS SP5 II confocal micro-
scope (Leica). An HC PL APO 40×/1.10 W CORR CS2 objective lens (Leica)
was used for imaging, and the signal was detected with a hybrid detector
(HyD, Leica) in a photon-counting mode, with the scanning frequency
fz = 8,000 Hz, and the number of pixels ξmax = 16. The size of the detection
area was Sx = 403 nm, comparable to the size of the diffraction-limited
point spread function (PSF) of the observation lens (wxy = 290 nm, and
wz = 1,450 nm).
Further, we employed scanning FCS (sFCS)^32 to minimize pho-
tobleaching and the effect of movement of the PAS itself.
In sFCS, a single line scanning data set was taken with a scanning
frequency fz. The intensity of each pixel ξ (1 ≤ ξ ≤ ξmax) is the integration
of fluorescent signals for a pixel dwell time Tx. The illumination volume
moves from the leftmost position ξ = 1 to the rightmost position ξmax,