European Journal of Cell Biology 97 (2018) 523–532

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European Journal of Cell Biology

journal homepage: www.elsevier.com/locate/ejcb

Research paper

ATG16 mediates the autophagic degradation of the 19S proteasomal T

subunits PSMD1 and PSMD2
Qiuhong Xionga,b, Sarah Fischera, Malte Karowa, Rolf Müllera, Susanne Meßlinga,
Ludwig Eichingera,
⁎

a
Center for Biochemistry, Medical Faculty, University of Cologne, Joseph-Stelzmann-Str. 52, 50931 Cologne, Germany.
b
Institute of Biomedical Sciences, Shanxi University, No. 92 Wucheng Road, 030006 Taiyuan, Shanxi, China

ARTICLE INFO ABSTRACT

Keywords: Autophagy and the ubiquitin proteasome system are the two major cellular processes for protein and organelle
Autophagy recycling and clearance in eukaryotic cells. Evidence is accumulating that these two pathways are interrelated
Proteasome through adaptor proteins. Here, we found that PSMD1 and PSMD2, both components of the 19S regulatory
Dictyostelium particle of the proteasome, directly interact with Dictyostelium discoideum autophagy 16 (ATG16), a core au-
UPS
tophagosomal protein. ATG16 is composed of an N-terminal domain, which is responsible for homo-dimerization
ATG16
PSMD1
and binding to ATG5 and a C-terminal β-propeller structure. Deletion analysis of ATG16 showed that the N-
PSMD2 terminal half of ATG16 interacted directly only with PSMD1, while the C-terminal half interacted with both,
PSMD1 and PSMD2. RFP-tagged PSMD1 as well as PSMD2 were enriched in large puncta, reminiscent of au-
tophagosomes, in wild-type cells. These puncta were absent in atg16‾ and atg9‾/16‾ cells and weaker and less
frequent in atg9‾ cells, showing that ATG16 was crucial and the autophagic process important for their for-
mation. Co-expression of ATG16-GFP or GFP-ATG8a(LC3) with RFP-PSMD1 or RFP-PSMD2, respectively, in
atg16‾ or wild-type cells revealed many instances of co-localization, suggesting that RFP-PSMD1 or RFP-PSMD2
positive puncta constitute autophagosomes. LysoTracker® labeling and a proteolytic cleavage assay confirmed
that PSMD1 and PSMD2 were present in lysosomes in wild-type cells. In vivo, ATG16 is required for their en-
richment in ATG8a positive puncta, which mature into autolysosomes. We propose that ATG16 links autophagy
and the ubiquitin proteasome system.

1. Introduction proteasome and, vice versa, proteasomal subunits were found to be

Cell homeostasis is maintained by a precisely regulated balance
between synthesis and degradation of cellular components. Autophagy
and the ubiquitin proteasome system (UPS) are the two major routes for
protein and organelle clearance in eukaryotic cells. In general, short-
lived, abnormal or damaged proteins are degraded by the UPS, while
most of the long-lived proteins, protein aggregates as well as cellular
organelles (mitochondria, peroxisomes, ribosomes, and infectious or-
ganisms) are cleared by autophagy (Kim et al., 2011; Schreiber and
Peter, 2014). There is increasing evidence that the two degradative
pathways, which are of utmost importance for the recycling of cellular
material, are interconnected (Driscoll and Chowdhury, 2012;
Korolchuk et al., 2009, 2010). For example, α-synuclein and other ag-
gregation-prone proteins are substrates of both pathways (Ravikumar
et al., 2002; Webb et al., 2003). Furthermore, the autophagosomal
marker protein ATG8 (LC3 in mammals) can be degraded by the 20S
degraded by lysosomes (Cuervo et al., 1995; Gao et al., 2010). More
recently proteaphagy, i.e. degradation of whole proteasomes by au-
tophagy, was reported in HeLa cells, the mouse-ear cress Arabidopsis
thaliana and in yeast (Cohen-Kaplan et al., 2016; Marshall et al., 2015,
2016). The autophagy adaptor protein p62/SQSTM1 delivers the ubi-
quitinated proteasome to the autophagy machinery through binding to
LC3 in mammals (Cohen-Kaplan et al., 2016). In A. thaliana the 19S
proteasomal subunit RPN10 mediates selective autophagic degradation
of the 26S proteasome (Marshall et al., 2015) and in yeast the autop-
hagic turnover of inactive 26S proteasomes is directed by the ubiquitin
receptor Cue5 and Hsp42 (Marshall et al., 2016).
The core autophagy protein ATG16 is a component of the tetrameric
ATG12-ATG5/ATG16 complex which consists of two ATG12-ATG5
conjugates bound to an ATG16 homodimer and is essential for autop-
hagosome formation (Kuma et al., 2002; Mizushima et al., 2003, 1999;
Noda and Inagaki, 2015). In the early phase of autophagosome

⁎
Corresponding author.
E-mail address: ludwig.eichinger@uni-koeln.de (L. Eichinger).

https://doi.org/10.1016/j.ejcb.2018.09.002
Received 27 February 2018; Received in revised form 6 August 2018; Accepted 10 September 2018
0171-9335/ © 2018 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Q. Xiong et al. European Journal of Cell Biology 97 (2018) 523–532

generation the ATG12-ATG5/ATG16 complex mainly resides on the

outer side of the phagophore in yeast or the isolation membrane in
higher eukaryotes and catalyzes the covalent linkage of ATG8/LC3 to
the phospholipid phosphatidylethanolamine (PE). In this complex, the
ATG12-ATG5 conjugate holds the catalytic activity while ATG16 is
required for the correct localization of the complex to the isolation
membrane (Fujita et al., 2008; Hanada et al., 2007; Suzuki et al., 2001).
Dictyostelium ATG16 is highly similar to the mammalian orthologue
ATG16L1 and is composed of 3 distinct regions: the N-terminal domain
which is responsible for binding to ATG5, followed by a coiled coil
domain (CCD) which mediates homo-dimerization and seven WD40
repeats in the C-terminal half of the protein, which are predicted to
form a β-propeller structure and are lacking in the yeast protein. The β-
propeller structure is found in a wide range of proteins involved in
distinct biological activities and generally acts as a protein-protein in-
teraction platform (Neer et al., 1994; Smith et al., 1999). In mammalian
ATG16L1 the β-propeller has been identified as an interface for the
transmembrane protein TMEM59 and Nod1 and Nod2, members of the
Nod-like receptor family, which mediate the autophagic response to
bacterial pathogens by recruiting ATG16L1 to the bacterial entry site
(Boada-Romero et al., 2013; Travassos et al., 2010). Furthermore, a
large subset of WD40 repeats was defined as a new class of ubiquitin-
binding domains that interact with ubiquitin in a similar fashion
(Pashkova et al., 2010). Along this line it was found that ubiquitin is
recognized by the autophagic machinery during infection of HeLa cells
with Salmonella typhimurium through adaptor proteins, including a di-
rect interaction between ubiquitin and Atg16L1 (Fujita et al., 2013).
ATG16 also appears to play a role in the link between autophagy
and the UPS. It is indirectly involved in Cullin-3-mediated ubiquitina-
tion of p62 followed by proteasomal degradation because in its absence
the E3 ubiquitin ligase Cullin-3 could not be neddylated which resulted
in the accumulation of p62 (Lee et al., 2012). In addition, the half-life
and cellular concentration of ATG16 itself is possibly regulated by the
UPS, since treatment of MEFs with proteasome inhibitors led to an in-
crease of ATG16 (Fujita et al., 2009). On the other hand, ATG16 is also
crucial for UPS function because the Dictyostelium atg16 knock-out
mutant displayed strongly reduced proteasomal activity (Xiong et al.,
2015).
The social amoeba D. discoideum is a well-established model or-
ganism for the investigation of the autophagic process (Mesquita et al.,
2017). In this organism novel conserved autophagy genes have been
discovered and the analysis of single or double knock-outs of core au-
tophagy genes revealed informative phenotypes (Calvo-Garrido and
Escalante, 2010; Calvo-Garrido et al., 2014; Messling et al., 2017;
Munoz-Braceras et al., 2015; Otto et al., 2004; Tung et al., 2010; Xiong
et al., 2015). Here, we identified PSMD1 and PSMD2, both components
of the 19S regulatory particle of the proteasome, as ATG16-interacting
proteins. We show that PSMD1 and PSMD2 are degraded in lysosomes
and that ATG16 has an essential function in this process. Our results
reveal a novel role for ATG16 in the crosstalk between autophagy and
the UPS.

2. Results
(caption on next page)
2.1. PSMD1 and PSMD2 interact with ATG16

To identify novel ATG16 binding proteins we incubated cell lysates

from Dictyostelium AX2 wild-type cells with recombinant GST-ATG16
followed by GST pull-down. Mass spectrometric analysis revealed a
number of proteins that specifically interacted with GST-ATG16, among
them were PSMD1 with 108 kDa and PSMD2 with 90 kDa (Fig. 1A).
PSMD1 and PSMD2 are two subunits of the regulatory 19S particle of
the proteasome. Pairwise alignments of the protein sequences of PSMD1
and PSMD2 with the corresponding orthologues of human and yeast
showed significantly higher sequence identity of Dictyostelium PSMD1
and PSMD2 with the human orthologues (Table 1).

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Q. Xiong et al.
Fig. 1. Identification and verification of PSMD1 and PSMD2 as new binding
partners of ATG16. (A) GST-ATG16 pulled down PSMD1 and PSMD2.
Recombinant GST-ATG16 was incubated with total cell lysates from AX2 cells
followed by GST pull-down assays. Precipitated proteins were separated by
SDS-PAGE and stained with Coomassie Brilliant Blue (CBB). Specific protein
bands were cut out and the identity of the proteins determined by mass spec-
trometry. The positions of PSMD1, PSMD2 and GST-ATG16 are marked by
black arrowheads. (B) GST-PSMD1 and GST-PSMD2 pulled down ATG16-GFP.
Recombinant GST-PSMD1 or GST-PSMD2 was incubated with total cell lysates
from atg16‾/atg16-gfp cells followed by GST pull-down assays. ATG16-GFP was
detected with a GFP specific antibody. Upper part, Western Blot; lower part
PonceauS stain. (C) ATG16 interacted with PSMD1 and PSMD2 in vivo. Total
cell lysates of atg16‾/atg16-gfp cells expressing RFP, RFP-PSMD1 or RFP-
PSMD2 were incubated with GFP-Trap®_A beads. Precipitated proteins were
separated by SDS-PAGE, transferred by Western blotting and ATG16-GFP, RFP-
PSMD1 and RFP-PSMD2 were detected by GFP or RFP specific antibodies. (*):
Degradation product of RFP-PSMD1.
Table 1
Protein sequence identities of human, yeast and Dictyostelium PSMD1 and 2.
Dictyostelium Yeast
PSMD1 Human 46 41
Dictyostelium 38
PSMD2 Human 51 42
Dictyostelium 35
Sequence identity was determined by aligning the corresponding protein se-
quences using the alignment program (bl2seq) at the NCBI (http://www.ncbi.
nlm.nih.gov/blast/Blast.cgi). Percentage values of sequence identities are
given.
We verified the interaction between ATG16 and the two protea-
somal subunits by utilizing recombinant GST-PSMD1 and GST-PSMD2
in pull-down assays with cell lysates from atg16‾/atg16-gfp cells. In
these assays both proteins, GST-PSMD1 and GST-PSMD2, could pull
down ATG16-GFP (Fig. 1B). We next ectopically expressed either RFP-
PSMD1 or RFP-PSMD2 in atg16‾/atg16-gfp cells, respectively. Im-
munoprecipitation (IP) experiments with GFP-Trap®A beads showed
that RFP-PSMD1 as well as RFP-PSMD2 could be immune-precipitated
by ATG16-GFP (Fig. 1C). These results suggest that PSMD1 and PSMD2
are interacting with ATG16 in vivo.
2.2. The interaction of ATG16 with PSMD1 and PSMD2 is direct
We next wanted to determine, if ATG16 directly interacts with
PSMD1 and PSMD2 and, if this is the case, which region of ATG16 is
responsible for the interaction. For this purpose we generated and
purified a series of GST tagged ATG16 fragments. These were, based on
the ATG16 domain structure, ATG161−309, the N-terminal half,
ATG16310–612, the C-terminal half with the seven WD-repeats,
ATG161–198, which contains the ATG5-interacting motif (AFIM) and the
coiled coil domain (CCD) and ATG16160–312, which contains the CCD
and the region between the CCD and the WD-repeats (Fig. 2A). In pull-
down assays we found that the N- and C-terminal halves of ATG16,
ATG161−309 and ATG16310–612, respectively, could both pull down
PSMD1 but only ATG16310-612 was able to also pull down PSMD2
(Fig. 2B). Further truncation analysis of the interaction of the N-term-
inal half of ATG16 with PSMD1 revealed that ATG161-198 but not
ATG16160-312 was able to pull down PSMD1 (Fig. 2C). We next gener-
ated constructs for the expression of ATG161−309-GFP, ATG16310-612-
GFP and ATG16156–612-GFP in Dictyostelium atg16‾ cells (Fig. 2A). Un-
fortunately, we were not able to obtain an ATG16310-612-GFP expressing
atg16‾ strain, therefore, we could only analyze the interaction of
ATG161−309-GFP and ATG16156–612-GFP with PSMD1 and PSMD2, re-
spectively. Consistent with the in vitro interaction studies we found that
ATG16156–612-GFP was pulled down by both proteins, GST-PSMD1 and
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European Journal of Cell Biology 97 (2018) 523–532
GST-PSMD2, while ATG161−309-GFP was only pulled down by GST-
PSMD1 (Fig. S1). Taken together, these results show that the C-terminal
half of ATG16, which harbors the seven WD repeats, can bind directly
to both proteasomal subunits, PSMD1 and PSMD2, while the N-terminal
half of ATG16 only interacts directly with PSMD1.
2.3. PSMD1 and PSMD2 puncta co-localize with ATG16 and ATG8a
To investigate possible co-localizations of PSMD1 or PSMD2 with
ATG16 and ATG8a (LC3) in vivo, we expressed either RFP-PSMD1 or
RFP-PSMD2, respectively, in atg16‾/atg16-gfp and AX2/gfp-atg8a cells
(Matthias et al., 2016; Xiong et al., 2015). Immunofluorescence analysis
was performed after treatment of the cells with NH4Cl for 4 h to slow
down the autophagic flux. We found that RFP-PSMD1 and RFP-PSMD2
were enriched in punctate structures in atg16‾/atg16-gfp cells and
partially co-localized with ATG16-GFP (Fig. 3A, B). RFP-PSMD1 and
RFP-PSMD2 positive punctate structures were only observed in
NH4Cl–treated but not in untreated AX2 cells. Since ATG16 belongs to
the core autophagy genes and associates with the isolation membrane
in the early phase of autophagosome biogenesis, we considered the
possibility that the PSMD1 and PSMD2 positive puncta might be au-
tophagosomes. To address this question, we analyzed the co-localiza-
tion of the autophagosomal marker GFP-ATG8a with RFP-PSMD1 and
RFP-PSMD2 in AX2 wild-type cells in vivo. Immunofluorescence ana-
lysis revealed many instances of partial co-localization of GFP-ATG8a
with RFP-PSMD1 or RFP-PSMD2. In addition, we found that the
punctate structures were frequently encircled to various degrees by
GFP-ATG8a (Fig. 3C, D). Furthermore, the GFP-ATG8a signal changed
position in tact with the RFP-PSMD1 puncta suggesting that the co-
localization is real (supplementary movie 1). Quantitative analysis
showed that 40.5 and 31.4% of RFP-PSMD1 or RFP-PSMD2 positive
puncta co-localized with ATG16-GFP, respectively. The corresponding
values for the co-localization with GFP-ATG8a were 12.1 and 23.9%,
respectively (Table 2). Taken together, these results show that a sub-
fraction of RFP-PSMD1 and RFP-PSMD2 co-localises with autophago-
somes.
2.4. The autophagosomal localization of PSMD1 and PSMD2 is dependent
on ATG16
Next we expressed RFP-PSMD1 also in AX2, atg16‾, atg9‾, atg9‾/
16‾ and atg9‾/16‾/atg16-gfp cells and RFP-PSMD2 also in AX2 and
atg16‾ cells (Figure S2 A). Immunofluorescence analysis showed that
tagged PSMD1 and PSMD2 were enriched in punctate structures upon
treatment with NH4Cl in AX2 wild-type cells but not in atg16‾ cells
(Fig. 4A). Similar results were obtained after treatment of the cells with
Bafilomycin A1, which inhibits lysosomal acidification (Fig. S2B).
Quantitative analysis revealed that 98% of AX2 cells contained RFP-
PSMD1 and 97% RFP-PSMD2 positive punctate structures, while such
RFP-PSMD1 or RFP-PSMD2 positive punctae were only seen in less than
1% of atg16‾ cells (Fig. 4B). After expression of ATG16-GFP in ATG16‾
cells we observed the re-appearance of RFP-PSMD1 and RFP-PSMD2
positive puncta in 86 and 94% of the cells, respectively (Fig. 4A, B).
Next, we performed immunofluorescence analysis of atg9‾ and atg9‾/
16‾ cells expressing RFP-PSMD1 and found that RFP-PSMD1 positive
puncta were only formed in 23% of the atg9‾ cells and were absent in
atg9‾/16‾ cells. Expression of ATG16-GFP in atg9‾/16‾ cells partially
rescued puncta formation to similar levels as in atg9‾ cells. However,
the punctate structures were significantly less prominent in atg9‾ and
atg9‾/16‾/atg16-gfp cells in comparison to AX2 (Fig. S2C, D). In pre-
vious studies it was shown that ATG16 binds to the isolation membrane
in an ATG5-dependent manner and more recently an AFIM was iden-
tified in ATG16L1 (Kim et al., 2015; Romanov et al., 2012). Sequence
alignments with ATG16 protein sequences showed that the AFIM is
nicely conserved in other ATG16 proteins as for example yeast, Danio
rerio, Mus musculus, Homo sapiens and Dictyostelium (Fig. 4C). To test
Q. Xiong et al.
whether the N-terminal region of ATG16 containing the AFIM is crucial
for mediating the formation of RFP-PSMD1 and RFP-PSMD2 punctate
structures we expressed ATG161−309-GFP or ATG16156-612-GFP, lacking
the AFIM, with RFP-PSMD1 or RFP-PSMD2, respectively in atg16‾ cells.
Immunofluorescence analysis showed that in atg16‾ cells expressing
ATG161−309-GFP, but not in those expressing ATG16156-612-GFP, RFP-
PSMD1 and RFP-PSMD2 were again enriched in punctate structures
(Fig. 4D, E). These results show that the enrichment of PSMD1 and
PSMD2 in punctate structures, which are presumably autophagosomes,
is dependent on the N-terminal 309 amino acids of ATG16, which
contain the AFIM. The results are consistent with the assumption that
ATG16 lacking the AFIM cannot be recruited to the autophagosome. We
conclude that the N-terminal 155 amino acids of ATG16, which contain
the AFIM motif, are required and the C-terminal half of ATG16, which
harbors the seven WD repeats, is dispensable for the formation of
PSMD1 and PSMD2 puncta in atg16‾ cells.
2.5. PSMD1 and PSMD2 are degraded by autophagy
Our results so far suggest that a subfraction of PSMD1 and PSMD2
positive puncta are autophagosomes, because these structures partially
co-localized with ATG8a, could only be detected when autophagosomal
degradation was slowed down and their formation was dependent on
ATG16. Autophagic degradation of substrates can be monitored with
the GFP cleavage assay (Calvo-Garrido et al., 2011). This assay is based
on the observation that the GFP moiety is often cleaved as a whole from
GFP-tagged autophagic substrates inside the autolysosome and accu-
mulates because of its resistance to further digestion. We expressed
GFP-PSMD1 and GFP-PSMD2 in AX2 and atg16‾ cells, respectively, and
found that upon treatment with NH4Cl the released free GFP was clearly
detected in AX2 but not in atg16‾ cells (Fig. 5A). We conclude that the
degradation of PSMD1 and PSMD2 is mediated by ATG16 and
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European Journal of Cell Biology 97 (2018) 523–532
Fig. 2. The interaction of ATG16 with PSMD1
and PSMD2 is direct. (A) Domain structure of
ATG16 and schematic representation of in-
vestigated ATG16 fragments. Amino acid po-
sitions are given above full length ATG16 and
each ATG16 fragment. The N-terminal domain
is depicted in green and the C-terminal β-pro-
peller domain in blue. CC, coiled coil domain;
WD1-7 depicts the seven WD repeats. (B) The N-
terminal half of ATG16 interacted directly with
PSMD1 and the C-terminal half with PSMD1
and PSMD2. GST-tagged ATG161−309 and
ATG16310-612 were purified from E. coli, bound
to Glutathione Sepharose 4B beads and in-
cubated with purified recombinant PSMD1 or
PSMD2. Proteins were separated by 12% SDS-
PAGE, transferred to nitrocellulose membrane
and stained with PonceauS. PSMD1 and
PSMD2 were detected by specific polyclonal
antibodies. Upper part, Western Blot (WB) with
anti PSMD1 (left) and anti PSMD2 (right) an-
tibodies; (*), unspecific binding of the PSMD2
antibody; lower part, WB with anti GST anti-
body. (C) ATG161-198 but not ATG16160-312
interacted directly with PSMD1. Experimental
procedure was as described for (B) (For inter-
pretation of the references to colour in this
figure legend, the reader is referred to the web
version of this article.).
presumably takes place in autolysosomes. To investigate the putative
localization of PSMD1 and PSMD2 in autolysosomes we expressed RFP-
GFP-PSMD1 or RFP-GFP-PSMD2 in AX2 cells (Fig. S3). The fluorescence
of GFP is very sensitive to the acidic environment of the lysosome, thus,
the presence of red puncta lacking the green fluorescence is an in-
dication of the acidification of the autophagosome through fusion with
the lysosome (Calvo-Garrido et al., 2011). We found that treatment
with NH4Cl or Bafilomycin A1, which both slow down autophagosomal
degradation, resulted in the appearance of red puncta in these cells. The
disappearance of the green fluorescence indicates the acidification of
PSMD1 and PSMD2 positive puncta through fusion with the lysosome
(Fig. 5B). Finally, to determine whether PSMD1 and PSMD2 are indeed
present in lysosomes, we stained live AX2 and atg16‾ cells expressing
RFP-PSMD1 or RFP-PSMD2 with LysoTracker. In AX2 cells we found
that many of the PSMD1 and PSMD2 positive puncta co-localized with
LysoTracker, while no such puncta were formed and hence no co-lo-
calization was seen in atg16‾ cells (Fig. 5C). Quantification of the co-
localization showed that approximately 50% of the RFP-PSMD1 or RFP-
PSMD2 positive puncta were also positive for LysoTracker. Interest-
ingly, the ratio of co-localization increased to nearly 90% for puncta
with a diameter larger than 1 μm (Table 3). We conclude that PSMD1
and PSMD2 are degraded by ATG16-mediated autophagy.
3. Discussion
The core autophagy protein ATG16 is crucial for the expansion of
the isolation membrane by acting in concert with the ATG12-5 con-
jugate as an E3 ligase in the ATG8(LC3) conjugation system (Noda and
Inagaki, 2015). The protein is composed of an N-terminal half with a
CCD for homo-dimerization and binding sites for ATG5, clathrin,
FIP200, and Rab33B (Gammoh et al., 2013; Itoh et al., 2008; Kuma
et al., 2002; Mizushima and Levine, 2010; Nishimura et al., 2013;
Q. Xiong et al. European Journal of Cell Biology 97 (2018) 523–532

Fig. 3. ATG16 and ATG8a (LC3) co-localize with RFP-PSMD1 and RFP-PSMD2 positive puncta. (A, B) Immunofluorescence microscopy of live atg16‾/atg16-gfp/rfp-
psmd1 and atg16‾/atg16-gfp/rfp-psmd2 cells. Arrows indicate partial co-localization of ATG16-GFP and RFP-PSMD1 and of ATG16-GFP and RFP-PSMD2. Scale bar:
2 μm. (C, D) Immunofluorescence microscopy of live AX2/gfp-atg8a/rfp-psmd1 and AX2/gfp-atg8a/rfp-psmd2 cells. RFP-PSMD1 or RFP-PSMD2, respectively, were
ectopically expressed in AX2 cells together with GFP-ATG8a. RFP-PSMD1 and RFP-PSMD2 puncta partially co-localized with the autophagosomal marker ATG8a
(LC3) (row 1). We often observed that the GFP-ATG8a signal expanded along the puncta to different degrees and sometimes completely encircled the puncta (row
2–4). Scale Bar: 2 μm.

Table 2 particle of the proteasome, as novel ATG16 interacting proteins and

Co-localization of RFP-PSMD1 and RFP-PSMD2 with ATG16-GFP and GFP- found that their interaction with ATG16 is direct (Figs. 1, 2 and S1). We
ATG8a, respectively. furthermore narrowed down the binding sites and could show that the
RFP-PSMD1 RFP-PSMD2 N-terminal 198 amino acids of ATG16 are sufficient for the binding to
PSMD1, while the C-terminal half (aa 310–612) directly interacts with
RFP puncta Co-localization RFP puncta Co-localization both proteins, PSMD1 and PSMD2 (Figs. 2B, C, S1). At present it is
with GFP with GFP
unclear how the interaction between ATG16 and PSMD1/2 is regulated.
# # % # # %
Possible elicitors could be e.g. environmental stress, non-stoichiometric
presence of PSMD1/2 with respect to other proteasome subunits or
ATG16-GFP 425 172 40.5 341 107 31.4 folding or activity problems.
GFP-ATG8a 1284 156 12.1 1013 242 23.9 The direct interaction raised the question whether ATG16 acts as an
autophagy receptor or adaptor for the degradation of PSMD1/2. Since
For ATG16-GFP the puncta from 5 and for GFP-ATG8a from 6 independent
experiments were counted. For each combination of tagged proteins at least 300 autophagy is a fast process we made use of the lysosomotropic com-
cells were analyzed. pound NH4Cl and of Bafilomycin A1, which both slow down the au-
tophagic flux by increasing the lysosomal pH, albeit by different me-
Ravikumar et al., 2010) and a C-terminal β-propeller structure, that chanisms (Aubry et al., 1993; Calvo-Garrido et al., 2011; Klionsky et al.,
binds NOD 1 and 2, TMEM59 and ubiquitin and appears to be crucial in 2016; Mesquita et al., 2013; Yoshimori et al., 1991). Treatment with
xenophagy (Boada-Romero et al., 2013; Fujita et al., 2013; Travassos these compounds led to the frequent detection of RFP-PSMD1 and RFP-
et al., 2010; Ver Heul et al., 2013). The numerous so far identified in- PSMD2 positive puncta in AX2 wild-type cells, however, we only very
teractors imply that ATG16 acts as a major hub in autophagy and we set rarely could detect such puncta in atg16‾ cells (Figs. 4A, B, S2B).
out to identify novel binding proteins in the Dictyostelium system. Using PSMD1 and PSMD2 puncta formation was rescued in atg16‾ cells ex-
GST-ATG16 as bait in pull down assays with total cell lysates, we pressing ATG16-GFP or ATG161−309-GFP but not ATG16156–612-GFP
identified PSMD1 and PSMD2, two subunits of the 19S regulatory (Fig. 4A, D). It was previously shown that the N-terminal half of ATG16

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Q. Xiong et al.
Fig. 4. The formation of PSMD1 and PSMD2 positive puncta is dependent on
ATG16. (A) Immunofluorescence analysis of live Dictyostelium cells expressing
RFP-PSMD1 or RFP-PSMD2, respectively. After treatment with NH4Cl, RFP-
PSMD1 (top) or RFP-PSMD2 (bottom) were enriched in punctate structures
(white arrows) in AX2 and atg16‾/atg16-gfp cells but not in atg16‾ cells. Scale
Bar: 2 μm. (B) Quantification of the percentage of AX2, atg16‾ and atg16‾/
atg16-gfp cells containing RFP-PSMD1 or RFP-PSMD2 positive puncta. For each
experiment and strain more than 200 cells were analyzed. Error bars represent
standard deviations of three independent experiments. (C) Sequence alignment
of the ATG5-interacting motif (AFIM) of ATG16 from different organisms. Sc,
Saccharomyces cerevisiae; Dr, Danio rerio; Hs, Homo sapiens; Mm, Mus musculus;
Dd, Dictyostelium discoideum. Numbers denote the position of the first and the
last amino acid of the AFIM in ATG16 of the different organisms. Red letters
highlight key residues for ATG5 binding. (D) Immunofluorescence analysis of
live Dictyostelium atg16‾ cells expressing either ATG161−309-GFP (left) or
ATG16156-312-GFP (right) and RFP-PSMD1 (top) or RFP-PSMD2 (bottom),
528
European Journal of Cell Biology 97 (2018) 523–532
respectively. RFP-PSMD1 or RFP-PSMD2 positive puncta were formed in atg16‾
cells after expression of ATG161−309-GFP (left images) but not after expression
of ATG16156-312-GFP, which lacks the AFIM (right images). Scale Bar: 2 μm.
Expression of tagged constructs was confirmed by Western blotting; 1, atg16‾/
rfp-psmd1/atg161-309-gfp; 2, atg16‾/rfp-psmd1/atg16156-312-gfp; 3, atg16‾/rfp-
psmd2/atg161-309-gfp; 4, atg16‾/rfp-psmd2/atg16156-312-gfp. PSMD1 and PSMD2
were detected with specific polyclonal antibodies, ATG161−309-GFP and
ATG16156-312-GFP were detected with a GFP specific monoclonal antibody.
Actin served as loading control and was detected with the Act1-7 anti actin
antibody (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.).
is required for the recruitment of the ATG12-5/16 complex to the au-
tophagosomal membrane (Romanov et al., 2012). In this region of
ATG16 the AFIM (Kim et al., 2015) is located between amino acids 43
and 60 (Fig. 4B). The ATG16156–612-GFP fusion protein lacks the AFIM
and hence is not able to rescue puncta formation in atg16‾ cells. Al-
though ATG161−309 was not able to interact with PSMD2, expression of
ATG161−309-GFP in atg16‾ rescued also the formation of RFP-PSMD2
puncta (Figs. 2C, 4 D, S1A). It was reported that PSMD1 and PSMD2
directly interact in S. cerevisiae (Rosenzweig et al., 2008). In in vitro
pull-down assays we could, however, not find a direct interaction be-
tween Dictyostelium PSMD1 and PSMD2 (Fig. S4A), nevertheless,
binding of PSMD1 to ATG161−309-GFP could mediate an indirect in-
teraction between ATG16 and PSMD2 in vivo.
We next wanted to determine the nature and function of the puncta.
They could constitute protein aggregates, which could have been caused
by the overexpression of RFP-PSMD1 or RFP-PSMD2 in AX2 and the
different transgenic strains. However, the expression level of RFP-PSMD1
in atg16‾ and atg9‾/16‾ cells, which did not form RFP-PSMD1 positive
puncta, was as high as in AX2, atg16‾/atg16-gfp and atg9‾/16‾/atg16-gfp
cells (Fig. S2A). Furthermore, we frequently observed ubiquitinated
protein aggregates in atg16‾ cells (Xiong et al., 2015) but hardly any
RFP-PSMD1 or RFP-PSMD2 positive puncta (Figs. 4, S2B, C). Taken to-
gether, these results strongly argue against the hypothesis that the puncta
are protein aggregates. We then considered the possibility that the
puncta are autophagosomes and/or autolysosomes since their formation
was dependent on ATG16 (Fig. 4A, B). We found that around 40% of the
RFP-PSMD1 and 30% of the RFP-PSMD2 positive puncta co-localized
with ATG16, and 12 and 24%, respectively, with the autophagosomal
marker ATG8a (Fig. 3 and Table 2), suggesting that a considerable
fraction of the puncta are indeed autophagosomes. At present it is un-
clear why RFP-PSMD2 co-localization is lower with ATG16 and higher
with ATG8a as compared to RFP-PSMD1 puncta. To further investigate
whether a subfraction or all of the remaining puncta could be auto-
lysosomes, we applied three approaches: i) in a proteolytic cleavage
assay (Calvo-Garrido et al., 2011) we detected the released free GFP from
the GFP-PSMD1 and GFP-PSMD2 fusion proteins in AX2 wild-type cells
but not in atg16‾ cells (Fig. 5A). ii) We were able to monitor the lyso-
somal localization of PSMD1 and PSMD2 through the expression of RFP-
GFP-PSMD1 and RFP-GFP-PSMD2. The acidic pH of the lysosome
quenches the GFP fluorescence, thus the emission of only red fluores-
cence indicates acidification of the autophagosome through fusion with a
lysosome (Fig. 5B). This strategy was originally described for RFP-GFP-
ATG8 which is often used to monitor autophagic flux (Mesquita et al.,
2013). iii) Finally, we investigated the co-localization of RFP-PSMD1 or
RFP-PSMD2 with LysoTracker and found that approximately 50% of the
puncta co-localized with LysoTracker (Fig. 5C, Table 3). These results
suggest that, dependent on ATG16, PSMD1 and PSMD2 get enriched in
autophagosomes, followed by degradation in autolysosomes. At present
it is not clear if only excess PSMD1 and PSMD2, tagged as well as en-
dogenous, or the whole 19S regulatory particle or even the 26S protea-
some get degraded in this way in AX2 wild-type cells. In the latter case,
we would expect an increase of the protein levels of the proteasomal
subunits and a concomitant increase in proteasomal activity in atg16‾
and other autophagy-compromised cells. However, based on protein
Q. Xiong et al. European Journal of Cell Biology 97 (2018) 523–532

Fig. 5. PSMD1 and PSMD2 are degraded in autolysosomes. (A) Proteolytic

cleavage assay to monitor autophagy. GFP-PSMD1 and GFP-PSMD2 were ex-
pressed in AX2 and atg16‾ cells, respectively, cells were either left untreated (-)
or treated (+) two times for two hours with 100 mM NH4Cl, lysed and total cell
lysates subjected to Western blotting. The cleaved free GFP was detected with a
GFP-specific monoclonal antibody in AX2 cells but not in atg16‾ cells. The
positions of GFP-PSMD1, GFP-PSMD2 and of GFP are indicated. (B)
Fluorescence analysis of AX2 cells expressing RFP-GFP-PSMD1 or RFP-GFP-
PSMD2. Treatment of these cells with NH4Cl or Bafilomycin A1 followed by
confocal microscopy revealed the formation of red punctate structures for both,
RFP-GFP-PSMD1 and RFP-GFP-PSMD2 (white arrows). Since the GFP fluores-
cence is rapidly lost in the acidic environment of the lysosome these puncta are
by definition autolysosomes. Scale bar: 2 μm. (C) Fluorescence analysis of
LysoTracker labelled AX2 and atg16‾ cells expressing RFP-PSMD1 or RFP-
PSMD2. RFP-PSMD1 and RFP-PSMD2 positive puncta co-localize in many cases
with LysoTracker in AX2 cells (for quantitative analysis see Table 3). No such
RFP-PSMD1 and RFP-PSMD2 positive puncta are present in atg16‾ cells and
hence there is no co-localization with LysoTracker. Scale Bar: 2 μm (For inter-
pretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.).

levels of subunits 4 and 5 (SU4 and SU5) of the 20S proteasome (Xiong
et al., 2015) and of PSMD1 and PSMD2 of the 19S regulatory particle
(Fig. S2A) there was no increase in the number of proteasomes in these
strains. Moreover, we previously found that proteasomal activity was
strongly reduced in different autophagy-compromised strains (Arhzaouy
et al., 2012; Messling et al., 2017; Xiong et al., 2015). We therefore
speculate that in atg16‾ and other autophagy-compromised cells pro-
teasomes with reduced activity, which are normally cleared by autop-
hagy, are not at all or only insufficiently removed. This then causes the
observed decrease in proteasomal activity (Arhzaouy et al., 2012;
Messling et al., 2017; Xiong et al., 2015).
For a long time autophagy and the UPS were thought to act in-
dependently but evidence is accumulating that these two major in-
tracellular pathways for protein and organelle clearance in eukaryotic
cells are interrelated (Bartel, 2015; Driscoll and Chowdhury, 2012;
Korolchuk et al., 2009, 2010; Kraft et al., 2010). An early finding was
that proteasomes accumulated in lysosomes upon nutrient starvation or
treatment with the lysosomal inhibitor leupeptin (Cuervo et al., 1995).
In colon cancer cells pharmacological inhibition of autophagy and also
downregulation of autophagy genes by RNAi resulted in an increase of
proteasomal subunits and proteasomal activity (Wang et al., 2013). In
contrast, inhibition of lysosomal activities by chloroquine in neuro-
blastoma cells led to an accumulation of ubiquitinated proteins and
reduced proteasomal activities (Qiao and Zhang, 2009). Furthermore,
inhibition of autophagy with Bafilomycin A1 or siRNA knock-down of
atg7 or atg12 resulted in an impaired clearance of UPS clients in HeLa
cells, consistent with reduced proteasomal activity (Korolchuk et al.,
2009). A quantitative proteomic analysis of autophagosomes showed,
that proteasomal proteins were abundant among the stimulus-in-
dependent common autophagosome-associated proteins (Dengjel et al.,
2012). Recently, it was reported that in Arabidopsis the degradation of
inactive 26S proteasomes is mediated by the proteasomal subunit
RPN10 (PSMD4) via ubiquitin and ATG8, and in S. cerevisiae via the
ubiquitin receptor Cue5 and the Hsp42 chaperone, a process the au-
thors termed proteaphagy (Marshall et al., 2015, 2016). Furthermore, it
was found that the autophagic clearance of proteasomes in yeast re-
quires the sorting nexin 4 and that upon starvation the proteaphagy
pathways for the core and regulatory particles of the proteasome are
different (Nemec et al., 2017; Waite et al., 2016). In mammals, stress-
induced proteaphagy was dependent on p62/SQSTM1 and ubiquitin
(Cohen-Kaplan et al., 2016). It appears therefore that proteaphagy
might be universal in eukaryotes, however, the mechanism and the
autophagy receptors appear to vary from organism to organism. In
Dictyostelium, impaired disposal of inactive proteasomes in autophagy-
compromised strains could cause the observed dramatic decrease in
proteasomal activity (Arhzaouy et al., 2012; Messling et al., 2017;

529
Q. Xiong et al.
Table 3
Co-localization of RFP-PSMD1 or RFP-PSMD2 with LysoTracker.
# of green # of red # of red and % of red puncta co-
puncta puncta green puncta localizing with green puncta > 1 μm
RFP-PSMD1 AX2 1318 968 483 49.9
atg16‾ 432 0 0 N/A
RFP-PSMD2 AX2 1403 1191 578 48.5
atg16‾ 447 0 0 N/A
Data from two independent experiments were quantified. Green (LysoTracker) and red (RFP-PSMD1 or RFP-PSMD2) puncta larger than 0.25 μm of 566 AX2/rfp-
psmd1, 322 atg16¯/rfp-psmd1, 607 AX2/rfp-psmd2 and 333 atg16¯/rfp-psmd2 cells were counted and the percentage of RFP-PSMD1 or RFP-PSMD2, respectively, co-
localizing with LysoTracker was calculated.
Xiong et al., 2015). These results and our identification of the 19S
regulatory particle subunits PSMD1 and PSMD2 as direct binding
partners of the core autophagy protein ATG16, suggest that ATG16 may
not only mediate the degradation of PSMD1 and PSMD2 but of the 19S
regulatory particle or even the complete 26S proteasome. Further work
is needed to solve the question whether ATG16 indeed acts as an au-
tophagy receptor or adaptor for proteaphagy in D. discoideum.
4. Material and methods
4.1. Dictyostelium strains
D. discoideum AX2 was used as wild-type strain. The generation and
phenotypes of atg9‾, atg16‾, atg16‾/atg16-gfp, atg9‾/16‾ and atg9‾/
16‾/atg16-gfp strains are described elsewhere (Tung et al., 2010; Xiong
et al., 2015). Expression plasmids for RFP-PSMD1, RFP-PSMD2, GFP-
PSMD1, GFP-PSMD2, RFP-GFP-PSMD1, RFP-GFP-PSMD2, ATG161−309-
GFP and ATG16156-612-GFP were used for transformation of different
strains by electroporation. AX2 and mutant cells were grown at 21 °C in
AX2 medium (for 1 L: 14.3 g bacteriological peptone, 7.15 g yeast ex-
tract, 18 g maltose, 0.62 g Na2HPO4 x 2H2O, 0.49 g KH2PO4, pH 6.7)
with shaking at 160 rpm in Erlenmeyer flasks or on SM agar plates with
Klebsiella aerogenes (Brink et al., 1990; Watts and Ashworth, 1970;
Williams and Newell, 1976). The different transgenic strains used in
this study are listed in table S1.
4.2. Vector construction and transformation
cDNAs encoding either full-length or parts of the ATG16 (gene ID:
DDB_G0275323, http://www.dictybase.org), of PSMD1
(DDB_G0287953) and PSMD2 (DDB_G0293752) were amplified by PCR
and cloned into the pGEX-6P-1 or pGEX-4T-1 GST expression vectors. For
ectopic expression of PSMD1 and PSMD2 fused C-terminally to RFP in
different Dictyostelium strains the p389-2-mRFPMars vector was used
(Fischer et al., 2004). The PSMD1 and PSMD2 coding sequences were
amplified by PCR. The PSMD1 cDNA was cloned into the p389-2-
mRFPMars vector via the BamHI and EcoRI restriction sites and the
PSMD2 cDNA via the EcoRI and ClaI restriction sites. For ectopic ex-
pression of PSMD1 and PSMD2 fused C-terminally to GFP the pBsr-N-GFP
vector was used (Blau-Wasser et al., 2009). The PSMD1 and PSMD2
coding sequences were cloned into the pBsr-N-GFP vector via the PstI and
XmaI restriction sites, respectively. For ectopic expression of
ATG161−309–GFP and ATG16156-612–GFP the pBsr-C1-GFP vector was
used (Blau-Wasser et al., 2009). Both coding sequences were amplified
by PCR and cloned into pBsr-C1-GFP via the PstI and XmaI restriction
sites. RFP-GFP-PSMD1 and RFP-GFP-PSMD2 expressing constructs were
generated by insertion of the GFP coding sequence, amplified by PCR
from the pBsr-N2-GFP vector, into the p389-2-mRFPMars/RFP-PSMD1
and p389-2-mRFPMars/RFP-PSMD2 plasmids (see above) via BamHI and
EcoRI restriction sites, respectively. All final expression constructs were
verified by sequencing and were introduced into bacterial cells by heat
shock and into Dictyostelium cells by electroporation. Transformants were
530
European Journal of Cell Biology 97 (2018) 523–532
# of red puncta # of red and green % of red puncta > 1 μm co-localizing
puncta > 1 μm with green puncta
69 60 87.0
0 0 N/A
128 108 84.4
0 0 N/A
selected in the presence of proper antibiotics. Dictyostelium cells expres-
sing RFP- and/or GFP-tagged proteins were identified by visual inspec-
tion under a fluorescence microscope and expression of fusion proteins
was verified by Western blotting. All primers used in PCR reactions in
this study are listed in table S2.
4.3. Protein quantification, antibody generation, SDS PAGE and western
blotting
For generation of PSMD1 and PSMD2 specific polyclonal antibodies
(pAbs), the cDNA sequences encoding a hydrophilic fragment (aa
263–312 for PSMD1 and aa 13–79 for PSMD2) were amplified by PCR
and cloned into the pGEX-6P-1 GST expression vector. The fusion
protein was expressed in E. coli XL1 blue, purified using GST-Sepharose
beads, released through cleavage with PreScission protease (GE
Healthcare Life Science) and used for the immunization of rabbits
(BioGenes GmbH, Germany). SDS-PAGE and Western blotting were
essentially performed as described (Laemmli, 1970; Towbin et al.,
1979). Dictyostelium total cell lysates were prepared and the proteins of
2 × 105 cells were separated per lane by SDS gel electrophoresis. The
generated PSMD1 pAbs were affinity purified and used for Western
blotting at a 1:2000 dilution. PSMD2 pAbs were used at a 1:2000 di-
lution. GFP was detected with monoclonal antibody K3-184-2 at a 1:20
dilution (Noegel et al., 2004). RFP was detected with monoclonal an-
tibody K64-372-1 at a 1:20 dilution (Fischer et al., 2004). Actin was
detected with monoclonal antibody Act1-7 at a 1:15 dilution (Simpson
et al., 1984). Secondary antibodies used were anti-mouse and anti-
rabbit lgG conjugated with peroxidase (POD) (Sigma, Germany) at a
1:10,000 dilution followed by chemiluminescence detection. Images
were recorded and analyzed using the Fluorchem SP imaging system
(Alpha Innotech, USA). Relative protein amounts were determined
densitometrically using the Spot Denso tool of the AlphaEaseFC soft-
ware (Alpha Innotech, USA).
4.4. GFP cleavage assay
The GFP cleavage assay was performed essentially as described
(Calvo-Garrido et al., 2011). Briefly, 1 mL AX2 or atg16‾ cells expressing
GFP-PSMD1 or GFP-PSMD2 in AX2 medium at a concentration of
1 × 106C/ml were deposited in the wells of a 6-well plate. The cells were
allowed to settle down for 15 min, washed twice with Soerensen buffer
(2.0 mM Na2PO4, 14.6 mM KH2PO4, pH 6.0), then treated with 100 mM
NH4Cl in Soerensen buffer for 2 h. The treatment with 100 mM NH4Cl in
Soerensen buffer was repeated for another 2 h. Proteins of total cell ly-
sates were separated in 12% SDS-PAGE gels, transferred by Western
blotting and GFP was detected with a specific monoclonal antibody.
4.5. Pulldown and co-immunoprecipitation (IP) assays
To identify new binding partners of ATG16, we used recombinant
GST-ATG16 fusion protein bound to Glutathione Sepharose 4B beads as
“bait” with a lysate from AX2 cells. Negative controls were GST-ATG16
Q. Xiong et al.
beads without AX2 Lysate and AX2 lysate with GST beads only. AX2
cells were lysed in 25 mM Tris/HCl pH 7.5, 5 mM EDTA, 150 mM NaCl,
10% Glycerol, 0.1% Triton X-100, 1 mM DTT and 1x Protease Inhibitor
Cocktail (Roche). Cell lysates were centrifuged at 20,000 xg for 10 min
at 4 °C and the supernatant containing the soluble proteins was in-
cubated with either GST or GST-ATG16 bound to Glutathione
Sepharose beads for 3 h at 4 °C on a rotating wheel. The beads were
collected by centrifugation at 500 x g for 15 s and then washed three
times with the same buffer. The bound proteins were eluted from the
beads by boiling in 1 x SDS buffer at 95 °C for 5 min, separated in 10%
and 15% SDS-PAGE gels and stained with the Imperial Protein Stain. 24
distinct bands in the size range between 400 and 15 kDa that were
missing in the negative controls were excised and proteins were in-gel
digested after reduction and alkylation with trypsin. Tandem mass
spectrometry (LCeMS/MS) was performed at the Center for Molecular
Medicine Cologne (CMMC) as described (Feldkirchner et al., 2012).
LCeMS data were acquired on a HCT ETD II ion-trap mass spectrometer
equipped with a nano ESI source (Bruker Daltonics). Data-dependent
acquisition of MS and MS/MS spectra was controlled by the Compass
3.0 software using previously published parameters (Albert et al.,
2010). Proteins were identified by searching the NCBInr release
20,080,210 (Dictyostelium) using a local installation of MASCOT 2.2
(Matrix Science Ltd). Searches were submitted via Proteinscape 2.0
(Bruker Daltonics) with the following parameter settings: enzyme
“trypsin”, species “Dictyostelium”, fixed modifications “carbamido-
methyl (Cys)”, optional modifications “Methionine oxidation” and
missed cleavages “1″. The mass tolerance was set to 0.3 Da for peptide
and fragment spectra. The identified proteins are listed in table S3.
Co-immunoprecipitation assays were performed with axenically
grown atg16‾/atg16-gfp/rfp-psmd1 or atg16‾/atg16-gfp/rfp-psmd2 cells
using GFP-Trap® (ChromoTek). Log phase cells (2–4 × 106C/ml) were
harvested, centrifuged at 500 xg for 4 min and washed twice with
Soerensen buffer. Cell pellets were frozen at −20 °C overnight and
lysed according to the manufacturer’s instructions (ChromoTek).
Recombinant PSMD1, PSMD2, full-length ATG16 and fragments of
ATG16 were expressed in E. coli XL1 blue or DH5α or ArcticExpress RIL
cells (Agilent Technologies) and purified using Glutathione Sepharose
beads. For pull-down assays of PSMD1 and PSMD2 with full-length
ATG16 and fragments of ATG16, PSMD1 and PSMD2 were released
from the beads through cleavage with PreScission protease, diluted
with RIPA buffer (50 mM Tris/HCl pH 7.5, 150 mM NaCl, 1% NP-40,
0.5% Na-deoxycholate) and then incubated with ATG16 and fragments
of ATG16 bound to Glutathione Sepharose beads for 3 h at 4 °C. Beads
were washed with RIPA buffer, bound proteins were eluted by boiling
the beads in SDS sample buffer, proteins were separated by SDS-PAGE
and then analyzed by western blotting. To test binding of ATG16 from
total Dictyostelium cell lysates to recombinant PSMD1 and PSMD2, cells
were lysed in 25 mM Tris/HCl pH 7.5, 5 mM EDTA, 150 mM NaCl, 10%
Glycerol, 0.1% Triton X-100, 1 mM DTT and 1x Protease Inhibitor
Cocktail (Roche). Cell lysates were centrifuged at 20,000 xg for 10 min
at 4 °C, the supernatant containing the soluble proteins was incubated
with GST-PSMD1 or GST-PSMD2 bound to Glutathione Sepharose beads
for 3 h at 4 °C. Beads were washed twice with the same buffer and
bound proteins were analyzed by western blotting.
4.6. Fluorescence microscopy
For live cell imaging, Dictyostelium cells ectopically expressing RFP
or GFP fusion proteins, RFP-GFP-PSMD1 or RFP-GFP-PSMD2, were
detached from the plastic surface of a cell culture dish (Ø 100 mm) and
transferred to a μ-Dish (Ø 35 mm, Ibidi). Cells were allowed to attach
for 15 min. Dictyostelium cells that expressed GFP fusion proteins were
washed once with Loflo (low fluorescence) medium (Formedium) and
pre-incubated overnight in Loflo medium to decrease the cytosolic
background fluorescence (Bloomfield et al., 2015). Approximately two
hours before treatment of the cells, the medium was replaced with fresh
531
European Journal of Cell Biology 97 (2018) 523–532
Loflo medium. Cells expressing RFP fusion proteins were analyzed
without pre-incubation in Loflo medium. GFP and/or RFP fusion pro-
teins expressing cells were washed once with Soerensen buffer followed
by an incubation with 100 mM NH4Cl or 5 μM Bafilomycin A1 (Enzo
life sciences) in Soerensen buffer for 2 h. Treatment with NH4Cl or
Bafilomycin A1 increases the lysosomal pH and thus slows autophagic
degradation of proteins (Aubry et al., 1993; Mesquita et al., 2013). The
cells were treated in the same way with 100 mM NH4Cl and with or
without 0.1 μM LysoTracker Deep Red (Molecular Probes) to visualize
lysosomes or with 5 μM Bafilomycin A1 for another 2 h. Delivery of
RFP-GFP-PSMD1 or RFP-GFP-PSMD2 from the autophagosome to the
autophagolysosome can be monitored in this way because the acidic
environment of the lysosome quenches the more sensitive fluorescence
of GFP, while the fluorescence of RFP is preserved. Thus, the presence
of red puncta lacking the green fluorescence indicates the presence of
RFP-GFP-tagged fusion proteins in autolysosomes (Mesquita et al.,
2013). Confocal images of cells were taken with a TCS SP5 laser
scanning microscope (Leica) with a 63x HCX PL APO lambda blue 1.40
oil UV immersion objective. Excitation of GFP was at 488 nm, emission
500–550 nm; of RFP at 561 nm, emission 570-620 nm; and of Lyso-
Tracker Deep Red at 647 nm, emission 646-727 nm. Images were pro-
cessed using the Leica Application Suite (LAS AF) software.
Author’s contributions
QX carried out the molecular lab work, performed data analysis and
drafted the manuscript; SF, MK, RM and SM contributed to the mole-
cular lab work; LE conceived of the study, designed and coordinated it
and finalised the manuscript. All authors gave final approval for pub-
lication.
Acknowledgments
We thank Dr. Angelika A. Noegel and Dr. Christoph Clemen for
helpful discussions, Dr. Ricardo Escalante for providing plasmids, Dr.
Salvatore Bozzaro for advice with LysoTracker and Maria Stumpf for help
with immunofluorescence studies. LE acknowledges support of this work
by the German Research Foundation (Deutsche Forschungsgemeinschaft:
CRC670, TP01) and by Köln Fortune. This work was supported by the
China scholarship council to QX.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.ejcb.2018.09.002.
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