Letters

https://doi.org/10.1038/s41556-019-0459-2

Selective autophagy degrades nuclear pore

complexes
Chia-Wei Lee1,6, Florian Wilfling   1,6, Paolo Ronchi2, Matteo Allegretti3, Shyamal Mosalaganti   3,
Stefan Jentsch1,7, Martin Beck   3,4* and Boris Pfander   5*

Nuclear pore complexes (NPCs) are very large proteinaceous
assemblies that consist of more than 500 individual pro-
teins1,2. NPCs are essential for nucleocytoplasmic transport of
different cellular components, and disruption of the integrity
of NPCs has been linked to aging, cancer and neurodegenera-
tive diseases3–7. However, the mechanism by which membrane-
embedded NPCs are turned over is currently unknown. Here
we show that, after nitrogen starvation or genetic interfer-
ence with the architecture of NPCs, nucleoporins are rapidly
degraded in the budding yeast Saccharomyces cerevisiae. We
demonstrate that NPC turnover involves vacuolar proteases
and the core autophagy machinery. Autophagic degradation
is mediated by the cytoplasmically exposed Nup159, which
serves as intrinsic cargo receptor and directly binds to the
autophagy marker protein Atg8. Autophagic degradation of
NPCs is therefore inducible, enabling the removal of individual
NPCs from the nuclear envelope.
The NPC is elaborately built from approximately 30 nucleoporins
(Nups) that can be allocated to distinct subcomplexes1,2 (Extended
Data Fig. 1a). Of these subcomplexes, only some peripheral com-
ponents have been shown to be dynamic8, whereas more integral
parts of the NPC appear to be remarkably stable. In particular, the
scaffold of the NPC exhibits an extremely long half-life in mam-
mals7,9, albeit with notable exceptions10, suggesting that NPC turn-
over is regulated by a specific, yet unknown, pathway. We therefore
investigated Nup degradation in the budding yeast Saccharomyces
cerevisiae, which is a simple eukaryotic model organism with a short
generation time. In dividing yeasts, Nups are also long-lived with
half-lives that exceed the generation time by several fold (Extended
Data Fig. 1b). We therefore screened for conditions that could
result in the degradation of Nup192—a core-scaffolding compo-
nent of the NPC inner ring. We found that enhanced green fluo-
rescent protein (eGFP)-tagged Nup192 as well as other members
of the NPC scaffold are degraded specifically under conditions of
nitrogen starvation or inhibition of mTOR (Fig. 1a, Extended Data
Fig. 1c). Nitrogen starvation triggers accelerated protein turnover to
restore intracellular amino acid homeostasis, mainly by upregula-
tion of macroautophagy (hereafter called autophagy), a process by
which cytoplasmic material is sequestered into a double-membrane
compartment called the autophagosome and then degraded in the
vacuole11,12. After nitrogen starvation, we found that eGFP accu-
mulated when either inner ring complex Nup192 or the Y-complex
Nup133 were tagged (Fig. 1a). This indicates degradation in the
vacuole, because the compact fold of eGFP renders the eGFP resis-
tant to vacuolar hydrolases13,14. Accordingly, simultaneous deletion
of PEP4 and PRB1, which encode two key vacuole-resident prote-
ases, abolished the formation of free eGFP and stabilized full-length
eGFP-tagged Nup192 (Fig. 1b).
It has previously been shown that NPC assembly is under sur-
veillance by the ESCRT-III–Vps4 complex and that defective assem-
bly intermediates are eliminated in a proteasome-dependent, but
Pep4-indepedent, manner15. Indeed, after nitrogen starvation, we
observed that Nups were stabilized when ESCRT function was
impaired by deletion of VPS4 or the proteasomal ubiquitin recep-
tor RPN10 (Fig. 1c). This stabilization was specific for deletion of
RPN10—deletions of other proteasomal ubiquitin receptors did not
stabilize Nup192–eGFP levels (Extended Data Fig. 1d). Notably, in
the rpn10∆ strain, a considerable fraction of Nup192–eGFP was still
degraded after nitrogen starvation and the appearance of free eGFP
suggested that there is an additional degradation pathway through
the vacuole. We hypothesized that autophagy could be a mechanism
for NPC degradation. To test this, we deleted the core autophagy
factor ATG8 and compared the degradation of Nup192–eGFP to
the degradation in the rpn10∆ strain. Deletion of ATG8 stabilized
Nup192–eGFP after nitrogen starvation to levels similar to the lev-
els observed in the rpn10∆ strain (Fig. 1d). Furthermore, vacuolar
degradation of Nup192–eGFP, as judged by the appearance of free
eGFP, was entirely blocked in atg8∆ mutant cells (Fig. 1d) as well as
in other mutants of the core autophagy machinery (Extended Data
Fig. 1e). Only when both pathways were impaired by double dele-
tion of ATG8 and RPN10, Nup192–eGFP levels were fully stabilized
(Fig. 1d). We also tested the temperature-sensitive mutant cim3-1,
which strongly impairs proteasome function at increased tempera-
tures. Interestingly, we found that the cim3-1 mutation as well as the
vps4∆ mutation strongly impaired autophagy under conditions of
nitrogen starvation (Extended Data Fig. 1f,g) and accordingly also
led to an almost complete stabilization of Nup192–eGFP (Extended
Data Fig. 1f,g). Although such secondary effects on autophagy after
nitrogen starvation warrant further examination, we conclude that,
after nitrogen starvation, turnover of a scaffold Nup is dependent
on at least two pathways that involve proteasomal degradation and
autophagy, respectively.
To investigate whether other Nups are degraded in a similar
manner, we tagged different Nups from different subcomplexes with
eGFP and followed their degradation after starvation (Fig. 2a). All of
the tested Nups, including the membrane-embedded Pom152,

1
Molecular Cell Biology, Max Planck Institute of Biochemistry, Martinsried, Germany. 2Electron Microscopy Core Facility (EMCF), European Molecular
Biology Laboratory, Heidelberg, Germany. 3Structural and Computational Biology Unit, Cell Biology and Biophysics Unit, European Molecular Biology
Laboratory, Heidelberg, Germany. 4Molecular Sociology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany. 5DNA Replication and
Genome Integrity, Max Planck Institute of Biochemistry, Martinsried, Germany. 6These authors contributed equally: Chia-Wei Lee, Florian Wilfling.
7
Deceased: Stefan Jentsch. *e-mail: martin.beck@biophys.mpg.de; bpfander@biochem.mpg.de

Nature Cell Biology | VOL 22 | FebruarY 2020 | 159–166 | www.nature.com/naturecellbiology 159

Letters NAturE CEll Biology
1.2
a Nup192–eGFP b

Relative Nup level

Inner ring complex Y-complex 1.0 SD-N (h): 0 24
0.8
SD-N (h): 0 2 4 8 16 24 40 SD-N (h): 0 2 4 8 16 24 40

∆
∆
0.6

b1
b1
(kDa) (kDa)

pr
pr
250 Nup192–eGFP 250 0.4

∆
4∆

p4
Nup133–eGFP

p
0.2

T
T
150 150

pe
pe

W
W
(kDa)
100 100 0 250
0 10 20 30 40 50 Nup192–eGFP
75 75 150
SD-N (h)
100
50 50 1.2
Nup133–eGFP 75

Relative Nup level

37 37 1.0
eGFP′ 0.8 50
25 25 eGFP′
0.6
37
25 Dpm1 25 Dpm1 0.4
0.2 eGFP′
25
0
0 10 20 30 40 50 Dpm1
25
SD-N (h)

c d SD-N (h): 0 24
SD-N (h): 0 24

∆
g8

g8
0∆

0∆

at

at
rp ∆

rp ∆
s4

s4
n1

n1

rp 10∆
0∆

rp 10∆
0∆
0 h SD-N 24 h SD-N
T

rp ∆

rp ∆
0 h SD-N 24 h SD-N
vp

vp
W

(kDa)

n1

n1
g8

g8
n

n
T

T
at

at
W

W
250 Nup192–eGFP P > 0.05 *** (kDa) ***
NS
*** 250 P > 0.05 **

P = 0.0004
150 120
P = 0.0002 Nup192–eGFP NS
**
P = 0.0002

Nup192–eGFP level (%)

Nup192–eGFP level (%)

150 120

P = 0.0032
100 100

P = 0.0043
75 100 100
80
75 80
50 60
50 60
37 40
37 40
25 eGFP′ 20 20
25 eGFP′
0 0
25 Dpm1
rp 4∆
rp ∆

0∆ 0∆
0∆

∆
0∆ 0∆
0∆

rp rp ∆

∆
T

T
Pgk1
s4

g8
g8

g8

g8
W

W
s

n1 n1
n1

n1 pn1
n1

rpn10∆ 37
vp
vp

WT

at
at

at

at
r
CHX (min):
30
60

30
60
0

rp
(kDa)
150 Ub-βgal

25 Dpm1

Fig. 1 | Nups are degraded after nitrogen starvation by both autophagy and the proteasome. a, Degradation of scaffold Nups (Nup192–eGFP (inner
ring complex) and Nup133–eGFP (Y-complex)) was measured after nitrogen starvation (using SD-N medium) using anti-eGFP immunoblotting (left).
eGFP′ denotes vacuolar eGFP remnant. Dpm1 was used as a loading control. Right: quantification of n = 3 biologically independent experiments. Data are
mean ± s.d. b, Analysis of Nup192–eGFP degradation in WT and vacuolar-protease-deficient (pep4∆ prb1∆) cells using anti-eGFP immunoblotting; n = 3
biologically independent experiments. c, Degradation of Nup192–eGFP in WT, ESCRT-deficient (vps4∆) and proteasome mutant (rpn10∆) cells (top left)
with quantification (top right) of Nup192–eGFP levels before and after nitrogen starvation for 24 h. Bottom: the Ub-βgal proteasome model substrate is
stabilized in rpn10∆ cells after treatment with cycloheximide (CHX; 80 µg ml−1). Data are mean ± s.d. of n = 3 biologically independent experiments.
d, Degradation of Nup192–eGFP in WT, autophagy-deficient (atg8∆) and proteasome mutants (rpn10∆), as well as in atg8∆ rpn10∆ double mutants, with
quantification of Nup192–eGFP levels before and after nitrogen starvation for 24 h. Pgk1 was used as a control. Data are mean ± s.d. of n = 3 biologically
independent experiments. For c and d, statistical analysis was performed using two-tailed Student’s t-tests; ***P ≤ 0.001, **P ≤ 0.005; NS, P ≥ 0.05. Source
data are available online.

underwent autophagosomal degradation, determined by the stabi- Furthermore, overexpression of Atg8 forced an interaction with
lization of the individual Nup–eGFP fusion proteins in the ATG8 Nups even without induction of nitrogen starvation (Extended Data
deletion background (Fig. 2a). We also quantified Nup levels and, Fig. 2b). To visualize intermediates of autophagosomal NPC degra-
specifically, the nuclear-envelope-associated pool by monitoring the dation, we deleted the vacuolar lipase ATG15, which is indispens-
fluorescence signal of Nup192–eGFP at the nuclear envelope before able for dissolving autophagosomal membranes in the vacuole16.
and after nitrogen starvation in individual cells trapped in a micro- After we induced nitrogen starvation in atg15∆ cells, we observed
fluidics device. We found that the volume of the nucleus increased that Nup192–eGFP-containing structures were trapped inside the
in cells that were deficient for Atg8 (atg8∆) when we switched to vacuole, most likely resembling autophagic bodies (Fig. 2d). We
medium lacking nitrogen, but not in WT cells (Extended Data Fig. 2a). also found that the vacuolar structures trapped by deletion of the
By contrast, the levels of Nup192–eGFP decreased during starva- ATG15 lipase not only contained Nup192–eGFP but also colocal-
tion, but remained constant in atg8∆ cells (Extended Data Fig. 2a). ized with Nups from different subcomplexes, such as Y-complex
Accordingly, we found that eGFP–Atg8 immunoprecipitates Nup84–mars, cytoplasmic filament Nup159–mars and, notably,
analysed using quantitative mass spectrometry (qMS) from nitro- transmembrane Nup Ndc1–mars (Fig. 2e), suggesting that these
gen starved pep4∆ cells were strongly enriched for Nups, includ- structures could be NPC-containing autophagosomes. The appear-
ing various scaffold components of the inner ring and Y-complex, ance of the Nup192–eGFP foci inside the vacuole was entirely
the cytoplasmically exposed Nup82-complex, the three transmem- dependent on the presence of the core autophagy machinery
brane Nups—Pom34, Pom152 and Ndc1—as well as the strongly (Fig. 2f, Extended Data Fig. 2c). By contrast, deletion of NVJ1
membrane-associated Nup53 (Fig. 2b). Analysis of eGFP–Atg8 implicated in piecemeal microautophagy of the nucleus17 did not
immunoprecipitates by western blotting confirmed these results influence the appearance of Nup-containing structures in the vacu-
(Fig. 2c), suggesting that nuclear-envelope-embedded NPCs ole (Extended Data Fig. 2c,d) or affect the turnover of Nup192–
are bound to Atg8 potentially during autophagic degradation. eGFP (Extended Data Fig. 2d).

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NAturE CEll Biology
All selective autophagy pathways need so-called cargo recep-
tor proteins (referred to as receptors hereafter) to facilitate
degradation of the specific cargo18–20. These receptors bind to
Atg8 and therefore enable loading of the autophagic cargo onto
autophagosomes. Recently, Atg39 and Atg40 were identified as
nucleophagy and ER-phagy receptors21,22 that are important for
autophagic degradation of proteins from these compartments.
However, when we tested single deletions of ATG39 or ATG40, or
the atg39∆ atg40∆ double mutant, we observed only mild effects
on the degradation of Nup192–eGFP (Extended Data Fig. 2e).
Deletions of other known Atg8-receptors, such as ATG19 or
CUE5 (refs. 23,24), also did not influence autophagic degradation
of Nup192–eGFP (Extended Data Fig. 2f,g), whereas deletion of
the autophagic scaffold protein Atg11 partially impaired the deg-
radation of Nup192–eGFP and Nup159–eGFP (Extended Data
Fig. 2g,h). Overall, these data indicate that autophagic degrada-
tion of the NPC is independent from known autophagy receptors
but requires the scaffold protein Atg11.
As autophagosomes are formed in the cytoplasm, we hypoth-
esized that the interaction between Atg8 and the NPC occurs at
the cytoplasmic face of the NPC. Nups of the cytoplasmic fila-
ments—which, in yeast, primarily consist of the members of the
Nup82 complex—might be most accessible for Atg8 binding. We
set out to test Nup159, which is the largest member of the Nup82
complex1 and contains a large intrinsically disordered domain that
could be accessible for direct interaction with Atg8. We therefore
incubated recombinantly expressed and purified His–Atg8 and
glutathione S-transferase (GST)-tagged Nup159 in  vitro and sub-
sequently enriched His–Atg8 using Ni-NTA purification (Fig. 3a).
Interestingly, GST–Nup159 was specifically eluted together with
His–Atg8 whereas GST alone was not, suggesting that there is
a direct interaction between both of these proteins (Fig. 3a).
Interactions between receptor proteins and Atg8 involve a small,
linear sequence motif called the Atg8-interacting motif (AIM) on
the receptor25 and two hydrophobic pockets (W-site and L-site)
on Atg8 (ref. 25). We used the atg8 mutant Y49A L50A, which has
mutations in both hydrophobic pockets, and tested its binding to
Nup159 tagged with 6× haemagglutinin (6HA) in yeast cell lysate.
The binding of Nup159–6HA to GST–Atg8Y49A L50A was strongly
decreased compared with wildtype (WT) Atg8 (Fig. 3b), suggest-
ing that Nup159 is an AIM-dependent Atg8 binding protein. We
noted from our in  vitro experiments that Atg8 did not recognize
the C-terminally truncated versions of Nup159 (Fig. 3a), suggesting
that the AIM is located within the C terminus. We therefore mutated
the five predicted AIMs by independently mutating the amino acids
Letters
at position 1 and 4 to alanine in each putative AIM (Extended Data
Fig. 3a) and tested binding to Atg8 using co-immunoprecipitation
(co-IP) experiments. The interaction between Atg8 and Nup159
(as well as other Nups) was decreased when AIM1 was mutated,
but was not decreased in the other potential AIM mutants (Fig. 3c,
Extended Data Fig. 3a). We next introduced this mutant in  vivo
and assessed the effect of the mutant on the degradation of differ-
ent Nups. Similar to the ATG8 deletion, we found that nup159AIM1
impaired the starvation-induced autophagic clearance of not only
Nup192–eGFP, but also Nup133–eGFP (Fig. 3d). By contrast,
the nup159AIM1 mutant had no effect on autophagy per  se, as
eGFP–Atg8 degradation was unchanged (Extended Data Fig. 3b).
Atg11 binds to most known autophagy receptors and this is an
important step in phagophore assembly at sites of cargo recogni-
tion19. To test whether Nup159 also follows this concept, we per-
formed immunoprecipitation experiments against eGFP-tagged
Atg11 and found that Nup159 indeed interacts with Atg11
(Extended Data Fig. 3c). These data therefore suggest that Nup159
acts as AIM-dependent autophagy receptor.
To visualize the interaction between Atg8 and Nup159 in vivo,
we used a bimolecular fluorescence complementation (BiFC) strat-
egy26. We labelled Atg8 and Nup159 with either the N-terminal
(VN) or C-terminal (VC) half of the Venus protein, respectively.
In this assay, a fluorescent signal is expected when Atg8 and
Nup159 interact and thereby reconstitute the full Venus protein.
Consistent with our Atg8 interaction data (Fig. 3a,b), we observed
a fluorescent BiFC signal for the Atg8–Nup159 interaction in the
absence of nitrogen starvation, when VN–Atg8 was overexpressed
(Fig. 3e). Notably, after nitrogen starvation, the BiFC signal was
enhanced in a focus, which co-localized with NPCs as indicated
by Nup170–mars signal (Fig. 3e). The BiFC signal was specific
for Nup159–VC and was absent for either inner ring Nup192–VC
(Fig. 3e) or nuclear basket Mlp1–VC (Extended Data Fig. 3d). It
was also strongly reduced when the interaction between Atg8 and
Nup159 was impaired by mutation of the AIM-binding pockets of
Atg8 (Atg8Y49A L50A; Extended Data Fig. 3e). We therefore conclude
that Atg8 interacts with Nup159 at the nuclear envelope, indicat-
ing that its function as an autophagy receptor may take place at the
nuclear envelope as well. Moreover, the focus-like accumulation
suggests that, after nitrogen starvation, NPCs cluster at specific sites
of the nuclear envelope when they are targeted by autophagy. To test
whether the Nup159–Atg8 interaction targets membrane-embed-
ded NPCs to autophagosomes, we counted the number of cells
with vacuolar Nup192–eGFP signal after nitrogen starvation in an
atg15∆ background and compared it with a strain in which the AIM

Fig. 2 | Nuclear-envelope-embedded NPCs are degraded by autophagy. a, Degradation of different Nup–eGFP fusion proteins from different
subcomplexes in WT and mutant cells deficient for the core autophagy machinery (atg8∆) after nitrogen starvation, measured as described in Fig. 1c,d.
Data are mean ± s.d. of n = 3 biologically independent experiments. Statistical analysis was performed using two-tailed Student’s t-tests. b, Quantitative
MS analysis of Atg8-interacting proteins determined using co-IP of eGFP–Atg8 under nitrogen-starvation conditions; n = 4 biologically independent
samples. pep4∆ cells expressing N-terminally eGFP-tagged or untagged Atg8 under the control of the ADH promoter were starved of nitrogen for 16 h
before immunoprecipitation analysis with GFP-Trap matrix. Data are log-transformed ratios of protein intensities versus −log-transformed P values of
two-tailed Student’s t-tests performed in quadruplicates. The hyperbolic curve separates specifically interacting proteins from background (square;
false-discovery-rate-adjusted P = 0.05; minimal fold change S0 = 0.5). The bait protein Atg8 and different Nups are highlighted by different colours.
c, Validation of Nup–Atg8 interactions identified in b using Co-IP followed by western blot (n = 2 biologically independent experiments) using anti-eGFP,
anti-Nup98 (reacts with multiple FG-Nups), anti-Nup84 and anti-HA (recognizes Nup192–HA (left), anti-Nup159–HA (top right) and anti-Pom152–6HA
(bottom right)) antibodies. d, eGFP-tagged Nup192 accumulates in the vacuole in the absence of the vacuolar lipase Atg15. Representative midsection
image of the localization of C-terminally eGFP-tagged Nup192 in Atg15-deficient (atg15∆) cells was measured using fluorescence microscopy after
nitrogen starvation for 24 h; n = 2 biologically independent experiments. Vph1–mars was used as a marker for the vacuolar membrane. The arrows indicate
autophagic bodies within the vacuole. Scale bar, 5 μm. e, In the absence of the vacuolar lipase Atg15, colocalization of eGFP-tagged Nup192 with outer
scaffold Nup84–mars, cytoplasmic filament Nup159–mars and transmembrane (Pom) Nup Ndc1–mars can be observed in focus-like accumulations,
which probably represent vacuole-engulfed autophagosomes; n = 2 biologically independent experiments. The arrows indicate autophagic bodies within
the vacuole. The dotted boxes indicate the regions magnified in the insets. Scale bars, 5 μm; 1 µm (inset). f, Focal Nup accumulations in the vacuole are
dependent on core autophagy machinery proteins, as monitored using Nup192–eGFP staining in WT, atg1∆ and atg7∆ cells; n = 2 biologically independent
experiments. The arrows indicate autophagic bodies within the vacuole. Scale bar, 5 μm. Source data are available online.

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Letters NAturE CEll Biology

of Nup159 was mutated (nup159AIM1). Interestingly, we observed a snapshots of these Nup-containing structures, we performed cor-
strong reduction of vacuolar focus accumulation when the interac- relative light and electron microscopy (CLEM) on a strain in which
tion between Nup159 and Atg8 was impaired (Fig. 3f). To obtain Nup159 was tagged with mars and ATG15 was deleted (atg15∆).

a Inner ring complex Y-complex Cytoplasm filament Nups Pore membrane Nups

SD-N (h): 0 24 SD-N (h): 0 24 SD-N (h): 0 24 SD-N (h): 0 24

∆
∆

W ∆

∆
W ∆

W ∆
g8
g8

g8

g8

g8
g8

g8

g8
T
T

T
T

T
T
at

W
at

at

at

at
W

W
at

at

at
W
W

(kDa) (kDa) (kDa) (kDa)

250 250 250
Nup192–eGFP 250 Nup159–eGFP Pom152–eGFP
Nup133–eGFP
150 150 150 150
100 100 100 100
75
75 75 75

50 50 50 50
37 37 37 37

25 eGFP′ 25 eGFP′ 25 eGFP′ 25 eGFP′

25 Dpm1 25 Dpm1 25 Dpm1 25 Dpm1

0 h SD-N 0 h SD-N P > 0.05 0 h SD-N 0 h SD-N

P > 0.05 P > 0.05
24 h SD-N P > 0.05 24 h SD-N NS 24 h SD-N 24 h SD-N
NS 120 120 120 NS
120 NS

Pom152–eGFP level (%)

Nup159–eGFP level (%)
Nup133–eGFP level (%)
Nup192–eGFP level (%)

100 100 100

100 P = 0.003
P = 0.004 P = 0.0022
P = 0.0003 80 80
80
***
80 ** ** **
60 60 60
60
40 40 40
40
20 20 20
20
0 0 0
0

∆
T

T
T
∆
∆

g8

g8

g8

g8
∆
∆

W
T

W
W
T

g8
g8
W

W
g8
g8
W

at

at

at

at
at
at
at
at

b 16 h SD-N pep4∆ c pep4∆, SD-N 16 h pep4∆, SD-N 16 h

bait: eGFP–Atg8 IP: eGFP Input IP: eGFP Input
9 Significant: Non-significant: eGFP–Atg8: eGFP–Atg8:
(kDa) (kDa)
Cytoplasm filament Nups: Basket Nups: 250 Nup192–6HA 250 Nup159–6HA
8 Gle2 Nup2 150
Atg8 Nup82 Nup60 Nup84 eGFP–Atg8
75
Nup159 37
7 Y-complex Nups: 150
Nup116
Pgk1
sec13 100 37
FG Nups:
Nsp1
6 75 Nup145N pep4∆, SD-N 16 h
Nup49
Nup57 IP: eGFP Input
Nup57
50 Nup49
5 Pore membrane Nups:
eGFP–Atg8:
(kDa) Pom152–6HA
P value

Ndc1 eGFP–Atg8
37 150
Pom34
4 Pgk1 eGFP–Atg8
Pom152 37 37
Pgk1
Inner ring Nups: 37
3 Nup53
Nup157

2
Nup170 d DIC Nup192–eGFP Vph1–mars Merge
Nup188
24 h SD-N, atg15∆

Nup192
1 Y-complex Nups:
Nic96
Nup84
0 Nup133
Nup145
–6 –4 –2 0 2 4 6 8 10 12 14

log2[eGFP–Atg8/untagged]
e DIC Nup192–eGFP Nup–mars Merge f DIC Nup192–eGFP Vph1–mars Merge
Nup84–mars

WT
SD-N 24 h, atg15∆

24 h SD-N, atg15∆
Nup159–mars

atg1∆
Ndc1–mars

atg7∆

Midsection Maximum intensity z-projection Midsection Maximum intensity z-projection

162 Nature Cell Biology | VOL 22 | FebruarY 2020 | 159–166 | www.nature.com/naturecellbiology

NAturE CEll Biology
The Nup159–mars fluorescence signal could indeed be tracked
to autophagic bodies within the vacuole, which were enriched for
nuclear vesicles with membrane-embedded NPCs, demonstrating
that membrane-embedded NPCs are indeed a target of Nup159-
dependent autophagic degradation (Fig. 3g, Extended Data Fig. 3f,
Supplementary Video 1). Interestingly, we found electron-dense
material in the nuclear vesicles that resembled the density seen
inside the nucleus, and nuclear vesicles were surrounded (within
the autophagic body) by material of apparent cytosolic origin that
contained ribosomes (Fig. 3g, Extended Data Fig. 3f).
Deletion of certain scaffold Nups, such as Nup120 and Nup133,
causes clustering of NPCs at one side of the nucleus27–29. It is thought
that clustering might protect daughter cells from inheriting mis-
assembled or damaged NPC species15. Whether and how these
clustered NPCs are removed is unknown at present. We therefore
tested whether autophagy targets NPC-clusters by comparing the
degradation of Nup192–eGFP in WT cells to mutant conditions
causing NPC clustering (nup120∆ or nup133∆). Interestingly, free
eGFP derived from either Nup192–eGFP or Nic96–eGFP appeared
more rapidly in both nup120∆ and nup133∆ cells compared with
WT cells (Fig. 4a, Extended Data Fig. 4a). The faster degradation
of Nups was specific to clustered NPC mutants, as impairment of
NPC assembly in nup116∆ cells did not lead to enhanced rates of
degradation but, rather, abolished autophagic degradation of Nups
(Extended Data Fig. 4b,c). This is consistent with other autophagy
pathways in which cargo clusters initially to be efficiently removed
by autophagy18,19.
In non-nitrogen-starved nup120∆ cells, the interaction between
Atg8 and Nups induced by eGFP–Atg8 overexpression and mea-
sured by eGFP co-IP was enhanced, while at the same time a reduc-
tion of Nups in the input levels was observed (Fig. 4b), indicating
that clustered NPCs are preferentially degraded by autophagy. In
contrast to the cluster mutants, in nup116∆ cells30, the cytosolic
filaments appeared to be dissociated from the NPCs, as eGFP–Atg8
pull-downs only enriched cytosolic filament proteins (Extended
Data Fig. 4d) and pull-down of the Y-complex protein Nup84–eGFP
failed to enrich members of the cytosolic filaments (Extended Data
Fig. 4e). These data further indicate that Nup159 is an autophagy
Letters
receptor and that the loss of Nup159 renders NPCs resistant to
autophagic degradation.
We also applied the Atg8–Nup159 BiFC assay in the background
of an NPC clustering mutant and observed the BiFC signal specifi-
cally at the site at which NPCs cluster even under normal growth
conditions (Fig. 4c). This signal collapsed into a dot like structure
after 24 h of nitrogen starvation, further suggesting that defective
NPCs are indeed degraded in an autophagy-dependent manner
(Fig. 4c). Finally, we introduced the nup159AIM1 mutant into our
cluster strains and found that degradation of Nups was strongly
impaired (tested using both Nup192–eGFP and Nic96–eGFP
strains) suggesting that receptor-mediated degradation of NPCs
occurs under these conditions (Fig. 4d, Extended Data Fig. 4f).
We therefore propose a model of NPC quality control, in
which the number of NPCs and/or their integrity is monitored
and controlled by the proteasome and the autophagy machinery,
respectively (Extended Data Fig. 4g). In the case of proteasomal
degradation, defective NPCs are first earmarked by the ESCRT
machinery15. In the case of autophagic degradation, it is unclear at
present whether earmarking by ESCRT is involved and at which
stage it may act. Notably, autophagic degradation of the NPC seems
to occur through a route that is distinct from piecemeal autophagy
or nucleophagy. Interestingly, autophagic degradation of the NPC is
also independent of previously characterized autophagy receptors
but, rather, involves Nup159—that is, a constitutive component of
the NPC—as an intrinsic receptor. Whether other receptors are also
involved in NPC degradation and whether Nup159 mediates the
degradation of proteins other than Nups requires further investiga-
tion, but interconnections to other autophagy pathways that act on
the nucleus31,32 seem to be likely.
Multiple lines of evidence suggest that Nup159-dependent
autophagy is specifically involved in the degradation of nuclear-
envelope-embedded NPCs rather than orphan subunits. First,
Nup159 controls the degradation of other Nups, including those
from other subcomplexes. Second, Nup159 is incorporated into
NPCs late during assembly33. Third, the interaction between Atg8
and NPCs occurs at the nuclear periphery. Finally, NPC-containing
vesicles can be visualized as degradation intermediates in the vacuole.

Fig. 3 | Nup159 is an autophagy receptor for NPC degradation. a, Purified Nup159 binds directly to Atg8 in vitro. Ni-pull-down assays were used to enrich
a Nup159–Atg8 complex, after recombinant His-tagged Atg8 (His–Atg8, 8 nM) was incubated with GST–Nup159 (at two different concentrations, 0.7 nM
and 1.4 nM) or with GST alone as a control; n = 2 biologically independent experiments. The asterisk indicates a C-terminal truncation in GST–Nup159,
which was present in the purification of GST–Nup159, that failed to associate with His–Atg8. b, AIM-dependent binding of Nup159 to Atg8. Lysates from
Nup159–6HA cells were incubated with recombinant His-tagged Atg8 or the Y49A L50A mutant (defective in AIM-dependent interactions), followed by
GST pull-down and immunoblotting using antibodies against HA; n = 2 biologically independent experiments. c, AIM1 of Nup159 is required for the
Atg8–Nup159 interaction. Schematics of the Nup159 protein domains, including AIM1 and the corresponding nup159AIM1 mutation, are shown (top)
(mutations of other putative AIM motifs are provided in Extended Data Fig. 3a). Bottom: co-IP of Nups with eGFP–Atg8 in WT or nup159AIM1 mutant cells,
followed by western blot using anti-eGFP, anti-Nup98 (multiple FG-Nups), anti-Nup84, anti-Nsp1 and anti-HA (detecting Nup159–6HA) antibodies; n = 3
biologically independent experiments. d, Degradation of scaffold Nups (Nup192–eGFP and Nup133–eGFP) in WT or nup159AIM1 mutant cells measured after
nitrogen starvation using anti-eGFP immunoblotting. Dpm1 was used as a loading control. Quantifications from n = 3 biologically independent experiments
are shown. Data are mean ± s.d. Statistical analysis was performed using two-tailed Student’s t-tests; ****P ≤ 0.0001. e, Nup159 interacts with Atg8 at the
nuclear envelope. Fluorescent micrographs of the BiFC signal arising from pADH::VN-ATG8 (N-terminal half of Venus (VN)) and NUP159-VC (C-terminal
half of Venus (VC)), or pADH::VN-ATG8 and NUP192-VC were examined before and after 24 h of nitrogen starvation; n = 2 biologically independent
experiments. Inner-ring nucleoporin Nup170–mars marks NPCs. Starvation-induced formation of VN–Atg8-, Nup159–VC- and Nup170-containing NPC
foci is indicated by arrows. The dotted box indicates the region magnified in the inset. Scale bars, 5 μm; 1 µm (inset). f, Focal accumulation of Nup192–eGFP
in the vacuoles of atg15∆ cells is dependent on Atg8–Nup159 interaction, as monitored by Nup192–eGFP staining in atg15∆ cells containing WT Nup159
or nup159AIM1. The arrows indicate autophagic bodies marked by Nup192–eGFP. Scale bar, 5 μm. Data are mean ± s.d.; n = 4 biologically independent
experiments, >250 cells were counted in each set in each replicate. Statistical anaylsis was performed using two-tailed Student’s t-tests. g, CLEM
visualization of vacuolar trapped autophagic bodies in atg15∆ cells loaded with NPC-containing nuclear vesicles. Mars-tagged Nup159 cells in which the
vacuolar lipase Atg15 was deleted were starved of nitrogen for 24 h and analysed using CLEM; n = 2 biologically independent experiments. The Nup159–mars
signal (top left), a single-plane image of an EM tomogram (top middle) and a merge of the two corresponding images (top right) are shown. Inset: an
autophagic body loaded with an NPC-containing vesicle with magnification (bottom). A manual segmentation of the corresponding tomogram is shown
(bottom right). NPCs are indicated in red; nuclear membrane in yellow; autophagosomal membrane in green; and nuclear content in cyan. Scale bars,
500 nm (top); 200 nm (bottom). Source data are available online.

Nature Cell Biology | VOL 22 | FebruarY 2020 | 159–166 | www.nature.com/naturecellbiology 163

Letters NAturE CEll Biology

This mechanism provides an elegant solution to the problem of how vesicles that we observed using CLEM. Whether this concept applies to
to control the number34,35 and integrity of nuclear-envelope-embed- organisms with open mitosis remains to be investigated in the future.
ded NPCs36 in organisms with closed mitosis, in which chromo- In this case, it would be appealing to explain NPC turnover in termi-
somes are segregated and nuclei divide without breakdown of the nally differentiated, non-dividing cells10. Notably, a very recent study
nuclear envelope. It also offers an escape route for nuclear material37, found evidence for turnover of entire NPCs in quiescent mammalian
which could become sequestered to NPCs during the vesicle-for- cells38, even though an initial experiment with autophagy inhibitors
mation step and appears to accumulate inside the NPC-containing did not provide supporting evidence for clearance by autophagy38.

a b c Unstructured
1 AIM1 1460
Ni-eluate Input GST PD
Nup159 β-Propeller FG repeats DID CC
His–Atg8 (8 nM):

GST–Atg8 Y49A L50A

GST (8 nM): 1 ...
1078 1081
YDKL ... 1460
GST–Nup159 (0.7 nM): AIM1
Nup159 β-Propeller FG repeats DID CC
GST–Nup159 (1.4 nM):
(kDa)

GST–Atg8
1078 1081
250 IP : eGFP Input ... ADKA ...
GST–
150
Nup159 AIM

Input

GST

1
100

1
IM

I M
59 A

59 A
T d

W gge
W gge
75 (kDa)
*

p1

p1
a

nt
nt

T
nu

nu
250

U
U
Nup159–6HA eGFP–Atg8:
50
(kDa)
250 Nup159–6HA
37 37 GST–Atg8
150
Nup116
25 GST 25 GST
75 Nup145N
20
His–Atg8 Coomassie Nup57
15
50 Nup49
Coomassie
100 Nsp1

75 Nup84
d Inner ring complex Y-complex
eGFP–Atg8
37
SD-N (h): 0 24 SD-N (h): 0 24
Pgk1
37
1

1
M

M
AI

AI

AI

AI
9

9
15

15

15

15

Split Venus
e
p

p
T

T
nu

nu

nu

nu
W

(kDa) DIC Nup170–mars Nup159–VC VN–Atg8 Merge

(kDa)
250 Nup192–eGFP 250
150 Nup133–eGFP
150
0 h SD-N

100 100
75 75

50 50
37 37
24 h SD-N

eGFP′ 25 eGFP′
25

25 Dpm1 25 Dpm1

1.2 1.2 Split Venus

eGFP′/Dpm1 (fold change)

DIC Nup170–mars Nup192–VC VN–Atg8 Merge

eGFP′/Dpm1 (fold change)

1.0 1.0
0.0001
0.0001

0.8 0.8
0 h SD-N

0.6 VI 0.6 VI
P
P

0.4 0.4
**** ****
0.2 0.2

0 0
1

24 h SD-N
M
T

M
T
AI

AI
W

W
9

9
15

15
p

p
nu

nu

f atg15∆
WT nup159
AIM1 g Nup159–mars EM CLEM

atg15∆
Maximum intensity
Nup192–eGFP

100
z-projection

V
Nup192–eGFP foci (%)

80
Cells with vacuolar

60 N
P = 0.0006

40

20
***
0
Midsection

Inset Segmentation
1
T

M
DIC

AI
9
p 15
nu

164 Nature Cell Biology | VOL 22 | FebruarY 2020 | 159–166 | www.nature.com/naturecellbiology

NAturE CEll Biology Letters
a WT nup120∆ nup133∆ b
IP: eGFP Input

P = 0.006
P = 0.016

P = 0.018
P = 0.003

P = 0.009
P = 0.039

P = 0.008
P = 0.002
P > 0.05

P > 0.05
1.2
WT nup120∆ nup133∆

T ed
T ed

∆
nt ∆
Nup192–eGFP/Dpm1
1.0

20
20

W gg
W gg
SD-N (h): 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8
*

p1
p1
*

a
a
nt
(kDa)

(fold change)

nu
nu
0.8

U
U
eGFP–Atg8:
250
Nup192–eGFP * NS * NS (kDa)
150 0.6 150 Nup116
0.4
* *
100 ** ** 75 Nup145N
0.2
75 Nup57
0 50 Nup49
50 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8
100 Nsp1
SD-N (h)
37
eGFP–Atg8
37

P = 0.007
P = 0.017
P = 0.049

P = 0.036

P = 0.029
P = 0.008
P = 0.026
P = 0.018
25 eGFP′ Pgk1
37
3.5
*

eGFP′/Dpm1 (fold change)

WT
3.0 nup120∆
25 Dpm1
nup133∆ *
2.5
**
2.0 *
1.5
*
1.0
0.5
* *
0
0 2 4 6 8 0 2 4 6 8 0 2 4 6 8
SD-N (h)

c Split Venus d nup120∆ nup159∆

YCplac111–
DIC Nup170–mars Nup159–VC VN–Atg8 Merge WT AIM1
Nup159:
SD-N (h): 0 2 4 8 16 0 2 4 8 16 nup120∆
(kDa)
nup120∆ nup159
AIM1
0 h SD-N

250 Nup192–eGFP 1.2

eGFP′/Dpm1 (fold change)

150

P = 0.028
P = 0.042
P = 0.023
P = 0.003
1.0
100
nup120∆

75 0.8

0.6
**
50
*
24 h SD-N

0.4
37 *
0.2
25 eGFP′ *
0
0 2 4 6 8 0 2 4 6 8
25 Dpm1
SD-N (h)

Fig. 4 | Selective autophagy is important for the clearance of aberrant NPCs. a, Mutations that cause NPC clustering trigger enhanced NPC degradation.
Starvation-induced degradation of Nup192–eGFP in WT, nup120∆ and nup133∆ mutant backgrounds, causing the appearance of NPC clusters27–29,
was analysed as described in Fig. 3d. The disappearance of the Nup192–eGFP band, as well as appearance of the free eGFP band, (both of which were
normalized to the loading control) was quantified. Data are mean ± s.d. of n = 3 biologically independent experiments. Statistical analysis was performed
using two-tailed Student’s t-tests; *P ≤ 0.05. b, Atg8–Nup interactions are enhanced in nup120∆ cells. Co-IP of Nups with eGFP–Atg8 in WT and nup120∆
cells; n = 2 biologically independent experiments. Immunoprecipitates were probed using anti-eGFP, anti-Nup98 (multiple FG-Nups) and anti-Nsp1
antibodies. Anti-Pgk1 antibodies were used as a control. c, Atg8 interacts with Nup159 at the site of the NPC cluster. Fluorescent micrographs of Atg8–
Nup159 BiFC assay, as described in Fig. 3e, were examined before and after nitrogen starvation for 24 h in the nup120∆ background; n = 2 biologically
independent experiments. Inner-ring nucleoporin Nup170–mars marks NPCs. Starvation-induced formation of Nup159–Atg8- and Nup170-containing foci is
indicated by arrows. The dotted box indicates the region magnified in the inset. Scale bars, 5 μm; 1 µm (inset). d, Degradation of Nup192–eGFP in the cluster
background (nup120∆) is strongly impaired in nup159AIM1-mutant cells. Starvation-induced degradation of Nup192–eGFP was analysed as described in Fig. 3d,
but in nup120∆ and nup120∆ nup159AIM1 cells. WT Nup159 or nup159AIM1-mutant proteins were expressed under endogenous promoter from centromeric
plasmid YCplac111. The appearance of the free eGFP band was quantified. Data are mean ± s.d. of n = 3 biologically independent experiments. Statistical
analysis was performed using two-tailed Student’s t-tests. Source data are available online.

Online content
Any methods, additional references, Nature Research reporting
summaries, source data, extended data, supplementary informa-
tion, acknowledgements, peer review information; details of author
contributions and competing interests; and statements of data and
code availability are available at https://doi.org/10.1038/s41556-
019-0459-2.
Received: 11 January 2019; Accepted: 17 December 2019;
Published online: 6 February 2020
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NAturE CEll Biology
Methods
Yeast strains and plasmids. A list of budding yeast (S. cerevisiae) strains and
plasmids used in this study is provided in Supplementary Tables 1 and 2,
respectively. All of the yeast strains were based on the DF5 background.
Yeast cell culture, starvation, Nup degradation and eGFP cleavage assay and
cloning. Standard protocols for transformation, mating, sporulation and tetrad
dissection were used for yeast manipulations39. Yeast cultures were inoculated from
overnight cultures and grown using standard growth conditions and medium40.
Cells were cultured at 30 °C in YPD medium (1% yeast extract, 2% peptone and 2%
glucose) unless indicated otherwise. For the Nup-degradation and eGFP-cleavage
assays, cells were grown to mid-log phase (optical density at 600 nm (OD600) of
1.0) then switched to SD-N medium (synthetic minimal medium lacking nitrogen;
0.17% YNB without amino acids and ammonium sulfate, and supplemented
with 2% glucose) and incubated for the indicated time. Alternatively, cells were
treated with either 80 µg ml−1 cycloheximide or 12 nM rapamycin for the indicated
time. Autophagy substrate proteins were tagged at the endogenous chromosomal
location with eGFP and their vacuolar degradation after starvation was monitored
by accumulation of the released eGFP moiety, which is largely resistant to vacuolar
degradation. Chromosomally tagged strains and knockout strains were constructed
using a PCR-based integration strategy41–43. Standard cloning and site-directed
mutagenesis techniques were used.
Immunoblot techniques. We collected 3 × 107 cells (OD 1.5) for the indicated time
points and total cell protein extracts were obtained using alkaline lysis (2 M NaOH
and 7.5% (v/v) 2-mercaptoethanol for 15 min on ice)41 followed by trichloroacetic
acid precipitation (to a final concentration of 22% for 10 min on ice). Protein pellets
were collected by centrifugation (14,000 r.p.m., 20 min), solubilized in HU loading
buffer (8 M urea, 5% SDS, 200 mM Tris-HCl pH 6.8, 20 mM dithiothreitol (DTT)
and bromophenol blue 1.5 mM) and disrupted by vortexing with an equal volume of
acid-washed glass beads (425−600 µm) for 6 min. The samples were then incubated
at 65 °C (1,400 r.p.m.) for 10 min. Proteins were separated using NuPAGE 4–12%
gradient gels (Invitrogen), transferred onto polyvinylidene fluoride membranes
(Immobilon-P) and then analysed using specific antibodies (see the ‘Antibodies’
section). To calculate degradation of eGFP-fused protein levels (%), the intensity of
the full-length eGFP-fused protein was normalized to the intensity of the respective
loading control, and shown as relative to WT cells at 0 h. To calculate degradation
of eGFP′ levels (fold change), the intensity of the free eGFP′ was divided by the
intensity of the Dpm1 loading control; values are shown relative to that in WT cells
(always normalized to the longest starvation time point of the WT cells).
Co-IP of Nups. Yeast lysates were prepared by cell disruption using a multitube
bead beater (MM301, Retsch) in lysis buffer (100 mM HEPES pH 7.4, 150 mM
NaCl, 1% NP-40, 10% glycerol, 50 mM NaF, 2 mM phenylmethylsulfonyl fluoride
and EDTA-free protease inhibitor cocktail (cOmplete Tablets, Roche)) with
zirconia/silica beads. The extracts were cleared by centrifugation at 8,000g
for 10 min and the supernatants were incubated with GFP-Trap_A matrix
(ChromoTek) or Ni-NTA agarose beads (Qiagen) for 2 h with head-over-tail
rotation at 4 °C and then washed five times using lysis buffer to remove non-
specific background binding. Bound proteins were eluted by adding HU loading
buffer and were incubated at 65 °C for 10 min.
Atg8 interactome and MS. To analyse the Atg8 interactome after nitrogen
starvation, yeast cells were grown in 200 ml YPD medium. At OD 1, cells were
shifted to SD-N medium and incubated for 16 h at 30 °C. Yeast cells were collected
by centrifugation and yeast lysates were prepared by cell disruption on a multitube
bead beater (MM301, Retsch) in yeast lysis buffer (20 mM HEPES pH 7.5, 150 mM
potassium acetate, 1% NP-40, 5% glycerol, 10 mM N-ethylmaleimide, 1 mg ml−1
Pefabloc SC (Roche) and EDTA-free protease inhibitor cocktail (cOmplete Tablets,
Roche)) with zirconia/silica beads. The extracts were cleared using centrifugation
at 2,000g for 10 min. The supernatants were incubated with GFP-Trap_M matrix
(ChromoTek) for 1 h on a rotary wheel at 4 °C. Magnetic beads were washed twice
with yeast lysis buffer and four times with washing buffer (50 mM Tris pH 7.5,
150 mM NaCl, 1 mg ml−1 Pefabloc SC (Roche) and EDTA-free protease inhibitor
cocktail (cOmplete Tablets, Roche)) to remove any residual detergent. The
supernatants were removed and beads were incubated with a buffer containing
4 M urea and 20 mM DTT in 25 mM Tris pH 8.0 buffer for 10 min followed by
incubation with 40 mM chloroacetamide for 20 min for alkylation of cysteines.
The sample was diluted to final concentration of 1 M urea with digestion buffer
(25 mM Tris pH 8.0) and vortexed. The sample was digestion for 2 h with 0.5 µg
of endoproteinase lysine-C (Wako chemicals) and then digested with 0.5 µg of
trypsin (Promega) overnight. The digested peptides were purified using StageTip44.
Peptides were loaded onto a 15 cm column (inner diameter, 75 μm) packed with
C18 reprosil 3 micron beads (Dr Maisch) and directly sprayed into an LTQ-
Orbitrap XL instrument operated in a data-dependent manner. Up to the top five
precursors were selected for fragmentation by CID and analysed in the iontrap.
The raw data were processed using MaxQuant45 v.1.6.0.15. Peak lists generated
were searched against a yeast open reading frame database using Andromeda
search engine45 built into Maxquant. Proteins were quantified using the MaxLFQ
Nature Cell Biology | www.nature.com/naturecellbiology
Letters
algorithm46. Analysis was performed using Perseus47 v.1.5.2.4. The MS proteomics
raw data have been deposited to the ProteomeXchange Consortium through the
PRIDE30 partner repository with the dataset identifier PXD011571.
Expression and purification of GST–Nup159, GST–Atg8, GST–Atg8(Y49A, L50A)
and His–Atg8. GST-fusion proteins and His–Atg8 were expressed in E. coli
Rosetta 2(DE3) (for GST-fusion proteins) or Rosetta 2(DE3)pLysS (for His–Atg8)
cells, respectively. Expression was induced using 1 mM IPTG in a 1 l culture of
Luria–Bertani for 20 h at 22 °C. Cells were collected using centrifugation and were
lysed in lysis buffer (GST-fusion proteins: 40 mM Tris pH 7.5, 150 mM NaCl, 5 mM
DTT, EDTA-free protease inhibitors cocktail (cOmplete Tablets, Roche), 1 mg ml−1
Pefabloc SC (Roche); His–Atg8: 40 mM Tris pH 7.5, 500 mM NaCl, 5 mM MgCl2,
5 mM 2-mercaptoethanol, 20 mM imidazole, 10% glycerol (w/v), EDTA-free
protease inhibitors cocktail (cOmplete Tablets, Roche), 1 mg ml−1 Pefabloc SC
(Roche)) using an EmulsiFlex C3 homogenizer (Avestin). DNA was digested using
SM DNase (final concentration, 75 U ml−1, 15 min on ice). Supernatant containing
soluble proteins was collected by centrifugation (20,000 r.p.m., 30 min). GST-fusion
proteins and His–Atg8 were affinity purified using Glutathione Sepharose 4 Fast Flow
(GE Healthcare) or Ni-NTA agarose (Qiagen), respectively (2 h on a rotary wheel
at 4 °C). The resins were recovered using gravity-flow chromatography. Resins were
subsequently washed three times with 25 ml lysis buffer, followed by three times with
washing buffer (GST-fusion proteins: 40 mM Tris pH 7.5, 450 mM NaCl, 5 mM DTT;
His–Atg8: 40 mM Tris pH 7.5, 500 mM NaCl, 5 mM MgCl2, 5 mM 2-mercaptoethanol,
70 mM imidazole, 10% glycerol (w/v)). Bound proteins were eluted from the
individual resin (GST-fusion proteins: 40 mM Tris pH 7.5, 150 mM NaCl, 5 mM DTT,
50 mM glutathione; His–Atg8: 40 mM Tris pH7.5, 500 mM NaCl, 5 mM MgCl2, 5 mM
2-mercaptoethanol, 270 mM imidazole, 10% glycerol (w/v)). The eluted proteins
were collected and dialysed (50 mM Tris pH 7.5, 150 mM NaCl, 20% glycerol (w/v),
overnight at 4 °C). Purified proteins were directly frozen in liquid nitrogen after
dialysis and stored in aliquots at −80 °C until further use. The identity of the different
proteins was confirmed by molecular-mass analysis using SDS–PAGE.
Atg8–Nup159 in vitro binding assay. The in vitro binding assay was performed
by incubating the indicated protein combinations in 1 ml assay buffer (50 mM Tris
pH 7.5, 150 mM NaCl, 5% glycerol (w/v), 20 mM imidazol, 0.1% Triton X-100) for
1 h at room temperature. 50 µl were used as input control and mixed with an equal
amount of HU loading buffer. The rest of the supernatant was added to 100 µl Ni-
NTA agarose (Qiagen) slurry and incubated for 2.5 h at 4 °C on a rotary wheel. The
resin was collected by centrifugation (800 r.p.m., 1 min) and washed six times with
1 ml washing buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol (w/v), 20 mM
imidazol, 1% Triton X-100). After the last washing step, the supernatant was
removed (27 gauge needle) and the proteins were eluted with 50 µl of elution buffer
(50 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol (w/v), 270 mM imidazol, 1% Triton
X-100). The eluate was transferred into a new tube (27 gauge needle), mixed with
an equal amount of HU loading buffer and analysed using SDS–PAGE. Proteins
separated by SDS–PAGE were stained with PageBlue Protein Staining Solution
(Thermo Scientific).
GST–Atg8 pull-down with cell extract. The indicated GST-fusion proteins were
incubated (125 µg) diluted in 1 ml assay buffer (50 mM Tris pH 7.5, 150 mM NaCl,
5% glycerol (w/v, 0.1% Triton X-100) and mixed with 50 µl Glutathione Sepharose
4 Fast Flow (GE Healthcare) slurry. The mixture was incubated for 2 h at 4 °C on
a rotary wheel. The yeast lysate was prepared as described above (MS, 20 µl were
kept as input control and mixed with 200 µl HU loading buffer). The Glutathione
Sepharose was collected using centrifugation (800 r.p.m., 1 min) and washed once
with washing buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol (w/v) and 1%
Triton X-100) then once with the yeast lysis buffer (20 mM HEPES pH 7.5, 150 mM
potassium acetate, 1% NP-40, 5% glycerol, 1 mg ml−1 Pefabloc SC (Roche) and
EDTA-free protease inhibitor cocktail (cOmplete Tablets, Roche)). Subsequently,
300 µl of the yeast lysate containing Nup159–6HA was mixed with 700 µl yeast
lysis buffer and added to the resin (2.5 h, 4 °C rotary wheel). The resin was washed
three times with yeast lysis buffer and finally the supernatant was removed using a
27 gauge needle. The bound proteins were denatured by adding 50 µl HU loading
buffer and incubating at 65 °C for 10 min.
Fluorescence microscopy. For fluorescence microscopy, yeast cells were grown
in synthetic growth medium supplemented with all essential amino acids and 2%
glucose. The next day, cells were diluted to OD 0.1 and grown until mid-log phase
(0.5–0.8 OD) before imaging. Microscopy slides were pretreated with 1 mg ml−1
concanavalin A solution. Widefield imaging was performed at the Imaging Facility
of the Max Planck Institute of Biochemistry (MPIB-IF) using a GE DeltaVision
Elite system comprising an Olympus IX-71 inverted microscope, an Olympus
(×100/1.40 NA UPLSAPO and ×60/1.42 NA PLAPON) objective and a PCO sCMOS
5.5 camera. The images were deconvolved using softWoRx (additive enhanced, 20
iterations). Image analysis was performed using ImageJ (https://imagej.nih.gov/ij/).
Microfluidics experiments. Microfluidics experiments were performed using
a CellASIC ONIX microfluidic platform (Merck) with the CellASIC ONIX
microfluidic plates for haploid yeast cells (Y04C-02-5PK). Cells were loaded from a
Letters
logarithmic-growing culture (OD 0.5–0.8) according to manufacturer’s manual and
imaged at 1 h intervals over a time course of 24 h. At each position for each time
point, a z-stack was recorded (z-height, 300 nm). Imaging was performed using a
constant medium flow rate of 1 psi. To rapidly switch medium, flow rate was set
to 5 psi for 5 min and then switched back to a flow rate of 1 psi. Cells were initially
cultured for 2 h in synthetic growth medium and then switched to SD-N medium
for 24 h. The microscope used is described in the ‘Fluorescence microscopy’
section. Nuclear signal was quantified using ImageJ (https://imagej.nih.gov/ij/),
complemented with all of the default plugins provided by Fiji (https://fiji.sc)
and the additional plugins provided on the ImageScience update website. Each
frame in the time-lapse videos following the variation of the signal over time was
preprocessed independently to determine the location and size of nuclei in the field
of view. Their segmentation was achieved by first applying a Laplacian of Gaussian
operation, and then using the Otsu threshold method to transform the images into
binary maps. Finally, nuclei boundaries were rounded by morphological opening.
Once the location and the extension of nuclei were determined, an interactive
script was used to select the same nuclei at different time frames and extract the
corresponding average intensity and volume.
CLEM. CLEM analysis was performed as previously described48,49. S. cerevisiae cells
were grown in YPD to OD 1.0, washed twice with SD-N medium and subsequently
resuspended in SD-N medium. After incubation in SD-N medium for 24 h, the
strain was high-pressure frozen (HPM010, AbraFluid) and freeze-substituted
(EM-AFS2, Leica) with 0.1% uranyl acetate in acetone for 72 h at −90 °C. The
temperature was then raised to −45 °C at 3 °C h−1 and the samples were incubated
for a further 5 h. After rinsing in acetone, the samples were infiltrated in Lowicryl
HM20 and the resin was polymerized under ultraviolet light. Then, 300 nm sections
were cut using a microtome (EM UC7, Leica) and placed on carbon-coated 200
mesh copper grids (S160, Plano). Fluorescence microscopy imaging of the sections
was performed as previously described48 using a widefield fluorescence microscope
(Olympus IX81) equipped with an Olympus PlanApo ×100/1.40 NA oil-immersion
objective and a CCD camera (Orca-ER; Hamamatsu Photonics). After fluorescence
imaging, grids were post-stained with uranyl acetate and lead citrate. Tilt series
of the cells of interest were acquired semi-automatically with a Tecnai F30
(Thermofisher, FEI) at 300 kV using SerialEM50. Tomograms were reconstructed
and manually segmented using IMOD51. Overlay of fluorescence spots and
tomograms was performed using the ec-CLEM Plugin52 in ICY53 by clicking
manually on corresponding pairs of notable features in the two imaging modalities.
Antibodies. Monoclonal antibodies against HA-epitope (1:1,000; clone F-7)
and eGFP (1:500; clone B-2) were purchased from Santa Cruz Biotechnology;
antibodies against Dpm1 (1:10,000; clone 5C5A7) and Pgk1 (1:15,000; clone
22C5D8) were obtained from Invitrogen; antibodies against Nsp1 (1:10,000; 32D6
catalogue ab4641) were obtained from Abcam; and antibodies against Nup98
(1:5,000; 2H10) were obtained from BioAcademia. Polyclonal antibodies against
Nup84 (1:500; SAB2501815) were obtained from Sigma. Mouse monoclonal
antibodies against β-galactosidase (1:500; Z378A) were purchased from Promega.
Statistics and reproducibility. Representative results of at least two independent
experiments were presented in all of the figure panels. P values for all graphs were
generated using two-tailed Student’s t-tests, as indicated in the figure legends; NS,
P ≥ 0.05; *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.001, ****P ≤ 0.0001. For all error bars, data
are mean ± s.d.
Reporting Summary. Further information on research design is available in the
Nature Research Reporting Summary linked to this article.
Data availability
Yeast strains and plasmids are available on request. The mass spectrometry
proteomics raw data have been deposited to the ProteomeXchange Consortium
through the PRIDE partner repository with the dataset identifier PXD011571.
All other data supporting the findings of this study are available from the
corresponding authors on reasonable request.
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Acknowledgements
We thank J. Sun (intern in the EMCF at EMBL) for tomogram manual segmentation
and N. Romanov for MS analysis; staff at the MPIB Imaging Facility, in particular
G. Cardone and M. Spitaler, for help with image analysis; W. Baumeister, J. Buchner,
F. U. Hartl, M. Hayer-Hartl, B. Schulman and B. Engel, and members of the Jentsch,
Beck and Pfander laboratories for discussions and comments on the manuscript; and
N. Nagaraj and S. Uebel of the Biochemistry Core Facility of the Max Planck Institute
of Biochemistry for MS analysis. The B.P. laboratory acknowledges funding by the Max
Planck Society and Deutsche Forschungsgemeinschaft. M.B. acknowledges funding
by the EMBL. Research in the S.J. laboratory was supported by Max Planck Society,
Deutsche Forschungsgemeinschaft, Center for Integrated Protein Science Munich,
Louis-Jeantet Foundation and a European Research Council (ERC) Advanced Grant.
F.W. was supported by an EMBO Long-Term Fellowship ALTF 764-2014. M.A. was
funded by an EMBO Long-Term Fellowship ALTF-1389–2016.
Author contributions
C.-W.L., F.W., S.J., M.B. and B.P. conceived the study. C.-W.L., F.W., S.J., M.B. and B.P.
designed experiments. P.R. performed the CLEM experiments. M.A. helped with the
experimental design for CLEM. S.M. performed the microscopy and GFP-cleavage assay
in the mammalian system that was not included in the current manuscript. C.-W.L.
and F.W. performed all of the other experiments. C.-W.L., F.W., M.A., S.J., M.B. and B.P.
analysed the data. C.-W.L., F.W., M.B. and B.P. wrote the manuscript.
Competing interests
The authors declare no competing interests.
Additional information
Extended data is available for this paper at https://doi.org/10.1038/s41556-019-0459-2.
Supplementary information is available for this paper at https://doi.org/10.1038/
s41556-019-0459-2.
Correspondence and requests for materials should be addressed to M.B. or B.P.
Reprints and permissions information is available at www.nature.com/reprints.
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Extended Data Fig. 1 | see figure caption on next page.

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Extended Data Fig. 1 | NPC scaffold proteins are degraded by both autophagy and the proteasome. a, Schematic representation of NPC architecture
in a cross-section view of the NPC. The central core of the NPC consists of one inner-ring complex (magenta) flanked on both sides by the outer NPC
scaffold (orange, consistent of Y complex and the heteromeric Nic96), together forming the NPC scaffold. Peripheral nucleoporins constitute cytoplasmic
filaments (dark brown) and the nuclear basket (navy blue). The permeability barrier is established by FG-nucleoporins (green). Three pore membrane
proteins (Poms; light brown) anchor the NPC scaffold at the outer nuclear membrane (ONM) and inner nuclear membrane (INM) joint. b, Degradation of
scaffold nucleoporins (Nup192–eGFP and Nup133–eGFP) was measured over 24 h after cycloheximide block (CHX; 80 μg ml–1) of translation by anti-eGFP
immunoblotting. Dpm1 serves as control. Quantifications from n=3 biologically independent experiments are shown as mean ± s.d.. c, Nup192–eGFP-
tagged cells were subjected to nitrogen starvation (SD-N), glucose starvation (YP medium lacking glucose), or rapamycin treatment (12 nM) for indicated
time (n=2 biologically independent experiments). The Nup192–eGFP western blot illustrates that nitrogen starvation or rapamycin treatment, but not
glucose starvation-induces a drop in Nup192–eGFP levels. Dpm1 serves as control. d, Degradation of Nup192–eGFP after nitrogen starvation as in Fig. 1c,
but in different ubiquitin-proteasome receptor mutants (rpn10∆, rpn13∆, rad23∆, dsk2∆, rad23∆ dsk2∆) (n=2 biologically independent experiments).
e, Starvation-induced Nup192–eGFP degradation was measured as in Fig. 1d, but in atg7∆, atg8∆, atg1∆, atg6∆, atg3∆ and atg4∆ cells deficient in the core
autophagy machinery (n=2 biologically independent experiments). Autophagy-deficient cells do not show appearance of the free eGFP band and lead
to stabilization of Nup192–eGFP. f, g Starvation-induced Nup192–eGFP degradation (f) was measured in wildtype and mutants cells, deficient in ESCRT
(vps4∆) or proteasome (cim3-1) as in Fig. 1c. (g) Atg8-degradation and vacuolar cleavage of eGFP-Atg8 was measured by immunoblotting in wildtype,
vps4∆, and cim3-1 mutant cells before and 2, 4, 8 h after induction of autophagy by nitrogen starvation. Quantifications from n=3 biologically independent
experiments are shown as mean ± s.d., **p ≤ 0.005, ***p ≤ 0.001, ****p ≤ 0.0001, two-tailed Student’s t-tests. Data available in Statistical Source Data
Extended Data Figure 1 and Unprocessed Blots Extended Data Figure 1.

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Extended Data Fig. 2 | see figure caption on next page.

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Letters NAturE CEll Biology
Extended Data Fig. 2 | The core autophagy machinery is important for NPC degradation in contrast to other previously characterized selective
autophagy pathways. a, Quantitative analysis of Nup192–eGFP fluorescence signal and nuclear volume before and after nitrogen starvation. Wildtype
or atg8∆ mutant cells expressing Nup192-eGFP were trapped in a microfluidic device and incubated for two hours with regular growth media before the
buffer stream was changed and cells were incubated with medium lacking any nitrogen source (SD-N) for 24 hours. Every hour a z-stack for the Nup192-
eGFP signal was recorded. The nuclear volume and the Nup192-eGFP intensity within this volume were followed in individual cells (n=100 individual
cells for each condition) before and after 24 hours of SD-N treatment. ****p ≤ 0.0001, two-tailed Student’s t-tests. b, eGFP-Atg8 interacting proteins
were measured by eGFP-Atg8 Co-IP as in Fig. 2c, with eGFP-Atg8 under control of the ADH promoter. For this analysis, cells with tagged Nup159-6HA
(upper right panel), Pom152-6HA (bottom right panel) or Nup192-6HA (left panel) were subjected to immunoprecipitation with GFP-Trap matrix
(n=2 biologically independent experiments). Immunoprecipitates were probed with antibodies directed against eGFP, HA (labeling Nup192-6HA and
Nup159-6HA, respectively), Nup84, Nup98 (multiple FG-nucleoporins) and Nsp1. Pgk1 serves as control. c, Fluorescent images of cells with Nup192-
eGFP-containing ATG8-dependent structures (marked by arrows) inside the vacuole after 24 h of nitrogen starvation (n=2 biologically independent
experiments). ATG15 was deleted in order to block vacuolar digestion of autophagic bodies16. Deletion of ATG8, but not NVJ1 abolished accumulation of
Nup192-containing structures inside the vacuole. Vph1-mars was used as marker of the vacuolar membrane. Scale bar, 5 μm. d, Degradation of Nup192-
eGFP after induction of autophagy by nitrogen starvation (24 h) was measured by immunoblotting against eGFP in wildtype and nvj1∆ mutant cells
as in Fig. 1c. Quantifications from n=3 biologically independent experiments are shown as mean ± s.d., NS: P ≥ 0.05, two-tailed Student’s t-tests.
e, Degradation of Nup192-eGFP was measured by immunoblotting against eGFP in wildtype, atg39∆, atg40∆, and atg39∆ atg40∆ mutant cells before and
after induction of autophagy by nitrogen starvation (24 h) (n=3 biologically independent experiments). f, Degradation of Nup192-eGFP was measured by
immunoblotting against eGFP in wildtype, and cue5∆ mutant cells before and after induction of autophagy by nitrogen starvation (24 h). g, h, Degradation
of Nup192-eGFP involves the Atg11 scaffold. (g) Degradation of Nup192-eGFP was measured by immunoblotting against eGFP in wildtype, atg11∆, and
atg19∆ mutant cells before and after induction of autophagy by nitrogen starvation (24 h). Depicted are mean ± s.d. from n=3 biologically independent
experiments, NS: P ≥ 0.05, ***p ≤ 0.001, two-tailed Student’s t-tests. (h) Degradation of Nup159-eGFP was measured by immunoblotting against eGFP
in wildtype and atg11∆ mutant cells before and after induction of autophagy by nitrogen starvation (24 h). Depicted are mean ± s.d. from n=3 biologically
independent experiments, ****p ≤ 0.0001, two-tailed Student’s t-tests. Data available in Statistical Source Data Extended Data Figure 2 and Unprocessed
Blots Extended Data Figure 2.

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Extended Data Fig. 3 | Nup159 is an AIM-dependent Atg8 receptor. a, Depiction of Nup159 protein domains including the AIMs and the corresponding
AIM mutations. Co-immunoprecipitation of eGFP-Atg8 with wildtype Nup159-6HA or different nup159AIM mutants, followed by Western blot with
the indicated antibodies (n=2 biologically independent experiments). Schematic overview of Nup159 domain architecture with the potential Atg8-
interacting motifs (AIM) depicted. b, Atg8 turnover is normal in nup159AIM1 cells. Degradation of eGFP-Atg8 measured by immunoblotting against eGFP
in wildtype cells or nup159AIM1 mutant cells. Quantifications from (n=3) experiments are shown as mean ± s.d.. c, Nup159 interacts with Atg11 in vivo.
Co-immunoprecipitation of Nup159-6HA with eGFP-Atg11 expressed under the control of the ADH promoter in wildtype cells (n=2 biologically
independent experiments). Immunoprecipitates were probed with anti-eGFP and anti-HA antibodies. Anti-Pgk1 serves as control. d, Mlp1 does not interact
with Atg8 at the nuclear envelope. Fluorescent micrographs of the BiFC signal arising from pADH::VN-ATG8 with MLP1-VC are examined before and after
24 h of nitrogen starvation (n=2 biologically independent experiments). Inner-ring nucleoporin Nup170-mars marks fully assembled NPCs. Scale bar, 5
μm. e, The VN-Atg8 Y49A, L50A mutant does not interact with Nup159-VC at the nuclear envelope. Fluorescent micrographs of the BiFC signal arising
from pADH::VN-ATG8 or pADH::VN-ATG8 Y49A, L50A mutant with NUP159-VC are examined at normal growth condition (n=2 biologically independent
experiments). Inner-ring nucleoporin Nup170-mars marks fully assembled NPCs. Scale bar, 5 μm. f, NPC-containing nuclear vesicles visualized by CLEM.
Additional examples of CLEM visualized vacuolar trapped autophagic bodies in atg15∆ cells loaded with NPC-containing nuclear vesicles (as in Fig. 3g).
Scale bar, 500 nm and 200 nm for inset. Data available in Statistical Source Data Extended Data Figure 3 and Unprocessed Blots Extended Data Figure 3.

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Extended Data Fig. 4 | see figure caption on next page.

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NAturE CEll Biology Letters
Extended Data Fig. 4 | Nup159-dependent autophagy is important for clearing aberrant, clustered NPCs. a, Nic96-eGFP degradation in wildtype and
nup120∆ cells was induced by nitrogen starvation for indicated time. Measurement and quantification of free eGFP band was done as in Fig. 3d. The ratio
of eGFP’/Dpm1 was normalized to 16 hours SD-N time point in wildtype. Dpm1 served as a control and was used for normalization (right). Depicted
are mean ± s.d. from n=3 biologically independent experiments, *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.001, two-tailed Student’s t-tests. b, Degradation of
Nup188-eGFP in Nup116-deficient cells (nup116∆) is strongly impaired. Starvation-induced degradation of Nup188-eGFP in nup116∆ mutant background,
was analysed as in Fig. 1a. Dpm1 served as a control and was used for normalization (right). Depicted are mean ± s.d. from n=3 biologically independent
experiments normalized against the 24 hours SD-N time point in wildtype, *p ≤ 0.05, **p ≤ 0.005, ****p ≤ 0.0001, two-tailed Student’s t-tests.
c, Degradation of Nup133-eGFP in Nup116-deficient cells (nup116∆) is strongly impaired. Starvation-induced degradation of Nup133-eGFP in nup116∆
mutant background, was analysed as in Fig. 1a. Dpm1 served as a control and was used for normalization (right). Depicted are mean ± s.d. from n=3
biologically independent experiments, *p ≤ 0.05, ***p ≤ 0.001, two-tailed Student’s t-tests. d, eGFP-Atg8 interacting proteins were measured by eGFP-
Atg8 Co-IP as in Fig. 2c, for wildtype or nup116∆ mutant cells. For this analysis, cells with tagged Nup159-6HA, were subjected to immunoprecipitation
of eGFP-Atg8 with GFP-Trap matrix (n=2 biologically independent experiments). Immunoprecipitates were probed with antibodies against eGFP tag,
HA tag, Nup84, Nup98 (multiple FG-nucleoporins), Nsp1 and Pgk1 as control. e, Nup159 does not interact with NPC in nup116∆ cells at non-permissive
temperature. Cells with tagged Nup159-6HA and Nup84-eGFP, were subjected to immunoprecipitation with GFP-Trap matrix and probed with antibodies
against eGFP tag, HA tag, Nup98 (multiple FG-nucleoporins) and Pgk1 as control (n=2 biologically independent experiments). f, Degradation of
Nic96-eGFP in the cluster background (nup120∆) is impaired in nup159AIM1 mutant cells. Starvation-induced degradation of Nic96-eGFP in nup120∆ nup159∆
mutant background complemented with wildtype NUP159 or the nup159AIM1 mutant, was analysed as in Fig. 3d. Dpm1 served as a control and was used
for normalization (right). Depicted are mean ± s.d. from n=3 biologically independent experiments, *p ≤ 0.05, ***p ≤ 0.001, two-tailed Student’s t-tests.
g, Hypothetical cartoon model of NPC turnover by selective autophagy. A fraction of fully assembled NPCs (I) clusters upon nitrogen starvation or even
more dramatic upon genetic perturbation (nup120∆) (II). This leads to interaction of the intrinsic receptor Nup159 with Atg8 and packaging of nuclear
vesicles into autophagosomes (III), which our data predicts as cellular intermediate. These autophagosomes will subsequently fuse with the vacuole
for degradation (IV). Color-code of NPC is analogue to the NPC model (Extended Data Fig. 1a). Data available in Statistical Source Data Extended Data
Figure 4 and Unprocessed Blots Extended Data Figure 4.

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