Introduction
Picornaviruses are single-stranded positive-sense RNA viruses that replicate on intracellular
membranes (1). Previous work has indicated that the 3A protein is responsible for
the reorganization of Golgi membranes to create viral replication organelles (2, 3).
The molecular basis of this reorganization has only recently become apparent through
the dual discoveries that enteroviral 3A directly interacts with Golgi-specific brefeldin
A resistance guanine nucleotide exchange factor 1 (GBF1) and that many RNA viruses
require the activity of phosphatidylinositol 4-kinase class III beta (PI4KB) for viral
replication (2, 3). However, many questions remain, as not all picornaviruses that
require PI4KB activity interact with GBF1, and the portion of GBF1 required for viral
replication appears independent of its known Arf1 guanine exchange activity (4).
Two protein-protein interaction screens recently demonstrated that the 3A proteins
of enteroviruses and kobuviruses both interact with the host Golgi adaptor protein
acyl coenzyme A (acyl-CoA) binding domain protein 3 (ACBD3/GCP60) to recruit PI4KB
to viral replication organelles (5, 6). ACBD3 is a highly conserved Golgi complex-associated
60-kDa protein among metazoans that contains a remarkably long N-terminal acyl-CoA
binding domain, a coiled-coil domain composed of a charged amino acid region (CAR)
and glutamine-rich region (Q-rich), and a highly conserved C-terminal Golgi dynamics
domain (GOLD domain) that interacts with the Golgi resident protein giantin/GOLGB1
(7). The level of expression of ACBD3 has been shown to be important for the maintenance
of the Golgi structure (7). The picornavirus 3A-ACBD3 interaction is required for
replication, as knockdown of ACBD3 significantly reduces poliovirus and Aichi virus
replication (5, 6). Furthermore, mutations that reduce binding of Aichi virus 3A to
ACBD3 sensitize virus to PI4KB inhibitors, suggesting that recruitment of PI4KB is
mediated by the 3A-ACBD3 interaction (5). Intriguingly, a recent yeast two-hybrid
screen of hantavirus nonstructural proteins also demonstrated a physical interaction
with ACBD3, suggesting that this protein may be broadly required for viral replication
due to its native association with PI4KB (8). Further study of the cellular function
of ACBD3 is warranted to better understand its role in viral replication and its potential
as a target for therapeutic intervention.
In this study, we used affinity purification-mass spectrometry (AP-MS) to identify
new protein-protein interactions of ACBD3. We discovered a new interaction with the
putative Rab33 GTPase-activating protein (GAP) TBC1D22A/B (9). Detailed mapping of
the ACBD3 interactome revealed a mutually exclusive interaction between PI4KB and
TBC1D22A/B for a highly conserved region in the coiled-coil domain of ACBD3. While
picornaviral 3A proteins bind to the C-terminal GOLD domain on ACBD3, we also find
that the enterovirus and kobuvirus 3A proteins interact with ACBD3 in a functionally
distinct manner based on differential displacement of TBC1D22A/B, isolation of an
ACBD3 mutant that displays differential binding between the 3A proteins, and different
ACBD3 binding sites on the 3A proteins.
RESULTS
AP-MS of Strep-tagged ACBD3 reveals interaction with TBC1D22A/B.
Based on its identification as a specific protein interaction partner for picornavirus
3A proteins, the multifunctional protein ACBD3 was hypothesized to be used by 3A as
an organizing-scaffold protein at the Golgi interface. To identify potential novel
interactions of ACBD3 relevant to the 3A system in host cells, we transiently overexpressed
and affinity purified N- and C-terminally StrepII-tagged ACBD3 in 293T cells and identified
interacting proteins by mass spectrometry (Table 1; see also Tables S1, Tab SI.3A
and SI.4 in the supplemental material). Nonspecific interacting proteins were defined
as the highest-frequency proteins identified in a background model of 550 AP-MS data
sets compiled from the structural and nonstructural genes from 12 different picornaviruses,
excluding the 3A protein itself (see Table S1, Tab SI.1).
TABLE 1
Interacting proteins identified by AP-MS for ACBD3, TBC1D22A, TBC1D22B, and PI4KIIIb
a
Bait
Ca2+
Accession no.
Gene designation
Protein name
Z
score
Replicate count sum
ACBD3 NS
−
15826852
ACBD3
Golgi resident protein GCP60
23.5
699
ACBD3 NS
−
22507409
TBC1D22A
TBC1 domain family member 22A
23.5
13
ACBD3 NS
−
154816184
TMEM55B
Transmembrane protein 55B isoform 2
19.5
11
ACBD3 NS
−
311771621
PI4KB
Phosphatidylinositol 4-kinase beta isoform 2
15.6
5
ACBD3 CS
−
15826852
ACBD3
Golgi resident protein GCP60
23.5
587
ACBD3 CS
−
154816184
TMEM55B
Transmembrane protein 55B isoform 2
19.5
6
ACBD3 CS
−
149944715
PPM1H
Protein phosphatase 1H
15.6
14
ACBD3 CS
−
22507409
TBC1D22A
TBC1 domain family member 22A
11.7
12
ACBD3 CS
−
311771621
PI4KB
Phosphatidylinositol 4-kinase beta isoform 2
7.8
6
ACBD3 NS
+
15826852
ACBD3
Golgi resident protein GCP60
23.5
701
ACBD3 NS
+
148596984
GOLGB1
Golgin subfamily B member 1
23.5
62
ACBD3 NS
+
83641874
CPVL
Probable serine carboxypeptidase CPVL precursor
23.5
21
ACBD3 NS
+
154816184
TMEM55B
Transmembrane protein 55B isoform 2
23.5
17
ACBD3 NS
+
11386135
BCKDHA
2-Oxoisovalerate dehydrogenase subunit alpha, mitochondrial isoform 1 precursor
15.6
27
ACBD3 NS
+
34101272
BCKDHB
2-Oxoisovalerate dehydrogenase subunit beta, mitochondrial precursor
15.6
13
ACBD3 NS
+
38026892
ALG6
Dolichyl pyrophosphate Man9GlcNAc2 alpha-1,3-glucosyltransferase precursor
15.6
12
ACBD3 NS
+
19923748
DLST
Dihydrolipoyllysine residue succinyltransferase component of 2-oxoglutarate dehydrogenase
complex, mitochondrial isoform 1 precursor
15.6
8
ACBD3 NS
+
124494254
PA2G4
Proliferation-associated protein 2G4
11.7
7
ACBD3 NS
+
7019485
PDCD6
Programmed cell death protein 6
11.7
7
ACBD3 NS
+
311771621
PI4KB
Phosphatidylinositol 4-kinase beta isoform 2
11.7
6
ACBD3 NS
+
6912582
PEF1
Peflin
11.7
6
ACBD3 NS
+
55741641
KIDINS220
Kinase D-interacting substrate of 220 kDa
7.8
4
ACBD3 NS
+
38679884
SRI
Sorcin isoform b
7.8
3
ACBD3 CS
+
15826852
ACBD3
Golgi resident protein GCP60
23.5
842
ACBD3 CS
+
83641874
CPVL
Probable serine carboxypeptidase CPVL precursor
23.5
24
ACBD3 CS
+
149944715
PPM1H
Protein phosphatase 1H
23.5
16
ACBD3 CS
+
311771621
PI4KB
Phosphatidylinositol 4-kinase beta isoform 2
19.5
18
ACBD3 CS
+
154816184
TMEM55B
Transmembrane protein 55B isoform 2
19.5
11
ACBD3 CS
+
56549147
STEAP3
Metalloreductase STEAP3 isoform b
15.6
14
ACBD3 CS
+
4504805
BLZF1
Golgin-45
15.6
10
ACBD3 CS
+
38026892
ALG6
Dolichyl pyrophosphate Man9GlcNAc2 alpha-1,3- glucosyltransferase precursor
11.7
12
ACBD3 CS
+
22507409
TBC1D22A
TBC1 domain family member 22A
11.7
7
ACBD3 CS
+
19923748
DLST
Dihydrolipoyllysine residue succinyltransferase component of 2-oxoglutarate dehydrogenase
complex, mitochondrial isoform 1 precursor
7.8
9
ACBD3 CS
+
34101272
BCKDHB
2-Oxoisovalerate dehydrogenase subunit beta, mitochondrial precursor
7.8
6
ACBD3 CS
+
40789249
DARS2
Aspartyl-tRNA synthetase, mitochondrial
7.8
3
ACBD3 CS
+
148839335
DPY19L1
Protein Dpy-19 homolog 1
7.8
2
ACBD3 CS
+
11559925
XPNPEP3
Probable Xaa–Pro aminopeptidase 3 isoform 1
7.8
2
TBC1D22A
−
22507409
TBC1D22A
TBC1 domain family member 22A
23.5
538
TBC1D22A
−
15826852
ACBD3
Golgi resident protein GCP60
23.5
42
TBC1D22A
−
4506583
RPA1
Replication protein A 70-kDa DNA binding subunit
11.7
36
TBC1D22A
−
4506587
RPA3
Replication protein A 14-kDa subunit
11.7
14
TBC1D22A
−
4506585
RPA2
Replication protein A 32-kDa subunit
11.7
6
TBC1D22A
−
17999541
VPS35
Vacuolar protein sorting- associated protein 35
7.8
10
TBC1D22A
−
124494254
PA2G4
Proliferation-associated protein 2G4
7.8
8
TBC1D22A
−
14211889
DPY30
Protein Dpy-30 homolog
7.8
6
TBC1D22A
−
17978519
VPS26A
Vacuolar protein sorting- associated protein 26A isoform 1
7.8
3
TBC1D22A
−
23397429
EIF3M
Eukaryotic translation initiation factor 3 subunit M
7.8
3
TBC1D22B
−
40068063
TBC1D22B
TBC1 domain family member 22B
23.5
1,575
TBC1D22B
−
15826852
ACBD3
Golgi resident protein GCP60
23.5
117
TBC1D22B
−
198041662
PYCRL
Pyrroline-5-carboxylate reductase 3
6.7
5
TBC1D22B
−
4505067
MAD2L1
Mitotic spindle assembly checkpoint protein MAD2A
6.7
5
TBC1D22B
−
150378533
USP7
Ubiquitin carboxyl-terminal hydrolase 7
6.7
4
TBC1D22B
−
51479145
ARFGEF1
Brefeldin A-inhibited guanine nucleotide exchange protein 1
6.7
4
PI4KB
−
311771621
PI4KB
Phosphatidylinositol 4-kinase beta isoform 2
23.5
1,686
PI4KB
−
15826852
ACBD3
Golgi resident protein GCP60
23.5
35
PI4KB
−
284807150
GBA
Glucosylceramidase isoform 2
18.8
23
PI4KB
−
294832006
PPP2R2A
Serine/threonine-protein phosphatase 2A 55-kDa regulatory subunit B alpha isoform
2
14.1
7
PI4KB
−
14141170
MTA2
Metastasis-associated protein MTA2
14.1
6
PI4KB
−
154350213
C10orf76
UPF0668 protein C10orf76
9.4
17
PI4KB
−
27363458
LRFN4
Leucine-rich repeat and fibronectin type III domain-containing protein 4 precursor
9.4
13
a
Interacting proteins identified by AP-MS for ACBD3, TBC1D22A, TBC1D22B, and PI4KIIIb
were weighted by Z score of the peptide counts. Proteins are listed here with replicate
Z scores and peptide counts in the experimental set with a minimum of n = 5 biological
replicates and were scored against a background set of 550 unrelated picornaviral
protein AP-MS experiments, excluding the 3A protein itself (Materials and Methods).
Shown here are the top-scoring proteins that appeared in at least two replicate experiments
and with >1 count in at least one experiment; a full Z score table is provided in
Table S1, Tab SI.4 in the supplemental material. The major interacting proteins for
ACBD3 included TBC1D22A and PI4KB, as well as PPM1H, TMEM55B isoform 2, CPVL, and
GOLGB1. Reciprocal AP-MS experiments with TBC1D22A, its closely related isoform TBC1D22B,
and PI4KB confirmed interaction with ACBD3. Proteins are C-terminally StrepII-tagged
(CS) unless otherwise noted as N-terminally tagged (NS).
Using this AP-MS approach combined with Z score ranking for interaction specificity,
we found that ACBD3 interacted with the TBC1 domain family member 22A protein (TBC1D22A)
in a highly specific manner and at levels that were comparable to those of PI4KB (Table 1;
full Z scores are provided in Table S1, Tab SI.4 in the supplemental material). Additional
proteins such as transmembrane protein 55B isoform 2 (TMEM55B) and protein phosphatase
1H (PPM1H) were also observed to be specific for ACBD3. The known ACBD3-interacting
Golgi protein giantin/GOLGB1 was identified in affinity purifications of N-terminally
tagged ACBD3 only in a buffer that included potassium chloride and divalent cations
(Table 1) (7).
TBC1D22A is a 58-kDa protein that is localized to the Golgi apparatus and involved
in Golgi membrane maintenance, along with its closely related isoform TBC1D22B (10).
Both contain a C-terminal TBC domain that is responsible for Rab GTPase activation
with a putative preference for Rab33A/B (9). Overexpression of TBC1D22B was shown
to cause disruption of the endoplasmic reticulum (ER)-Golgi intermediate compartment
(ERGIC), which was dependent on its GTPase-activating protein (GAP) activity (10).
Based on the localization of ACBD3 and picornavirus replication to ERGIC-associated
membranes, TBC1D22A/B merited further investigation.
Affinity purification of TBC1D22A/B or PI4KB captures ACBD3 and 14-3-3 proteins.
To confirm the ACBD3-TBC1D22A interaction, reciprocal affinity purification was undertaken
with N- and C-terminally Strep-tagged TBC1D22A and TBC1D22B (see Table S1, Tab SI.3A
and SI.3B in the supplemental material). The top-ranking interacting protein for both
TBC1D22A and TBC1D22B was ACBD3 (Table 1). Multiple 14-3-3 isoforms were also found
in high abundance in affinity purifications for both TBC1D22A and TBC1D22B (see Table S1,
Tab SI.5). 14-3-3 proteins are 27-kDa adaptor proteins that bind phosphoserine residues
on a multitude of cellular proteins involved in diverse signaling pathways (11). TBC1D22A/B
phosphopeptides are reported in Table S1, Tab SI.6 and include a putative 14-3-3 recognition
site at serine 167 (TBC1D22A) or serine 154 (TBC1D22B; spectra are provided in Fig. S1).
No peptides for PI4KB were recovered in any AP-MS experiments on TBC1D22A or TBC1D22B,
suggesting that ACBD3-TBC122A/B-containing complexes do not contain PI4KB.
To further confirm the PI4KB-ACBD3 interaction, affinity purification of Strep-tagged
PI4KB was performed. The top-ranking protein was ACBD3, while a 14-3-3 isoform was
ranked third (Table 1; see also Table S1, Tab SI.3D in the supplemental material).
The 14-3-3 proteins have been shown to bind PRKD1-phosphorylated serine 294 of PI4KB
and influence its catalytic activity (12). This phosphorylated site and others were
identified in our affinity purifications as well, as reported in Table S1, Tab SI.6
(spectrum is provided in Fig. S1). Several additional proteins of unknown significance
(C10orf76, GBA, and MTA2) discovered in a recent Flag tag AP-MS experiment on PI4KB
also ranked highly in our screen (13). Finally, as in the reciprocal case above, no
peptides for TBC1D22A or TBC1D22B were recovered in any AP-MS experiments on PI4KB.
ACBD3-TBC1D22A/B interaction maps to coiled-coil region and overlaps with PI4KB-interacting
region of ACBD3.
To further query the hypothesis that TBC1D22A/B binding and PI4KB binding may represent
distinct, potentially competitive binding states of ACBD3, a mammalian two-hybrid
reporter assay was used to map the sites of interaction on ACBD3 by deletion mutagenesis
(Fig. 1). N-terminal truncations of the acyl-CoA binding domain, C-terminal truncations
of the GOLD domain, and alanine scanning through the entirety of the CAR domain did
not affect ACBD3 binding to TBC1D22A/B, while deletions and point mutations located
in the conserved glutamine-rich coiled-coil region between residues 246 and 321 significantly
reduced TBC1D22A/B binding (see Fig. S2 and S3 in the supplemental material; also
Fig. 1). This region has previously been implicated in binding PI4KB (6). Mapping
of PI4KB on ACBD3 revealed significant overlap with the region bound by TBC1D22A/B
(Fig. 1A). Three mutants with mutations in the glutamine-rich region (VQF255AAA, PGN267AAA,
and EQHY281AAAA) significantly reduced ACBD3 binding to both PI4KB and TBC1D22A/B
(Fig. 1B; see also Fig. S2B). However, we could isolate no mutant of ACBD3 that retained
wild-type levels of PI4KB binding while disrupting TBC1D22A/B binding, or vice versa,
including mutation of a recovered phosphorylation site, SS344 (see Table S1, Tab SI.6
). Two single point mutants in the glutamine-rich region (F258A and Y285A) did demonstrate
a significant difference in PI4KB binding versus TBC1D22B and merit further investigation
(Fig. S3). Although we have mapped the critical binding region of TBC1D22A/B to the
Q-rich region of ACBD3, we cannot discount the role of the GOLD domain, as it appears
to have an influence on the binding of these proteins as shown in Fig. 1A. We also
note that TBC1D22A/B was not detected in any PI4KB AP-MS experiment nor was PI4KB
detected in any TBC1D22A/B AP-MS experiment (see Table S1, Tab SI.3B–SI.3D). Together,
these data support the notion that PI4KB and TBC1D22A/B participate in a mutually
exclusive relationship with ACBD3.
FIG 1
TBC1D22A/B interaction on ACBD3 is localized to the coiled-coil region and overlaps
the PI4KB-interacting region. (A) Mapping of TBC1D22A/B and PI4KB binding localizes
to the glutamine-rich (Q) region on ACBD3 by mammalian two-hybrid screening. The three
proteins demonstrate similar binding values for all of the mutants tested, with a
slight preference of TBC1D22A over TBC1D22B and PI4KB. (B) Alanine mutants across
the C-terminal half of ACBD3 demonstrate that TBC1D22A/B and PI4KB binding to ACBD3
is disrupted only by mutations in the glutamine-rich region and not by mutations in
the C-terminal GOLD domain. Binding values for the protein-protein interaction reporter
(firefly luciferase) are plotted as a percentage of the transfection control values
(Renilla luciferase). The graphical representation of each construct is colored based
on the binding value. Low binding is depicted in black while high binding is depicted
in red. The critical region defined by the collection of constructs is demarcated
by the yellow box with the dashed outline (ACBD, acyl-CoA binding domain; CAR, charged
amino acid region; Q, glutamine-rich region; GOLD, Golgi dynamics domain).
The N terminus of TBC1D22A/B is required for ACBD3 interaction.
To understand whether the RabGAP domain or some other region of TBC1D22A/B was responsible
for the interaction with ACBD3, both mammalian two-hybrid mapping and AP-Western blotting
were performed on deletion mutants of TBC1D22A/B. By both of these methods, the ACBD3-interacting
region was localized to a predicted alpha-helix between residues 90 and 105 of TBC1D22A,
demonstrating that the TBC domain was not required for binding (Fig. 2A; see also
Fig. S4A in the supplemental material). Alanine scanning mutagenesis of this region
revealed a valine-leucine residue pair that was required for ACBD3 binding along with
a minor involvement of residues VVME93 (Fig. 2B; see also Fig. S4B).
FIG 2
ACBD3 interaction localizes to the N terminus on TBC1D22A and TBC1D22B and is disrupted
by the same valine-leucine mutation. (A) Deletion mutagenesis of TBC1D22A specifically
localizes its interaction with ACBD3 by mammalian 2-hybrid screening to a predicted
N-terminal helix near residues 90 to 105. (B) Alanine scanning reveals a critical
dependence on residues VL101 with a contribution from VVME93. (C) Deletion mutagenesis
of TBC1D22B also localizes its interaction with ACBD3 to a predicted N-terminal helix,
as in TBC1D22A, despite an amino acid identity of <40% outside the RabGAP domain.
(D) Alanine scanning across the TBC1D22B helix demonstrates that the ACBD3 interaction
is significantly disrupted by the VL100AA mutation, as well as a contribution from
upstream residues LNS88.
TBC1D22A and TBC1D22B share 78% amino acid identity in their respective RabGAP domains
yet share <40% amino acid identity outside the RabGAP domain. Deletion mapping of
TBC1D22B similarly localized the ACBD3 interaction to a predicted alpha-helix with
conserved sequence to TBC1D22A, including the valine-leucine pair (Fig. 2C; see also
Fig. S4C in the supplemental material). Alanine scanning of this region on TBC1D22B
also revealed substantial defects (≥100-fold decrease) in binding, especially at VL100
(Fig. 2D). Mutation of phosphorylation sites on TBC1D22B discovered by mass spectrometry,
all of which reside outside the identified interacting region, to glutamic acid as
a phosphomimetic did not affect binding to ACBD3 (see Fig. S4D and Table S1, Tab SI.6).
Mammalian two-hybrid mapping on TBC1D22A/B was confirmed by AP-Western blotting and
AP-MS (see Fig. S4A to C). Interestingly, six total AP-MS experiments with full-length
TBC1D22A and TBC1D22B and deletion mutants of TBC1D22A, all of which retained ACBD3
binding, showed that they copurified with ARFGEF1, a trans-Golgi membrane-localized
Sec7 domain containing the guanine exchange factor for Arf1 (14). This interaction
is highly specific, as peptides to ARFGEF1 were detected in only those six deletion
and full-length TBC1D22A/B AP-MS experiments out of a total of >2,100 AP-MS runs in
our lab (spectra are provided in Fig. S1).
Deletion mapping, alanine mutagenesis, and AP-Western blotting of TBC1D22A revealed
a significant involvement of serines 165 and 167 for 14-3-3 protein recruitment. This
site matches the canonical type I 14-3-3 binding motif of R-[SFYW]-X-pS-X-P, suggesting
that serine 167 is the phosphorylated serine responsible for 14-3-3 recruitment (15).
TBC1D22A deletion and point mutants that disrupted 14-3-3 binding retained the ability
to bind ACBD3, while the TBC1D22A VL101AA mutant that disrupted ACBD3 interaction
was still able to bind 14-3-3 isoforms, similarly to wild-type TBC1D22A (Fig. 2A;
see also Fig. S4B in the supplemental material). While the significance of TBC1D22A
interaction with 14-3-3 proteins remains unknown, these data suggest that TBC1D22A
interaction with ACBD3 is not mediated by 14-3-3 isoforms or vice versa.
PI4KB-ACBD3 interaction maps to the N terminus of PI4KB.
We employed both AP-Western blotting and mammalian two-hybrid mapping in conjunction
with deletion mutants to localize the region of PI4KB required for interaction with
ACBD3 (Fig. 3). By N-terminal and C-terminal deletion, a region of 19 amino acids,
extending from residues 53 to 70, of PI4KB was necessary for interaction with ACBD3
by both assays, indicating that the catalytic domain, the lipid kinase unique (LKU)
region, and the Hom2 region of PI4KB were dispensable (16) (Fig. 3A; see also Fig. S5A,
B, and C in the supplemental material). Analogously to what was found in TBC1D22A/B,
alanine scanning of this narrow region in PI4KB revealed that a valine-leucine pair
(VL67) was required for ACBD3 interaction with additional contribution from upstream
residues (Fig. 3B), suggesting that these two proteins bind ACBD3 via similar motifs.
These results were also confirmed by AP-Western blotting (see Fig. S5A to C). Two
mutants, VL67 and IDP55, that retained the ability to bind alternative PI4KB partners,
C10orf76 and Rab11B, were identified, suggesting that the ACBD3 binding deficit was
specific (Fig. 3B). We note that the QE65AA mutant demonstrated no ability to bind
C10orf76 or Rab11B but bound to ACBD3 at levels 6-fold greater than those for the
wild type according to the mammalian two-hybrid reporter (Fig. 3B), although it is
not necessarily clear whether these differences in binding are due to increased binding
to one factor or to an inability to bind another competing factor. Finally, mutation
of PI4KB phosphorylation sites discovered by mass spectrometry, all of which reside
outside the critical interaction region, to glutamic acid did not affect binding to
ACBD3, C10orf76, or Rab11B (see Fig. S5D).
FIG 3
ACBD3 interaction localizes to the N terminus on PI4KB and is disrupted by the same
valine-leucine mutation. (A) Deletion mapping of PI4KB localizes its interaction with
ACBD3 to the far N terminus on PI4KB by mammalian 2-hybrid screening. (B) Alanine
scanning of the putative alpha-helix on PI4KB demonstrated a significant contribution
of residues VL67 and IDP55 to its interaction with ACBD3. Binding of PI4KB interactors
Rab11B and C10orf76 was used to control for global protein defects. Mutant QE65AA
abrogated binding of PI4KB to C10orf76 and Rab11B but increased binding to ACBD3 by
6-fold.
TBC1D22A/B competes with PI4KB for ACBD3 binding in vitro.
The combined AP-MS and fine-scale mapping data for TBC1D22A/B and PI4KB suggest a
mutually exclusive binding relationship with ACBD3. If this were the case, we would
expect there to be a competitive binding relationship between these two proteins and
ACBD3. To qualitatively test this hypothesis in a competition ex-periment, increasing
amounts of pro-tein lysate from 293T cells expressing TBC1D22A-Flag were added to
premixed 293 lysate from PI4KB-V5- and ACBD3-Strep-expressing cells (Fig. 4A). Affinity
purification of ACBD3-Strep revealed decreasing amounts of copurified PI4KB-V5 as
a function of increasing amounts of TBC1D22A-Flag (Fig. 4B). This competition was
independent of RabGAP activity, as only the N terminus of TBC1D22A was required (Fig. 4C).
293T cell lysate that did not express TBC1D22A-Flag could not compete off PI4KB-V5
(Fig. 4D).
FIG 4
The N terminus of TBC1D22A can compete with PI4KB for binding of ACBD3. (A) Experimental
setup to test for binding competition. V5-tagged PI4KB expressing 293T lysate was
premixed with Strep-tagged ACBD3 expressing 293T lysate and divided seven ways. Increasing
amounts of Flag-tagged TBC1D22A expressing 293T lysate were added to each tube, and
lysates were affinity purified for Strep-tagged ACBD3 and assayed by Western blotting
with anti-V5, anti-Strep, and anti-Flag antibodies. (B) Addition of increasing amounts
of full-length TBC1D22A-Flag reduces the amount of PI4KB-V5 bound by ACBD3-Strep.
(C) A Flag-tagged N-terminal fragment (1 to 128) of TBC1D22A is sufficient to compete
off PI4KB-V5 from ACBD3-Strep. (D) Untransfected lysate is unable to compete PI4KB-V5
from ACBD3-Strep.
3A binding region of ACBD3 localizes to the GOLD domain and associates with ACBD3
self-binding.
Previously, we and others demonstrated that enteroviral and kobuviral 3A proteins
interact with ACBD3 by AP-MS and AP-Western blotting (5, 6). To test whether other
picornavirus 3A proteins bound ACBD3 more transiently, we tested hepatovirus, klassevirus,
parechovirus, cardiovirus, and aphthovirus 3A proteins in the mammalian two-hybrid
assay. In this assay, both kobuvirus and enterovirus 3A proteins bound most strongly
to ACBD3 while the 3A proteins of hepatovirus, klassevirus, and parechovirus all demonstrated
a significant but lesser (approximately 10-fold) ability to bind ACBD3 (Fig. 5A).
Cardiovirus and aphthovirus 3A proteins did not demonstrate binding to ACBD3 above
background.
FIG 5
Picornaviral 3A proteins interact with the GOLD domain of ACBD3 and demonstrate that
the far C terminus is critical for ACBD3 self-binding. (A) Picornavirus 3A proteins
were tested for their ability to bind ACBD3 by mammalian two-hybrid screening. In
addition to kobuvirus and enterovirus, 3A proteins from hepatitis A virus, klassevirus,
and human parechovirus 1 (HPeV1) demonstrated an intermediate affinity for ACBD3.
Cardiovirus and aphthovirus showed no ability to bind ACBD3 above background. CVB3,
coxsackievirus B3; TMGDVII, Theiler's murine encephalomyelitis virus (strain GDVII);
FMDV, foot-and-mouth disease virus. (B) Poliovirus and Aichi virus 3A proteins interact
with the C-terminal GOLD domain of ACBD3. (C) Mutations in the GOLD domain disrupt
picornavirus 3A binding and ACBD3 self-binding. One ACBD3 mutant (SYS511AAA) demonstrated
a subtle ability to distinguish between poliovirus and Aichi virus 3A proteins. (D)
Picornavirus 3A proteins and ACBD3 demonstrate a critical dependence on the presence
of residue 525 for binding to ACBD3.
Aichi virus 3A interaction with ACBD3 was previously mapped to the GOLD domain of
ACBD3 (6). Given the broad range of picornavirus 3A proteins interacting with ACBD3,
we used the mammalian two-hybrid reporter system to investigate whether these 3A proteins
also interacted with the C-terminal GOLD domain. Both poliovirus and Aichi virus 3A
proteins mapped to the C-terminal half of ACBD3 (Fig. 5B), consistent with deletion
mapping by AP-Western blotting. To further refine the deletion mapping, we found that
alanine mutations in the coiled-coil region of ACBD3 that did not bind TBC1D22A/B
and PI4KB (VQF255AAA and EQHY281AAAA) retained picornavirus 3A binding. However, all
alanine mutations in the GOLD domain disrupted binding of both picornaviral 3A proteins
(Fig. 5C). One mutation near the C terminus (SYS511AAA or Y512A) demonstrated a slight
difference in preference of poliovirus 3A over Aichi virus 3A, suggesting that the
binding between the two proteins may be only subtly different (Fig. 5C; see also Fig.
S3B in the supplemental material).
Fine-scale mapping indicated that binding of the 3A proteins was critically dependent
on the presence of Tyr residue 525, only three amino acids from the C-terminus of
wild-type ACBD3 (Fig. 5D). Mapping of the hepatitis A virus (HAV) and klassevirus
3A interactions with ACBD3 revealed an interaction profile similar to those of Aichi
virus and poliovirus 3A (see Fig. S6 in the supplemental material), suggesting that
these diverse picornaviruses bind ACBD3 in an analogous manner despite their wide
sequence divergence (17–19).
The critical dependence of multiple GOLD domain-interacting proteins on the presence
of Tyr-525 for binding to ACBD3 suggested that a potential intramolecular ACBD3 interaction
was required. We tested whether ACBD3 could bind itself in the context of the mammalian
two-hybrid assay. ACBD3 demonstrated an interaction with itself that mapped to GOLD
domain as well as the threshold effect at Tyr-525 (Fig. 5B to D). Mutations in GOLD
domain that disrupted interaction with ACBD3-interacting partners similarly disrupted
ACBD3 homomerization. However, one mutant (SYL414AAA) specifically disrupted ACBD3
homomerization while only slightly decreasing binding to poliovirus 3A and Aichi virus
3A, demonstrating that 3A binding to ACBD3 is not dependent on ACBD3 homomerization,
at least for these viral species, as klassevirus 3A and hepatitis A virus 3A did not
bind the ACBD3 SYL414AAA mutant above background (see Fig. S6B in the supplemental
material). While these results are suggestive of a possible interplay between ACBD3
homomerization and binding by viral 3A proteins, additional biochemical characterization
will be required to explore this dynamic.
ACBD3 binding regions on picornavirus 3A are distinct.
To further investigate the possibility of differential ACBD3 interactions with Aichi
virus and poliovirus 3A proteins, we extended our deletion and mammalian two-hybrid
mapping of the ACBD3-interacting region for both of these proteins. Deletion of the
C-terminal half of poliovirus 3A reduced ACBD3 binding by 100-fold, while a comparable
deletion in Aichi virus 3A retained ACBD3 binding (Fig. 6A and B), consistent with
previous results in which alanine scanning the entirety of Aichi virus 3A revealed
only two point mutations that affected ACBD3 binding (5). N-terminal deletions in
the GBF1 binding domain of poliovirus 3A significantly increased ACBD3 binding relative
to that of the wild type by more than 10-fold (Fig. 6A). Alanine scanning of much
of the N-terminal half of poliovirus 3A revealed several point mutants C terminal
to the GBF1 binding site that were critical for binding ACBD3 by AP-Western blotting
(D29A, R34A, KKGW42-KGA, and R54A) (see Fig. S9C in the supplemental material). These
results implicate multiple portions of Aichi virus 3A for binding to ACBD3, while
poliovirus 3A appears to contain a defined ACBD3 binding domain that is C-terminal
to its GBF1 binding region and dimerization region (20).
FIG 6
Mapping of poliovirus and Aichi virus 3A interaction with ACBD3 reveals differences
in 3A-ACBD3 interaction architecture. (A) Deletions of poliovirus 3A illustrate that
a region C terminal to the GBF1 binding region and N terminal of the predicted transmembrane
region is required for binding ACBD3. (B) Deletions of Aichi virus 3A reveal the presence
of multiple ACBD3 binding regions across the 3A protein, with the bulk of ACBD3 binding
mapping to the N-terminal half of Aichi virus 3A.
Kobuviral 3A proteins prevent TBC1D22A binding to ACBD3, while enterovirus 3A proteins
do not.
Given that TBC1D22A/B appears to compete with PI4KB for ACBD3 binding and that picornaviruses
are dependent on PI4KB for activity, we hypothesized that picornavirus 3A interaction
with ACBD3 could influence the outcome of this competition. We note that in the picornavirus
3A AP-MS data that we have published previously (5), peptides to TBC1D22A/B were recovered
in multiple enteroviral 3A AP-MS experiments while no peptides to TBC1D22A/B were
ever identified in kobuviral 3A AP-MS experiments where a stable ACBD3-PI4KB complex
was identified (see Table S1, Tab SI.4 in the supplemental material) (5). To test
this hypothesis, we examined whether the presence of different 3A proteins affected
the TBC1D22A-ACBD3 interaction. We coexpressed kobuvirus 3A and enterovirus 3A proteins
together with TBC1D22A and then assayed affinity-purified TBC1D22A by Western blotting.
Whereas the expression of enterovirus 3A proteins did not affect the recovery of affinity-purified
TBC1D22A, we observed an apparent affect on the expression of TBC1D22A in the presence
of kobuvirus 3A (see Fig. S7).
We investigated whether the presence of Aichi virus 3A or poliovirus 3A could affect
the endogenous level of TBC1D22A mRNA. Using HEK293T cells transfected with Aichi
virus 3A-Flag or poliovirus 3A-Flag, we measured the level of TBC1D22A mRNA by quantitative
reverse transcription-PCR (qRT-PCR). There was no significant difference in the level
of TBC1D22A mRNA in the presence of poliovirus 3A and only modest changes (<14%) in
the presence of TBC1D22A (see Fig. S8 in the supplemental material).
To investigate the effect of picornavirus 3A protein interaction with respect to PI4KB
and TBC1D22A interaction with ACBD3, and to avoid the apparent effect of coexpression
differences in the presence of 3A proteins, we separated the expression of the proteins
by independently transfecting Flag-tagged picornavirus 3A proteins and TBC1D22A-V5
into different plates of HEK293T cells. We then immunoprecipitated TBC1D22A-V5 complexes
onto beads and added exogenous HEK293T lysates containing overexpressed poliovirus
3A-Flag and Aichi virus 3A-Flag. Finally, the TBC1D22A-V5 beads were recovered and
assayed for ACBD3 and 3A-Flag recruitment by Western blotting (Fig. 7A). Poliovirus
3A-Flag could be readily detected in TBC1D22A-ACBD3-containing complexes, while Aichi
virus 3A-Flag could not (Fig. 7B). Quantification of this difference demonstrated
a >7-fold increase in poliovirus 3A recovery from the TBC1D22A-V5 beads over that
of Aichi virus 3A (Fig. 7C). Collectively, these results support a model whereby the
kobuviral and enteroviral 3A proteins may differentially modulate the interaction
of PI4KB and TBC1D22A/B with ACBD3.
FIG 7
Aichi virus 3A does not occupy the same ACBD3 as does TBC1D22A, while poliovirus 3A
proteins can occupy the same ACBD3 as does TBC1D22A. (A) Experimental setup to test
the influence of 3A proteins on the interaction of TBC1D22A with ACBD3. V5-tagged
TBC1D22A, Flag-tagged Aichi virus 3A, and Flag-tagged poliovirus 3A were each singly
transfected into a 15-cm plate of HEK293T cells. TBC1D22A-V5 was immunoprecipitated
with anti-V5 beads, washed 4 times, and left on the bead, to which Flag-tagged picornaviral
3A protein lysate was added and left to incubate overnight at 4°C. This lysate was
again centrifuged with the anti-V5 beads and washed 4 times, and then captured proteins
were boiled off the beads in SDS sample buffer to examine whether 3A-Flag could occupy
the same ACBD3 as TBC1D22A by Western blotting. (B) Flag-tagged Aichi virus 3A does
not copurify with TBC1D22A-V5 and bound ACBD3, while Flag-tagged poliovirus 3A copurifies
with TBC1D22A-V5 and its captured ACBD3 by Western blotting. (C) Quantification of
bound poliovirus 3A-Flag relative to Aichi virus-Flag, adjusted for the amount of
ACBD3 pulled down, reveals a 7-fold difference in binding.
DISCUSSION
In this study, we have identified TBC1D22A/B as a new interacting partner of ACBD3,
a protein of central importance in Golgi organization and picornaviral replication.
TBC1D22A/B is a Golgi membrane-localized putative Rab33 RabGAP (9). Altered ER-Golgi
morphology has been associated with overexpression of TBC1D22B and is dependent on
the presence of TBC1D22B RabGAP activity (10). We found that the interaction between
TBC1D22A/B and ACBD3 was dependent on a narrow region that maps to a predicted helix
in the N terminus of TBC1D22A/B. This interaction may determine localization of these
RabGAPs to the Golgi membrane and their ability to affect Golgi morphology. TBC1D22A/B
bound to the same region on ACBD3 as did PI4KB, and we found that the same residues
that were critical for TBC1D22A/B binding were also critical for PI4KB binding. Together
with our finding that TBC1D22A/B directly competed with PI4KB for ACBD3 binding, this
suggests that, in addition to putatively regulating Rab33 GTP binding at the Golgi
membrane, these RabGAPs may in part determine the Golgi membrane localization of PI4KB,
thereby influencing cellular phosphatidylinositol 4-phosphate (PI4P) levels and Golgi
morphology. It is also of note that other RNA viruses and intracellular pathogens
use TBC domain-containing proteins and their binding partners to manipulate Rab dynamics
at the ER-Golgi interface for their replication, including hepatitis C virus, Legionella
and Shigella species, and pathogenic Escherichia coli (21–24).
Given that both TBC1D22A/B and PI4KB share very similar binding sequences and that
no ACBD3 mutant could be found that discriminates between these two partners, it remains
an open question whether direct regulation of either of these proteins produces modified
binding to ACBD3. Mutation of the main phosphorylation site on ACBD3 (SS344) to alanine
or glutamic acid had no differential effect on TBC1D22A/B versus PI4KB binding. Conversion
of the identified phosphorylation sites on TBC1D22B and PI4KB to glutamic acid also
did not disrupt interaction with ACBD3. TBC1D22A/B and PI4KB are organized in similar
fashions, with an N-terminal ACBD3 binding domain, a 14-3-3 binding phosphorylation
site in the middle, and a C-terminal enzymatic domain. Because 14-3-3 does not bind
to phosphomimetic mutations, we were unable to test the effect of 14-3-3 binding on
interaction with ACBD3 without phosphorylation (15). At present, in the absence of
three-dimensional structure information for each of these proteins, it is not clear
what regulates the binding of TBC1D22A/B versus PI4KB to the glutamine-rich region
of ACBD3.
The picornaviral 3A proteins may inform the cellular biology that regulates whether
TBC1D22A/B or PI4KB is bound to ACBD3. An open question in picornavirus biology is
why enteroviruses retain a specific GBF1 recruitment domain and are sensitive to brefeldin
A while kobuviruses do not bind GBF1 and are insensitive to brefeldin A. Given that
multiple picornaviruses rely on ACBD3 and PI4KB, direct competition by TBC1D22A/B
suggests that viruses have evolved a mechanism to subvert cellular regulation of these
two proteins. Our data show that the kobuviral 3A proteins appear to abrogate the
binding of TBC1D22A/B to ACBD3, thus favoring the formation of a stable 3A-ACBD3-PI4KB
complex that remains even after 25 to 30 min of washing. This stable complex may obviate
the need for GBF1 recruitment and activity and allow kobuviruses to directly influence
PI4KB localization and activity with respect to viral replication in a manner similar
to, yet distinct from, that of enterovirus 3A. It is also remarkable that a protein
that binds entirely to the GOLD domain can manipulate the binding of proteins in other
domains of ACBD3, suggesting cross talk between ACBD3’s various domains. Although
we have mapped the critical binding region of TBC1D22A/B to the Q-rich region of ACBD3,
we cannot discount the role of the GOLD domain, as it appears to have an influence
on the binding of these proteins as shown in Fig. 1A.
It is not clear if or how enteroviruses manipulate the TBC1D22A/B-ACBD3 interaction,
given that enterovirus 3A and TBC1D22A/B appear to occupy the same ACBD3 in our affinity
purifications. The ACBD3-PI4KB-TBC1D22A/B system may provide a clue as to why enterovirus
3A proteins directly bind to and require GBF1 for replication. Mapping of the ACBD3-interacting
region on the poliovirus 3A protein demonstrated an ACBD3 binding domain C-terminal
to the region responsible for binding GBF1, suggesting that poliovirus 3A’s recruitment
of GBF1 to Golgi membranes might be for recruitment of GBF1 to ACBD3. GBF1-Arf1 dynamics
at the membrane may be responsible for recruiting another regulator that determines
whether TBC1D22A/B or PI4KB is bound to ACBD3. Our affinity purification conditions
may not promote the formation of a 3A-GBF1-ACBD3-PI4KB complex, thus allowing TBC1D22A/B
to compete off PI4KB from ACBD3.
The reliance on overexpressed proteins in these protein-protein interaction studies
is an important caveat. For example, nonphysiological levels of ACBD3 and/or TBC1D22A/B
could produce an interaction that would be unlikely to occur at native levels. While
our attempts to address this have been hampered by the lack of immunoprecipitation-competent
antibodies to the untagged version of TBC1D22A/B, we have previously published experiments
which indicate that ACBD3 and TBC1D22A/B interact at native expression levels. Specifically,
we note that native TBC1D22A/B copurified with native ACBD3 in the presence of affinity-purified
enterovirus 3A proteins, suggesting that the interaction is not entirely an artifact
of overexpressing ACBD3 or TBC1D22A/B (see Table S7 in reference 5).
Future experiments with the PI4KB mutants isolated in this study will help illuminate
whether different PI4KB mutants can rescue enterovirus replication after depletion
of native PI4KB. However, it is of note that enteroviruses selected for resistance
to enviroxime family PI4KB inhibitors contain mutations in the ACBD3 binding region
of 3A (V45A and H57Y) (25). How these mutants affect ACBD3 binding by 3A and whether
PI4KB can compete TBC1D22A/B off ACBD3 are important outstanding questions.
In conclusion, multiple picornaviruses coopt the same central ACBD3-PI4KB axis for
replication but utilize different cellular mechanisms to manipulate the system. The
kobuviruses and enteroviruses may reflect convergent evolutionary strategies to manipulate
this key lipid regulator. The mutants obtained in this study will be useful tools
to test whether PI4KB localization to ACBD3 is required for picornavirus replication;
to test the effect of ACBD3, TBC1D22A/B, and GBF1 on PI4P levels in the cell; and
to discover regulatory mechanisms that govern the ACBD3 interactome.
MATERIALS AND METHODS
Cells, plasmids, and cloning.
293T cells were maintained in Dulbecco modified Eagle medium (DMEM)-H21 medium supplemented
with 10% fetal bovine serum (FBS) and penicillin-streptomycin. All genes for transient
transfections were cloned into a modified pCDNA4-TO vector containing an N-terminal
or C-terminal 2× Strep II-tag as described previously using primers from Table S1
in the supplemental material (26). Accession numbers used for genes were as follows:
ACBD3, NM_0022735; PI4KB, NM_002651; TBC1D22A, NM_014346; TBC1D22B, NM_017772; Rab11B.
The human C10orf76 gene was synthesized commercially (BioBasic Inc., Canada).
Transient transfections were performed in a 15-cm plate of 293T cells at 50 to 60%
confluency using 10 µg of total plasmid and a 3:1 ratio of TransIT-LT1 transfection
reagent to plasmid (Mirus Bio). Protein lysates were prepared in 0.5% NP-40 in a background
buffer of either 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA or 50 mM HEPES-KOH
(pH 6.8), 150 mM potassium acetate (KOAc), 2 mM magnesium acetate (MgOAc), 1 mM CaCl2,
15% glycerol, 1× Roche EDTA-free protease inhibitor cocktail. Affinity purifications
and Western blotting assays were otherwise performed as described previously (5, 26).
For the ACBD3 binding competition assay, transient transfections of ACBD3-Strep, TBC1D22A-Flag,
and PI4KB-V5 in individual 15-cm plates of 293T cells were performed as described
above. Lysates from each plate were prepared in 2 ml of EDTA-containing buffer. PI4KB-V5
and ACBD3-Strep lysate were premixed in a 15-ml Falcon tube for 4 h at 4°C. The lysate
was then divided into 7 aliquots of 500 µl each, and 0, 3, 10, 30, 100, 300, and 1,000 µl
of lysate from the TBC1D22A-Flag-transfected plate or an untransfected plate was added
to the aliquots along with 35 µl of StrepTactin resin (IBA Life Sciences) and incubated
overnight at 4°C. ACBD3-Strep was affinity purified, and copurified TBC1D22A-Flag
and PI4KB-V5 were detected by Western staining using anti-Flag and anti-V5 antibodies
(Sigma). For the 3A-TBC1D22A cotransfection experiments (see Fig. S8 in the supplemental
material), 2.5 µg of TBC1D22A-Strep and 7.5 µg of picornavirus 3A-Flag were cotransfected
using Mirus TransIT-LT1 transfection reagent as described above.
Mammalian two-hybrid screening.
Interaction mapping was performed by mammalian two-hybrid screening using the Checkmate
system (Promega, Madison, WI). Bait proteins were cloned into the pAct and pBind plasmids
using the KpnI and EcoRV restriction sites and primers as described in Table S1 in
the supplemental material. Thirty-three nanograms each of pAct, pBind, and pG5Luc
plasmids was transfected into 15,000 293T cells plated 24 h previously in each well
of a 96-well plate. Firefly and Renilla luciferase levels were measured using the
dual-luciferase assay kit 40 to 48 h after transfection (Promega). The level of binding
is expressed as firefly luciferase values as a percentage of transfection control
Renilla luciferase values, measured on a Veritas microplate luminometer.
qRT-PCR.
For qRT-PCR, 1 µg of Aichi virus 3A-Flag or poliovirus 3A-Flag was transfected into
a 6-well plate of HEK293T cells in log phase and harvested 48 h later. Total RNA was
extracted using the RNeasy kit (Qiagen). Two micrograms of total RNA from HEK293T
cells was reverse transcribed using SuperScript III reverse transcriptase and oligo(dT)20,
and qRT-PCR was performed using the 480 DNA SYBR Green I master mix (Roche) on a LightCycler
(Roche). Primers used were the TBC1D22A-CDS4 and hRPL19 set at a melting temperature
(Tm
) of 50°C and with an extension of 1 min at 72°C (see Table S1 in the supplemental
material).
Protein identification by mass spectrometry.
Protein identification from affinity-purified samples was performed using peptide
sequencing by mass spectrometry as previously reported (5). Affinity-purified samples
were reduced and alkylated with dithiothreitol (DTT) and iodoacetamide, respectively,
and then subjected to trypsin digestion either in solution or in excised SDS-PAGE
gel bands. Peptide sequencing was performed using an LTQ-FT, an LTQ-Orbitrap XL, or
an LTQ-Velos (Thermo) mass spectrometer, each equipped with 10,000-psi system nanoACUITY
(Waters) ultra-high-performance liquid chromatography (UPLC) instruments for reversed-phase
C18 chromatography and using the same data acquisition and processing methods as those
previously reported (5).
Database searches were performed against the Homo sapiens plus Virus subset of the
NCBInr RefSeq database (14 January 2012), to which were added virus clone sequences
missing from the public database, totaling 131,457 entries. This database was concatenated
with a fully randomized set of 131,457 entries for estimation of the false discovery
rate (27). Data were searched with a parent mass tolerance of 20 ppm and fragment
mass tolerances of 0.6 Da.
Using peptide counts as an approximation of protein abundance, Z scores were calculated
to represent prey-bait specificity as reported previously (5). Z scores for proteins
interacting with individual virus or human bait proteins were calculated using a minimum
of five replicate experiments together with a background model of 550 control, nonhuman
bait data sets. These nonhuman data sets were compiled from the genes from 12 different
picornaviruses, excluding the 3A gene itself (VP0/VP2, VP4, VP1, VP3, L, 2A, 2B, 2C,
3C, and 3D), which serve as an unbiased set of proteins that should be orthogonal
to the human protein-protein interactions in this network. The nonspecific interacting
proteins most commonly identified in this background set, analogous to “frequent fliers”
reported as common contaminants in Flag-AP-MS experiments, are reported in Table S2
in the supplemental material (28).
SUPPLEMENTAL MATERIAL
Figure S1
Tandem mass spectrometry spectra for phosphopeptides from ACBD3, TBC1D22A, TBC1D22B,
and PI4KB and for two unique peptides from ARFGEF1. Shown here are tandem mass spectrometry
spectra for phosphorylation of the following peptides: THTDSSEKELEPEAAEEALENGPK at
either position 344 or 345 in ACBD3 (A), SQSLPHSATVTLGGTSDPSTLSSSALSEREASR at position
165 or 167 in TBC1D22A (B), QQSLPLRPIIPLVAR at position 154 in TBC1D22B (C), and TASNPKVENEDEPVR
at position 294 in PI4KB (D). Also provided is a spectrum for the peptide SVDIHDSIQPR
in ARFGEF1 (E). Download
Figure S1, PDF file, 0.3 MB
Figure S2
ACBD3 interaction with TBC1D22B is localized to the glutamine-rich region by AP-Western
blotting. Confirmation of the mammalian two-hybrid mapping of ACBD3-TBC1D22A/B interaction
was performed by AP-Western blotting. (A) Deletion constructs of Strep-tagged ACBD3
were cotransfected with Flag-tagged TBC1D22B, affinity purified with StrepTactin resin,
and blotted with anti-Flag and anti-Strep antibodies. (B) Point mutants of ACBD3-Strep
in the glutamine-rich region were cotransfected with TBC1D22B-Flag or PI4KB-Flag,
affinity purified with StrepTactin resin, and blotted with anti-Flag and anti-Strep
antibodies. (C) Point mutants of ACBD3-Strep in the charged amino acid region (CAR)
were transfected into HEK293T cells, affinity purified with StrepTactin resin, and
blotted with anti-TBC1D22A and anti-Strep antibodies. No mutants in the CAR affected
TBC1D22A binding by ACBD3. (D) Point mutants of ACBD3-Strep in the glutamine-rich
region affect TBC1D22A binding. Download
Figure S2, PDF file, 1.2 MB
Figure S3
Single alanine mutants in ACBD3 reveal critical residues for interaction with TBC1D22B,
PI4KB, poliovirus 3A, and Aichi virus 3A. Residues that had been shown to break binding
to ACBD3’s interactors in a 3-mer and 4-mer alanine scan were singly mutated to alanine
and tested in the mammalian two-hybrid assay. (A) ACBD3 single mutants demonstrated
an interaction profile similar to those of TBC1D22B and PI4KB; however, two ACBD3
mutants (F258A and Y285A) demonstrated a significantly higher reported interaction
with TBC1D22B than with PI4KB. Point mutant H284A seemed to be responsible for the
disruption of the interaction between ACBD3 and TBC1D22B/PI4KB for EQHY282AAAA. (B)
ACBD3 single mutants demonstrated an interaction profile similar to those of poliovirus
3A and Aichi virus 3A; however, two ACBD3 mutants (F433A and Y512A) demonstrated a
significantly higher reported interaction with poliovirus 3A than with Aichi virus
3A. The Y512A mutant appears to be the discriminating residue in the SYS511AAA mutant
that demonstrates higher binding to poliovirus 3A than Aichi virus 3A. Download
Figure S3, PDF file, 0.1 MB
Figure S4
TBC1D22A/B interaction with ACBD3 is localized to the N-terminus of TBC1D22A/B by
AP-Western blotting. Confirmation of the mammalian two-hybrid mapping of TBC1D22A/B-ACBD3
interaction was performed by AP-Western blotting. (A and B) Deletion and point mutants
of Strep-tagged TBC1D22A were transfected into HEK293T cells, affinity purified with
StrepTactin resin, and blotted with anti-ACBD3, anti-14-3-3, and anti-StrepII antibodies.
AP-Western blotting indicated that the binding site of ACBD3 maps to the N terminus
of TBC1D22A with significant contribution by the VL101 residues. Because 14-3-3 binding
was not amenable to mammalian two-hybrid analysis, mapping of the 14-3-3 binding site
on TBC1D22A was performed by AP-Western blotting. Deletion mutants and individual
point mutants mapped the 14-3-3 binding site to serines 165 and 167. The canonical
14-3-3 binding site of R-[SFYW]-X-pS-X-P suggests serine 167 as the phosphorylated
residue responsible for 14-3-3 recruitment. (C) Deletion mutants of TBC1D22B-Strep
were transfected into HEK293T cells, affinity purified, and blotted with anti-ACBD3
and anti-Strep antibodies. AP-Western blotting indicated that the binding site of
ACBD3 on TBC1D22B maps to a similar location as that on TBC1D22A. (D) Phosphorylation
sites discovered by AP-MS of TBC1D22B were converted to the phosphomimetic amino acid
glutamic acid by site-directed mutagenesis, cloned into the pBind plasmid, and tested
for binding to ACBD3 in the mammalian two-hybrid assay. Where the individual residue
that was phosphorylated could not be identified due to runs of serines or threonines,
all sites that could be modified were changed to glutamic acid. Phosphomimetic mutations
did not affect the binding of TBC1D22B to ACBD3, suggesting that single phosphorylations
themselves do not alter the binding to ACBD3. Download
Figure S4, PDF file, 1.3 MB
Figure S5
PI4KB interaction with ACBD3 is localized to the N terminus of PI4KB by AP-Western
blotting. Confirmation of the mammalian two-hybrid mapping of PI4KB-ACBD3 interaction
was performed by AP-Western blotting. (A to C) Deletion and point mutants of Flag-tagged
PI4KB were cotransfected with Strep-tagged ACBD3, affinity purified with StrepTactin
resin, and blotted with anti-Flag and anti-Strep antibodies. The interaction mapped
to the N-terminal region of PI4KB by AP-Western blotting and demonstrated a significant
dependence on residues VL67 but not EV58. (D) Phosphorylation sites discovered by
AP-MS of PI4KB were converted to the phosphomimetic amino acid glutamic acid by site-directed
mutagenesis, cloned into the pBind plasmid, and tested for binding to ACBD3 in the
mammalian two-hybrid assay. PI4KB interactors C10orf76 and Rab11B were included as
positive controls to ensure that overall protein structure was not altered. Where
the individual residue that was phosphorylated could not be identified due to runs
of serines or threonines, all sites that could be modified were changed to glutamic
acid. Phosphomimetic mutations did not affect the binding of PI4KB to ACBD3, suggesting
that single phosphorylations themselves do not alter the binding to ACBD3. Download
Figure S5, PDF file, 1.7 MB
Figure S6
Mapping of the HAV and klassevirus 3A protein binding sites on ACBD3. Mammalian two-hybrid
analysis of HAV and klassevirus 3A on deletion (A) and point (B) mutants of ACBD3
was performed. Both proteins demonstrated a significant, above-background interaction
with full-length ACBD3 that localized to the C-terminal GOLD domain, similarly to
poliovirus and Aichi virus 3A proteins. However, both HAV and klassevirus 3A did not
demonstrate an above-background interaction with the SYL414AAA ACBD3 mutant, unlike
poliovirus and Aichi virus 3A proteins. Download
Figure S6, PDF file, 0.1 MB
Figure S7
Kobuvirus 3A proteins prevent the association of TBC1D22A with ACBD3, while enterovirus
3A proteins can occupy the same ACBD3 as TBC1D22A. (A) Experimental setup to test
the influence of 3A proteins on the interaction of TBC1D22A with ACBD3. Strep-tagged
TBC1D22A was cotransfected with Flag-tagged 3A, and TBC1D22A was affinity purified
with StrepTactin resin and assayed with anti-Strep, anti-Flag, and anti-ACBD3 antibodies.
(B) Flag-tagged Aichi virus 3A does not copurify with TBC1D22A and reduces the overexpression
of TBC1D22A. Enterovirus 3A affinity purifies with TBC1D22A and does not affect the
expression levels of TBC1D22A. (C) Porcine and bovine kobuvirus 3A proteins demonstrate
similar reductions in expression levels of Strep-tagged TBC1D22A relative to poliovirus
3A and an inability of TBC1D22A to copurify with ACBD3, consistent with a sequestration
of ACBD3 by the kobuviral 3A proteins. Figure 7 demonstrates that the inability of
TBC1D22A and kobuviral 3A proteins to occupy the same ACBD3 is independent of the
effects of kobuviral 3A proteins on the expression levels of cotransfected Strep-tagged
TBC1D22A. Download
Figure S7, PDF file, 1.1 MB
Figure S8
Transfection of picornavirus 3A genes does not result in large-scale changes of TBC1D22A
mRNA expression. (A) 293 cells were transfected with no construct or poliovirus 3A-Flag
or Aichi virus 3A-Flag and harvested after 48 h. qRT-PCR was performed with TBC1D22A
and ribosomal RPL19 housekeeping gene primers. TBC1D22A transcript levels relative
to RPL19 transcript levels are displayed. Only a subtle decrease in endogenous TBC1D22A
transcript expression is seen when Aichi virus 3A-Flag is transfected (<14% decrease).
(B) Western blotting confirms expression of transfected poliovirus 3A-Flag and Aichi
virus 3A-Flag on cells on which qRT-PCR was performed. Download
Figure S8, PDF file, 1 MB
Figure S9
AP-Western blotting confirms that picornavirus 3A interaction with ACBD3 is localized
to the C terminus of ACBD3. (A) Deletions of ACBD3-Strep were cotransfected with Flag-tagged
picornavirus 3A constructs, affinity purified with StrepTactin resin, and blotted
with anti-Strep and anti-Flag antibodies. C-terminal deletion of ACBD3 (1 to 321)
fails to copurify with picornaviral 3A proteins while N-terminal deletions of ACBD3
(175 to 528 and 260 to 528) copurify with picornaviral 3A proteins. Residue numbers
indicate the portion of ACBD3 that is present in the construct. (B) Aichi virus 3A
qualitatively competes with poliovirus 3A for ACBD3. The experimental setup is the
same as that for Fig. 4, except that increasing amounts of Aichi virus 3A-Flag lysate
are added to premixed to poliovirus 3A-Flag prebound to ACBD3-Strep. These data are
consistent with the mapping data showing that Aichi virus 3A and poliovirus 3A bind
to the same region of ACBD3. (C) Alanine scanning of poliovirus 3A interactions by
AP-Western blotting confirms mammalian two-hybrid mapping results from Fig. 6. Point
mutants in the N terminus of poliovirus 3A disrupt GBF1 while point mutants C terminal
to the DLL dimerization motif of poliovirus 3A disrupt ACBD3 binding. Download
Figure S9, PDF file, 0.9 MB
Table S1
Supplemental Tables.
Table S1, XLSX file, 0.4 MB.
Text S1
Supplemental table legends. Download
Text S1, DOC file, 0.1 MB