Structural	O
insights	O
and	O
in	B-experimental_method
vitro	I-experimental_method
reconstitution	I-experimental_method
of	O
membrane	O
targeting	O
and	O
activation	O
of	O
human	B-species
PI4KB	B-protein
by	O
the	O
ACBD3	B-protein
protein	O

Phosphatidylinositol	B-protein
4	I-protein
-	I-protein
kinase	I-protein
beta	I-protein
(	O
PI4KB	B-protein
)	O
is	O
one	O
of	O
four	O
human	B-species
PI4K	B-protein_type
enzymes	O
that	O
generate	O
phosphatidylinositol	B-chemical
4	I-chemical
-	I-chemical
phosphate	I-chemical
(	O
PI4P	B-chemical
),	O
a	O
minor	O
but	O
essential	O
regulatory	O
lipid	O
found	O
in	O
all	O
eukaryotic	B-taxonomy_domain
cells	O
.	O

To	O
convert	O
their	O
lipid	O
substrates	O
,	O
PI4Ks	B-protein_type
must	O
be	O
recruited	O
to	O
the	O
correct	O
membrane	O
compartment	O
.	O

PI4KB	B-protein
is	O
critical	O
for	O
the	O
maintenance	O
of	O
the	O
Golgi	O
and	O
trans	O
Golgi	O
network	O
(	O
TGN	O
)	O
PI4P	B-chemical
pools	O
,	O
however	O
,	O
the	O
actual	O
targeting	O
mechanism	O
of	O
PI4KB	B-protein
to	O
the	O
Golgi	O
and	O
TGN	O
membranes	O
is	O
unknown	O
.	O

Here	O
,	O
we	O
present	O
an	O
NMR	B-experimental_method
structure	B-evidence
of	O
the	O
complex	O
of	O
PI4KB	B-protein
and	O
its	O
interacting	O
partner	O
,	O
Golgi	B-protein_type
adaptor	I-protein_type
protein	I-protein_type
acyl	B-protein
-	I-protein
coenzyme	I-protein
A	I-protein
binding	I-protein
domain	I-protein
containing	I-protein
protein	I-protein
3	I-protein
(	O
ACBD3	B-protein
).	O

We	O
show	O
that	O
ACBD3	B-protein
is	O
capable	O
of	O
recruiting	O
PI4KB	B-protein
to	O
membranes	O
both	O
in	O
vitro	O
and	O
in	O
vivo	O
,	O
and	O
that	O
membrane	O
recruitment	O
of	O
PI4KB	B-protein
by	O
ACBD3	B-protein
increases	O
its	O
enzymatic	B-evidence
activity	I-evidence
and	O
that	O
the	O
ACBD3	B-complex_assembly
:	I-complex_assembly
PI4KB	I-complex_assembly
complex	O
formation	O
is	O
essential	O
for	O
proper	O
function	O
of	O
the	O
Golgi	O
.	O

Phosphatidylinositol	B-protein
4	I-protein
-	I-protein
kinase	I-protein
beta	I-protein
(	O
PI4KB	B-protein
,	O
also	O
known	O
as	O
PI4K	B-protein
IIIβ	I-protein
)	O
is	O
a	O
soluble	O
cytosolic	O
protein	O
yet	O
its	O
function	O
is	O
to	O
phosphorylate	O
membrane	O
lipids	O
.	O

It	O
is	O
one	O
of	O
four	O
human	B-species
PI4K	B-protein_type
enzymes	O
that	O
phosphorylate	O
phosphatidylinositol	B-chemical
(	O
PI	B-chemical
)	O
to	O
generate	O
phosphatidylinositol	B-chemical
4	I-chemical
-	I-chemical
phosphate	I-chemical
(	O
PI4P	B-chemical
).	O

PI4P	B-chemical
is	O
an	O
essential	O
lipid	O
found	O
in	O
various	O
membrane	O
compartments	O
including	O
the	O
Golgi	O
and	O
trans	O
-	O
Golgi	O
network	O
(	O
TGN	O
),	O
the	O
plasma	O
membrane	O
and	O
the	O
endocytic	O
compartments	O
.	O

In	O
these	O
locations	O
,	O
PI4P	B-chemical
plays	O
an	O
important	O
role	O
in	O
cell	O
signaling	O
and	O
lipid	O
transport	O
,	O
and	O
serves	O
as	O
a	O
precursor	O
for	O
higher	O
phosphoinositides	B-chemical
or	O
as	O
a	O
docking	O
site	O
for	O
clathrin	B-protein_type
adaptor	O
or	O
lipid	O
transfer	O
proteins	O
.	O

A	O
wide	O
range	O
of	O
positive	B-taxonomy_domain
-	I-taxonomy_domain
sense	I-taxonomy_domain
single	I-taxonomy_domain
-	I-taxonomy_domain
stranded	I-taxonomy_domain
RNA	I-taxonomy_domain
viruses	I-taxonomy_domain
(+	O
RNA	B-taxonomy_domain
viruses	I-taxonomy_domain
),	O
including	O
many	O
that	O
are	O
important	O
human	B-species
pathogens	O
,	O
hijack	O
human	B-species
PI4KA	B-protein
or	O
PI4KB	B-protein
enzymes	O
to	O
generate	O
specific	O
PI4P	B-chemical
-	O
enriched	O
organelles	O
called	O
membranous	O
webs	O
or	O
replication	O
factories	O
.	O

These	O
structures	B-evidence
are	O
essential	O
for	O
effective	O
viral	B-taxonomy_domain
replication	O
.	O

Recently	O
,	O
highly	O
specific	O
PI4KB	B-protein
inhibitors	O
were	O
developed	O
as	O
potential	O
antivirals	O
.	O

PI4K	B-protein_type
kinases	B-protein_type
must	O
be	O
recruited	O
to	O
the	O
correct	O
membrane	O
type	O
to	O
fulfill	O
their	O
enzymatic	O
functions	O
.	O

Type	B-protein_type
II	I-protein_type
PI4Ks	I-protein_type
(	O
PI4K2A	B-protein
and	O
PI4K2B	B-protein
)	O
are	O
heavily	B-protein_state
palmitoylated	I-protein_state
and	O
thus	O
behave	O
as	O
membrane	B-protein
proteins	I-protein
.	O

In	O
contrast	O
,	O
type	B-protein_type
III	I-protein_type
PI4Ks	I-protein_type
(	O
PI4KA	B-protein
and	O
PI4KB	B-protein
)	O
are	O
soluble	O
cytosolic	O
proteins	O
that	O
are	O
recruited	O
to	O
appropriate	O
membranes	O
indirectly	O
via	O
protein	O
-	O
protein	O
interactions	O
.	O

The	O
recruitment	O
of	O
PI4KA	B-protein
to	O
the	O
plasma	O
membrane	O
by	O
EFR3	B-protein
and	O
TTC7	B-protein
is	O
relatively	O
well	O
understood	O
even	O
at	O
the	O
structural	O
level	O
,	O
but	O
,	O
the	O
actual	O
molecular	O
mechanism	O
of	O
PI4KB	B-protein
recruitment	O
to	O
the	O
Golgi	O
is	O
still	O
poorly	O
understood	O
.	O

Acyl	B-protein
-	I-protein
coenzyme	I-protein
A	I-protein
binding	I-protein
domain	I-protein
containing	I-protein
protein	I-protein
3	I-protein
(	O
ACBD3	B-protein
,	O
also	O
known	O
as	O
GCP60	B-protein
and	O
PAP7	B-protein
)	O
is	O
a	O
Golgi	O
resident	O
protein	O
.	O

Its	O
membrane	O
localization	O
is	O
mediated	O
by	O
the	O
interaction	O
with	O
the	O
Golgi	O
integral	O
protein	O
golgin	B-protein
B1	I-protein
/	O
giantin	B-protein
.	O

ACBD3	B-protein
functions	O
as	O
an	O
adaptor	O
protein	O
and	O
signaling	O
hub	O
across	O
cellular	O
signaling	O
pathways	O
.	O

ACBD3	B-protein
can	O
interact	O
with	O
a	O
number	O
of	O
proteins	O
including	O
golgin	B-protein
A3	I-protein
/	O
golgin	B-protein
-	I-protein
160	I-protein
to	O
regulate	O
apoptosis	O
,	O
Numb	B-protein_type
proteins	I-protein_type
to	O
control	O
asymmetric	O
cell	O
division	O
and	O
neuronal	O
differentiation	O
,	O
metal	B-protein_type
transporter	I-protein_type
DMT1	B-protein
and	O
monomeric	B-oligomeric_state
G	B-protein_type
protein	I-protein_type
Dexras1	B-protein
to	O
maintain	O
iron	B-chemical
homeostasis	O
,	O
and	O
the	O
lipid	B-protein_type
kinase	I-protein_type
PI4KB	B-protein
to	O
regulate	O
lipid	O
homeostasis	O
.	O

ACBD3	B-protein
has	O
been	O
also	O
implicated	O
in	O
the	O
pathology	O
of	O
neurodegenerative	O
diseases	O
such	O
as	O
Huntington	O
’	O
s	O
disease	O
due	O
to	O
its	O
interactions	O
with	O
a	O
polyglutamine	B-structure_element
repeat	I-structure_element
-	O
containing	O
mutant	B-protein_state
huntingtin	B-protein
and	O
the	O
striatal	O
-	O
selective	O
monomeric	B-oligomeric_state
G	B-protein_type
protein	I-protein_type
Rhes	B-protein
/	O
Dexras2	B-protein
.	O

ACBD3	B-protein
is	O
a	O
binding	O
partner	O
of	O
viral	B-taxonomy_domain
non	B-protein_type
-	I-protein_type
structural	I-protein_type
3A	I-protein_type
proteins	I-protein_type
and	O
a	O
host	O
factor	O
of	O
several	O
picornaviruses	B-taxonomy_domain
including	O
poliovirus	B-taxonomy_domain
,	O
coxsackievirus	B-taxonomy_domain
B3	I-taxonomy_domain
,	O
and	O
Aichi	B-taxonomy_domain
virus	I-taxonomy_domain
.	O

We	O
present	O
a	O
biochemical	B-experimental_method
and	I-experimental_method
structural	I-experimental_method
characterization	I-experimental_method
of	O
the	O
molecular	O
complex	O
composed	O
of	O
the	O
ACBD3	B-protein
protein	O
and	O
the	O
PI4KB	B-protein
enzyme	O
.	O

We	O
show	O
that	O
ACBD3	B-protein
can	O
recruit	O
PI4KB	B-protein
to	O
model	O
membranes	O
as	O
well	O
as	O
redirect	O
PI4KB	B-protein
to	O
cellular	O
membranes	O
where	O
it	O
is	O
not	O
naturally	O
found	O
.	O

Our	O
data	O
also	O
show	O
that	O
ACBD3	B-protein
regulates	O
the	O
enzymatic	B-evidence
activity	I-evidence
of	O
PI4KB	B-protein
kinase	B-protein_type
through	O
membrane	O
recruitment	O
rather	O
than	O
allostery	O
.	O

ACBD3	B-protein
and	O
PI4KB	B-protein
interact	O
with	O
1	O
:	O
1	O
stoichiometry	O
with	O
submicromolar	O
affinity	O

In	O
order	O
to	O
verify	O
the	O
interactions	O
between	O
ACBD3	B-protein
and	O
PI4KB	B-protein
we	O
expressed	B-experimental_method
and	I-experimental_method
purified	I-experimental_method
both	O
proteins	O
.	O

To	O
increase	O
yields	O
of	O
bacterial	B-experimental_method
expression	I-experimental_method
the	O
intrinsically	B-structure_element
disordered	I-structure_element
region	I-structure_element
of	O
PI4KB	B-protein
(	O
residues	O
423	B-residue_range
–	I-residue_range
522	I-residue_range
)	O
was	O
removed	B-experimental_method
(	O
Fig	O
.	O
1A	O
).	O

This	O
internal	O
deletion	B-experimental_method
does	O
not	O
significantly	O
affect	O
the	O
kinase	B-protein_type
activity	O
(	O
SI	O
Fig	O
.	O
1A	O
)	O
or	O
interaction	O
with	O
ACBD3	B-protein
(	O
SI	O
Fig	O
.	O
1B	O
,	O
C	O
).	O

In	O
an	O
in	B-experimental_method
vitro	I-experimental_method
binding	I-experimental_method
assay	I-experimental_method
,	O
ACBD3	B-protein
co	B-experimental_method
-	I-experimental_method
purified	I-experimental_method
with	I-experimental_method
the	I-experimental_method
NiNTA	I-experimental_method
-	I-experimental_method
immobilized	I-experimental_method
N	O
-	O
terminal	O
His6GB1	B-protein_state
-	I-protein_state
tagged	I-protein_state
PI4KB	B-protein
(	O
Fig	O
.	O
1B	O
,	O
left	O
panel	O
),	O
suggesting	O
a	O
direct	O
interaction	O
.	O

Using	O
a	O
mammalian	B-experimental_method
two	I-experimental_method
-	I-experimental_method
hybrid	I-experimental_method
assay	I-experimental_method
Greninger	O
and	O
colleagues	O
localized	B-evidence
this	O
interaction	O
to	O
the	O
Q	B-structure_element
domain	I-structure_element
of	O
ACBD3	B-protein
(	O
named	O
according	O
to	O
its	O
high	O
content	O
of	O
glutamine	B-residue_name
residues	O
)	O
and	O
the	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
of	O
PI4KB	B-protein
preceding	O
its	O
helical	B-structure_element
domain	I-structure_element
.	O

We	O
expressed	B-experimental_method
the	O
Q	B-structure_element
domain	I-structure_element
of	O
ACBD3	B-protein
(	O
residues	O
241	B-residue_range
–	I-residue_range
308	I-residue_range
)	O
and	O
the	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
of	O
PI4KB	B-protein
(	O
residues	O
1	B-residue_range
–	I-residue_range
68	I-residue_range
)	O
in	O
E	B-species
.	I-species
coli	I-species
and	O
using	O
purified	O
recombinant	O
proteins	O
,	O
we	O
confirmed	O
that	O
these	O
two	O
domains	O
are	O
sufficient	O
to	O
maintain	O
the	O
interaction	O
(	O
Fig	O
.	O
1B	O
,	O
middle	O
and	O
right	O
panel	O
).	O

Because	O
it	O
has	O
been	O
reported	O
that	O
ACBD3	B-protein
can	O
dimerize	B-oligomeric_state
in	O
a	O
mammalian	B-experimental_method
two	I-experimental_method
-	I-experimental_method
hybrid	I-experimental_method
assay	I-experimental_method
,	O
we	O
were	O
interested	O
in	O
determining	O
the	O
stoichiometry	O
of	O
the	O
ACBD3	B-complex_assembly
:	I-complex_assembly
PI4KB	I-complex_assembly
protein	O
complex	O
.	O

The	O
sedimentation	B-evidence
coefficients	I-evidence
of	O
ACBD3	B-protein
and	O
PI4KB	B-protein
alone	B-protein_state
,	O
or	O
ACBD3	B-complex_assembly
:	I-complex_assembly
PI4KB	I-complex_assembly
complex	O
were	O
determined	O
by	O
analytical	B-experimental_method
ultracentrifugation	I-experimental_method
and	O
found	O
to	O
be	O
3	O
.	O
1	O
S	O
,	O
4	O
.	O
1	O
S	O
,	O
and	O
5	O
.	O
1	O
S	O
.	O
These	O
values	O
correspond	O
to	O
molecular	B-evidence
weights	I-evidence
of	O
approximately	O
55	O
kDa	O
,	O
80	O
kDa	O
,	O
and	O
130	O
kDa	O
,	O
respectively	O
.	O

This	O
result	O
suggests	O
that	O
both	O
proteins	O
are	O
monomeric	B-oligomeric_state
and	O
the	O
stoichiometry	O
of	O
the	O
ACBD3	B-complex_assembly
:	I-complex_assembly
PI4KB	I-complex_assembly
protein	O
complex	O
is	O
1	O
:	O
1	O
(	O
Fig	O
.	O
1C	O
,	O
left	O
panel	O
).	O

Similar	O
results	O
were	O
obtained	O
for	O
the	O
complex	O
of	O
the	O
Q	B-structure_element
domain	I-structure_element
of	O
ACBD3	B-protein
and	O
the	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
of	O
PI4KB	B-protein
(	O
Fig	O
.	O
1C	O
,	O
right	O
panel	O
).	O

We	O
also	O
determined	O
the	O
strength	O
of	O
the	O
interaction	O
between	O
recombinant	O
full	B-protein_state
length	I-protein_state
ACBD3	B-protein
and	O
PI4KB	B-protein
using	O
surface	B-experimental_method
plasmon	I-experimental_method
resonance	I-experimental_method
(	O
SPR	B-experimental_method
).	O

SPR	B-experimental_method
measurements	O
revealed	O
a	O
strong	O
interaction	O
with	O
a	O
Kd	B-evidence
value	O
of	O
320	O
+/−	O
130	O
nM	O
(	O
Fig	O
.	O
1D	O
,	O
SI	O
Fig	O
.	O
1D	O
).	O

We	O
concluded	O
that	O
ACBD3	B-protein
and	O
PI4KB	B-protein
interact	O
directly	O
through	O
the	O
Q	B-structure_element
domain	I-structure_element
of	O
ACBD3	B-protein
and	O
the	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
of	O
PI4KB	B-protein
forming	O
a	O
1	O
:	O
1	O
complex	O
with	O
a	O
dissociation	B-evidence
constant	I-evidence
in	O
the	O
submicromolar	O
range	O
.	O

Structural	B-experimental_method
analysis	I-experimental_method
of	O
the	O
ACBD3	B-complex_assembly
:	I-complex_assembly
PI4KB	I-complex_assembly
complex	O

Full	B-protein_state
length	I-protein_state
ACBD3	B-protein
and	O
PI4KB	B-protein
both	O
contain	O
large	O
intrinsically	B-structure_element
disordered	I-structure_element
regions	I-structure_element
that	O
impede	O
crystallization	O
.	O

We	O
used	O
hydrogen	B-experimental_method
-	I-experimental_method
deuterium	I-experimental_method
exchange	I-experimental_method
mass	I-experimental_method
spectrometry	I-experimental_method
(	O
HDX	B-experimental_method
-	I-experimental_method
MS	I-experimental_method
)	O
analysis	O
of	O
the	O
complex	O
to	O
determine	O
which	O
parts	O
of	O
the	O
complex	O
are	O
well	B-protein_state
folded	I-protein_state
(	O
SI	O
Fig	O
.	O
2	O
).	O

However	O
,	O
we	O
were	O
unable	O
to	O
obtain	O
crystals	B-evidence
even	O
when	O
using	O
significantly	O
truncated	B-protein_state
constructs	O
that	O
included	O
only	O
the	O
ACBD3	B-protein
Q	B-structure_element
domain	I-structure_element
and	O
the	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
of	O
PI4KB	B-protein
.	O

For	O
this	O
reason	O
,	O
we	O
produced	O
an	O
isotopically	B-protein_state
labeled	I-protein_state
ACBD3	B-protein
Q	B-structure_element
domain	I-structure_element
and	O
isotopically	B-protein_state
labeled	I-protein_state
ACBD3	B-protein
Q	B-structure_element
domain	I-structure_element
:	O
PI4KB	B-protein
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
protein	O
complex	O
and	O
used	O
NMR	B-experimental_method
spectroscopy	I-experimental_method
for	O
structural	O
characterization	O
.	O

As	O
the	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
protein	O
complex	O
was	O
prepared	O
by	O
co	B-experimental_method
-	I-experimental_method
expression	I-experimental_method
of	O
both	O
proteins	O
,	O
the	O
samples	O
consisted	O
of	O
an	O
equimolar	O
mixture	O
of	O
two	O
uniformly	O
15N	B-chemical
/	O
13C	B-chemical
labelled	B-protein_state
molecules	O
.	O

Comprehensive	O
backbone	O
and	O
side	O
-	O
chain	O
resonance	O
assignments	O
for	O
the	O
free	B-protein_state
ACBD3	B-protein
Q	B-structure_element
domain	I-structure_element
and	O
the	O
complex	O
,	O
as	O
illustrated	O
by	O
the	O
2D	B-experimental_method
15N	I-experimental_method
/	I-experimental_method
1H	I-experimental_method
HSQC	I-experimental_method
spectra	B-evidence
(	O
SI	O
Figs	O
3	O
and	O
4	O
),	O
were	O
obtained	O
using	O
a	O
standard	O
combination	O
of	O
triple	B-experimental_method
-	I-experimental_method
resonance	I-experimental_method
experiments	I-experimental_method
,	O
as	O
described	O
previously	O
.	O

Backbone	O
amide	O
signals	O
(	O
15N	B-chemical
and	O
1H	B-chemical
)	O
for	O
the	O
free	B-protein_state
ACBD3	B-protein
Q	B-structure_element
domain	I-structure_element
were	O
nearly	O
completely	O
assigned	O
apart	O
from	O
the	O
first	O
four	O
N	O
-	O
terminal	O
residues	O
(	O
Met1	B-residue_range
-	I-residue_range
Lys4	I-residue_range
)	O
and	O
Gln44	B-residue_name_number
.	O

Over	O
93	O
%	O
of	O
non	O
-	O
exchangeable	O
side	O
-	O
chain	O
signals	O
were	O
assigned	O
for	O
the	O
free	B-protein_state
ACBD3	B-protein
Q	B-structure_element
domain	I-structure_element
.	O

Apart	O
from	O
the	O
four	O
N	O
-	O
terminal	O
residues	O
,	O
the	O
side	O
-	O
chain	O
assignments	O
were	O
missing	O
for	O
Gln	B-residue_name
(	O
Hg3	O
),	O
Gln	B-residue_name
(	O
Ha	O
/	O
Hb	O
/	O
Hg	O
),	O
Gln44	B-residue_name_number
(	O
Ha	O
/	O
Hb	O
/	O
Hg	O
)	O
and	O
Gln48	B-residue_name_number
(	O
Hg	O
)	O
mainly	O
due	O
to	O
extensive	O
overlaps	O
within	O
the	O
spectral	O
regions	O
populated	O
by	O
highly	O
abundant	O
glutamine	B-residue_name
side	O
-	O
chain	O
resonances	O
.	O

The	O
protein	O
complex	O
yielded	O
relatively	O
well	O
resolved	O
spectra	B-evidence
(	O
SI	O
Fig	O
.	O
4	O
)	O
that	O
resulted	O
in	O
assignment	O
of	O
backbone	O
amide	O
signals	O
for	O
all	O
residues	O
apart	O
from	O
Gln	B-residue_name
(	O
ACBD3	B-protein
)	O
and	O
Ala2	B-residue_name_number
(	O
PI4KB	B-protein
).	O

The	O
essentially	O
complete	O
15N	B-chemical
,	O
13C	B-chemical
and	O
1H	B-chemical
resonance	O
assignments	O
allowed	O
automated	O
assignment	O
of	O
the	O
NOEs	B-evidence
identified	O
in	O
the	O
3D	B-experimental_method
15N	I-experimental_method
/	I-experimental_method
1H	I-experimental_method
NOESY	I-experimental_method
-	I-experimental_method
HSQC	I-experimental_method
and	O
13C	B-experimental_method
/	I-experimental_method
1H	I-experimental_method
HMQC	I-experimental_method
-	I-experimental_method
NOESY	I-experimental_method
spectra	B-evidence
that	O
were	O
subsequently	O
used	O
in	O
structural	B-experimental_method
calculation	I-experimental_method
.	O

Structural	B-evidence
statistics	I-evidence
for	O
the	O
final	O
water	O
-	O
refined	O
sets	O
of	O
structures	B-evidence
are	O
shown	O
in	O
SI	O
Table	O
1	O
.	O

This	O
structure	B-evidence
revealed	O
that	O
the	O
Q	B-structure_element
domain	I-structure_element
forms	O
a	O
two	B-structure_element
helix	I-structure_element
hairpin	I-structure_element
.	O

The	O
first	O
helix	B-structure_element
bends	O
sharply	O
over	O
the	O
second	O
helix	B-structure_element
and	O
creates	O
a	O
fold	O
resembling	O
a	O
three	B-structure_element
helix	I-structure_element
bundle	I-structure_element
that	O
serves	O
as	O
a	O
nest	O
for	O
one	O
helix	B-structure_element
of	O
the	O
PI4KB	B-protein
N	O
-	O
terminus	O
(	O
residues	O
44	B-residue_range
–	I-residue_range
64	I-residue_range
,	O
from	O
this	O
point	O
on	O
referred	O
to	O
as	O
the	O
kinase	B-structure_element
helix	I-structure_element
)	O
(	O
Fig	O
.	O
2A	O
).	O

Preceding	O
the	O
kinase	B-structure_element
helix	I-structure_element
are	O
three	O
ordered	O
residues	O
(	O
Val42	B-residue_name_number
,	O
Ile43	B-residue_name_number
,	O
and	O
Asp44	B-residue_name_number
)	O
that	O
also	O
contribute	O
to	O
the	O
interaction	O
(	O
Fig	O
.	O
2B	O
).	O

The	O
remaining	O
part	O
of	O
the	O
PI4KB	B-protein
N	O
-	O
termini	O
,	O
however	O
,	O
is	O
disordered	O
(	O
SI	O
Fig	O
.	O
5	O
).	O

Almost	O
all	O
of	O
the	O
PI4KB	B-complex_assembly
:	I-complex_assembly
ACBD3	I-complex_assembly
interactions	B-bond_interaction
are	I-bond_interaction
hydrophobic	I-bond_interaction
with	O
the	O
exception	O
of	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
between	O
the	O
side	O
chains	O
of	O
ACBD3	B-protein
Tyr261	B-residue_name_number
and	O
PI4KB	B-protein
His63	B-residue_name_number
,	O
and	O
between	O
the	O
sidechain	O
of	O
ACBD3	B-protein
Tyr288	B-residue_name_number
and	O
the	O
PI4KB	B-protein
backbone	O
(	O
Asp44	B-residue_name_number
)	O
(	O
Fig	O
.	O
2B	O
).	O

Interestingly	O
,	O
we	O
noted	O
that	O
the	O
PI4KB	B-protein
helix	B-structure_element
is	O
amphipathic	B-protein_state
and	O
its	O
hydrophobic	B-site
surface	I-site
leans	O
on	O
the	O
Q	B-structure_element
domain	I-structure_element
(	O
Fig	O
.	O
2C	O
).	O

To	O
corroborate	O
the	O
structural	B-evidence
data	I-evidence
,	O
we	O
introduced	B-experimental_method
a	O
number	O
of	O
point	B-experimental_method
mutations	I-experimental_method
and	O
validated	O
their	O
effect	O
on	O
complex	O
formation	O
using	O
an	O
in	B-experimental_method
vitro	I-experimental_method
pull	I-experimental_method
-	I-experimental_method
down	I-experimental_method
assay	I-experimental_method
(	O
Fig	O
.	O
2D	O
).	O

Wild	B-protein_state
type	I-protein_state
ACBD3	B-protein
protein	O
co	B-experimental_method
-	I-experimental_method
purified	I-experimental_method
together	O
with	O
the	O
NiNTA	O
-	O
immobilized	O
His6	B-protein_state
-	I-protein_state
tagged	I-protein_state
wild	B-protein_state
type	I-protein_state
PI4KB	B-protein
as	O
well	O
as	O
with	O
the	O
PI4KB	B-protein
V42A	B-mutant
and	O
V47A	B-mutant
mutants	B-protein_state
,	O
but	O
not	O
with	O
mutants	B-protein_state
within	O
the	O
imminent	O
binding	B-site
interface	I-site
(	O
I43A	B-mutant
,	O
V55A	B-mutant
,	O
L56A	B-mutant
).	O

As	O
predicted	O
,	O
wild	B-protein_state
type	I-protein_state
PI4KB	B-protein
interacted	O
with	O
the	O
ACBD3	B-protein
Y266A	B-mutant
mutant	B-protein_state
and	O
slightly	O
with	O
the	O
Y285A	B-mutant
mutant	B-protein_state
,	O
but	O
not	O
with	O
the	O
F258A	B-mutant
,	O
H284A	B-mutant
,	O
and	O
Y288A	B-mutant
mutants	B-protein_state
(	O
Fig	O
.	O
2D	O
).	O

ACBD3	B-protein
efficiently	O
recruits	O
the	O
PI4KB	B-protein
enzyme	O
to	O
membranes	O

We	O
next	O
sought	O
to	O
determine	O
if	O
the	O
ACBD3	B-complex_assembly
:	I-complex_assembly
PI4KB	I-complex_assembly
interaction	O
drives	O
membrane	O
localization	O
of	O
the	O
PI4KB	B-protein
enzyme	O
.	O

To	O
do	O
this	O
,	O
we	O
first	O
established	O
an	O
in	B-experimental_method
vitro	I-experimental_method
membrane	I-experimental_method
recruitment	I-experimental_method
system	I-experimental_method
using	O
Giant	B-experimental_method
Unilamellar	I-experimental_method
Vesicles	I-experimental_method
(	O
GUVs	B-experimental_method
)	O
containing	O
the	O
PI4KB	B-protein
substrate	O
–	O
the	O
PI	B-chemical
lipid	O
.	O

We	O
observed	O
that	O
PI4KB	B-protein
kinase	B-protein_type
was	O
not	O
membrane	O
localized	B-evidence
when	O
added	O
to	O
the	O
GUVs	B-experimental_method
at	O
600	O
nM	O
concentration	O
,	O
whereas	O
non	O
-	O
covalent	O
tethering	O
of	O
ACBD3	B-protein
to	O
the	O
surface	O
of	O
the	O
GUVs	B-experimental_method
,	O
using	O
the	O
His6	O
tag	O
on	O
ACBD3	B-protein
and	O
the	O
DGS	B-chemical
-	I-chemical
NTA	I-chemical
(	I-chemical
Ni	I-chemical
)	I-chemical
lipid	I-chemical
,	O
led	O
to	O
efficient	O
PI4KB	B-protein
membrane	O
localization	O
(	O
Fig	O
.	O
3A	O
).	O

We	O
hypothesized	O
that	O
if	O
ACBD3	B-protein
is	O
one	O
of	O
the	O
main	O
Golgi	O
localization	B-evidence
signals	I-evidence
for	O
PI4KB	B-protein
,	O
overexpression	B-experimental_method
of	O
the	O
Q	B-structure_element
domain	I-structure_element
should	O
decrease	O
the	O
amount	O
of	O
the	O
endogenous	O
kinase	B-protein_type
on	O
the	O
Golgi	O
.	O
Indeed	O
,	O
we	O
observed	O
loss	O
for	O
endogenous	O
PI4KB	B-protein
signal	O
on	O
the	O
Golgi	O
in	O
cells	O
overexpressing	B-experimental_method
the	O
GFP	B-experimental_method
–	O
Q	B-structure_element
domain	I-structure_element
construct	O
(	O
Fig	O
.	O
3B	O
upper	O
panel	O
).	O

We	O
attribute	O
the	O
loss	O
of	O
signal	B-evidence
to	O
the	O
immunostaining	O
protocol	O
-	O
the	O
kinase	B-protein_type
that	O
is	O
not	O
bound	O
to	O
Golgi	O
is	O
lost	O
during	O
the	O
permeabilization	O
step	O
and	O
hence	O
the	O
“	O
disappearance	O
”	O
of	O
the	O
signal	B-evidence
because	O
overexpression	B-experimental_method
of	O
GFP	B-experimental_method
alone	O
or	O
a	O
non	B-protein_state
-	I-protein_state
binding	I-protein_state
Q	B-structure_element
domain	I-structure_element
mutant	B-protein_state
has	O
no	O
effect	O
on	O
the	O
localization	B-evidence
of	O
the	O
endogenous	O
PI4KB	B-protein
(	O
Fig	O
.	O
3B	O
).	O

Given	O
this	O
result	O
,	O
overexpression	B-experimental_method
of	O
the	O
Q	B-structure_element
domain	I-structure_element
should	O
also	O
interfere	O
with	O
the	O
PI4KB	B-protein
dependent	O
Golgi	O
functions	O
.	O

Ceramide	B-chemical
transport	O
and	O
accumulation	O
in	O
Golgi	O
is	O
a	O
well	O
-	O
known	O
PI4KB	B-protein
dependent	O
process	O
.	O

We	O
have	O
used	O
fluorescently	B-protein_state
labeled	I-protein_state
ceramide	B-chemical
and	O
analyzed	O
its	O
trafficking	O
in	O
non	O
-	O
transfected	O
cells	O
and	O
cell	O
overexpressing	B-experimental_method
the	O
Q	B-structure_element
domain	I-structure_element
.	O

As	O
expected	O
,	O
the	O
Golgi	O
accumulation	O
of	O
ceramide	B-chemical
was	O
not	O
observed	O
in	O
cells	O
expressing	B-experimental_method
the	O
wt	B-protein_state
Q	B-structure_element
domain	I-structure_element
while	O
cells	O
expressing	O
RFP	B-experimental_method
or	O
the	O
mutant	B-protein_state
Q	B-structure_element
domain	I-structure_element
accumulated	O
ceramide	B-chemical
normally	O
(	O
Fig	O
.	O
3C	O
)	O
suggesting	O
that	O
ACBD3	B-complex_assembly
:	I-complex_assembly
PI4KB	I-complex_assembly
complex	O
formation	O
is	O
crucial	O
for	O
the	O
normal	O
function	O
of	O
Golgi	O
.	O

We	O
further	O
analyzed	O
the	O
function	O
of	O
ACBD3	B-complex_assembly
:	I-complex_assembly
PI4KB	I-complex_assembly
interaction	O
in	O
membrane	O
recruitment	O
of	O
PI4KB	B-protein
in	O
living	O
cells	O
using	O
fluorescently	B-protein_state
tagged	I-protein_state
proteins	O
.	O

We	O
used	O
the	O
rapamycin	B-chemical
-	O
inducible	O
heteromerization	O
of	O
FKBP12	B-protein
(	O
FK506	B-protein
binding	I-protein
protein	I-protein
12	I-protein
)	O
and	O
FRB	B-structure_element
(	O
fragment	B-structure_element
of	O
mTOR	B-protein
that	O
binds	O
rapamycin	B-chemical
)	O
system	O
.	O

We	O
fused	B-experimental_method
the	O
FRB	B-structure_element
to	O
residues	O
34	B-residue_range
–	I-residue_range
63	I-residue_range
of	O
the	O
mitochondrial	B-structure_element
localization	I-structure_element
signal	I-structure_element
from	O
mitochondrial	B-protein
A	I-protein
-	I-protein
kinase	I-protein
anchor	I-protein
protein	I-protein
1	I-protein
(	O
AKAP1	B-protein
)	O
and	O
CFP	B-experimental_method
.	O

The	O
ACBD3	B-protein
Q	B-structure_element
domain	I-structure_element
was	O
then	O
fused	B-experimental_method
to	I-experimental_method
FKBP12	B-protein
and	O
mRFP	B-experimental_method
(	O
Fig	O
.	O
3D	O
).	O

We	O
analyzed	O
localization	B-evidence
of	O
the	O
ACBD3	B-protein
Q	B-structure_element
domain	I-structure_element
and	O
GFP	B-experimental_method
–	O
PI4KB	B-protein
before	O
and	O
after	O
the	O
addition	O
of	O
rapamycin	B-chemical
.	O

As	O
a	O
control	O
we	O
used	O
H284A	B-mutant
mutant	B-protein_state
of	O
the	O
ACBD3	B-protein
Q	B-structure_element
domain	I-structure_element
that	O
does	O
not	O
significantly	O
bind	O
PI4KB	B-protein
kinase	B-protein_type
.	O

In	O
every	O
case	O
the	O
ACDB3	B-protein
Q	B-structure_element
domain	I-structure_element
was	O
rapidly	O
(	O
within	O
5	O
minutes	O
)	O
recruited	O
to	O
the	O
mitochondrial	O
membrane	O
upon	O
addition	O
of	O
rapamycin	B-chemical
,	O
but	O
only	O
the	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
protein	O
effectively	O
directed	O
the	O
kinase	B-protein_type
to	O
the	O
mitochondria	O
(	O
Fig	O
.	O
3E	O
,	O
Movie	O
1	O
and	O
2	O
).	O

Notably	O
,	O
we	O
observed	O
that	O
when	O
the	O
GFP	B-experimental_method
-	O
PI4KB	B-protein
kinase	B-protein_type
is	O
co	B-experimental_method
-	I-experimental_method
expressed	I-experimental_method
with	O
the	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
ACDB3	B-protein
Q	B-structure_element
domain	I-structure_element
it	O
loses	O
its	O
typical	O
Golgi	O
localization	B-evidence
(	O
Fig	O
.	O
3E	O
upper	O
panel	O
).	O

However	O
,	O
PI4KB	B-protein
retains	O
it	O
Golgi	O
localization	B-evidence
when	O
co	B-experimental_method
-	I-experimental_method
expressed	I-experimental_method
with	O
the	O
non	B-protein_state
-	I-protein_state
interacting	I-protein_state
Q	B-structure_element
domain	I-structure_element
mutant	B-protein_state
(	O
Fig	O
.	O
3E	O
lower	O
panel	O
).	O

ACBD3	B-protein
increases	O
PI4KB	B-protein
enzymatic	B-evidence
activity	I-evidence
by	O
recruiting	O
PI4KB	B-protein
to	O
close	O
vicinity	O
of	O
its	O
substrate	O

To	O
test	O
whether	O
ACBD3	B-protein
can	O
stimulate	O
PI4KB	B-protein
kinase	B-protein_type
enzymatic	B-evidence
activity	I-evidence
we	O
performed	O
a	O
standard	O
luminescent	B-experimental_method
kinase	I-experimental_method
assay	I-experimental_method
using	O
PI	B-chemical
-	O
containing	O
micelles	O
as	O
the	O
substrate	O
.	O

We	O
observed	O
no	O
effect	O
on	O
the	O
kinase	B-protein_type
activity	O
of	O
PI4KB	B-protein
(	O
Fig	O
.	O
4A	O
)	O
suggesting	O
that	O
ACBD3	B-protein
does	O
not	O
directly	O
affect	O
the	O
enzyme	O
(	O
e	O
.	O
g	O
.	O
induction	O
of	O
a	O
conformation	O
change	O
).	O

However	O
,	O
in	O
vivo	O
ACBD3	B-protein
is	O
located	O
at	O
the	O
Golgi	O
membranes	O
,	O
whereas	O
in	O
this	O
experiment	O
,	O
ACBD3	B-protein
was	O
located	O
in	O
the	O
solution	O
and	O
PI	B-chemical
is	O
provided	O
as	O
micelles	O
.	O

For	O
this	O
,	O
we	O
again	O
turned	O
to	O
the	O
GUV	B-experimental_method
system	O
with	O
ACBD3	B-protein
localized	B-evidence
to	O
the	O
GUV	B-experimental_method
membrane	O
.	O

The	O
GUVs	B-experimental_method
contained	O
10	O
%	O
PI	B-chemical
to	O
serve	O
as	O
a	O
substrate	O
for	O
PI4KB	B-protein
kinase	B-protein_type
.	O

The	O
buffer	O
also	O
contained	O
CFP	B-experimental_method
-	O
SidC	B-protein
,	O
which	O
binds	O
to	O
PI4P	B-chemical
with	O
nanomolar	O
affinity	O
.	O

This	O
enabled	O
visualization	O
of	O
the	O
kinase	B-protein_type
reaction	O
using	O
a	O
confocal	B-experimental_method
microscope	I-experimental_method
.	O

We	O
compared	O
the	O
efficiency	O
of	O
the	O
phosphorylation	B-ptm
reaction	O
of	O
the	O
kinase	B-protein_type
alone	B-protein_state
with	O
that	O
of	O
kinase	B-protein_type
recruited	O
to	O
the	O
surface	O
of	O
the	O
GUVs	B-experimental_method
by	O
ACBD3	B-protein
.	O

Reaction	O
was	O
also	O
performed	O
in	O
the	O
absence	B-protein_state
of	I-protein_state
ATP	B-chemical
as	O
a	O
negative	O
control	O
(	O
Fig	O
.	O
4B	O
).	O

These	O
experiments	O
showed	O
that	O
PI4KB	B-protein
enzymatic	B-evidence
activity	I-evidence
increases	O
when	O
ACBD3	B-protein
is	O
membrane	O
localized	O
(	O
Fig	O
.	O
4C	O
,	O
SI	O
Fig	O
.	O
6	O
).	O

Membrane	O
recruitment	O
of	O
PI4KB	B-protein
enzyme	O
is	O
crucial	O
to	O
ensure	O
its	O
proper	O
function	O
at	O
the	O
Golgi	O
and	O
TGN	O
.	O

However	O
,	O
the	O
molecular	O
mechanism	O
and	O
structural	O
basis	O
for	O
PI4KB	B-protein
interaction	O
with	O
the	O
membrane	O
is	O
poorly	O
understood	O
.	O

In	O
principle	O
,	O
any	O
of	O
the	O
binding	O
partners	O
of	O
PI4KB	B-protein
could	O
play	O
a	O
role	O
in	O
membrane	O
recruitment	O
.	O

To	O
date	O
,	O
several	O
PI4KB	B-protein
interacting	O
proteins	O
have	O
been	O
reported	O
,	O
including	O
the	O
small	B-protein_type
GTPases	I-protein_type
Rab11	B-protein
and	O
Arf1	B-protein
,	O
the	O
Golgi	O
resident	O
acyl	B-protein
-	I-protein
CoA	I-protein
binding	I-protein
domain	I-protein
containing	I-protein
3	I-protein
(	O
ACBD3	B-protein
)	O
protein	O
,	O
neuronal	B-protein
calcium	I-protein
sensor	I-protein
-	I-protein
1	I-protein
(	O
NCS	B-protein
-	I-protein
1	I-protein
also	O
known	O
as	O
frequenin	B-protein
in	O
yeast	B-taxonomy_domain
)	O
and	O
the	O
14	B-protein_type
-	I-protein_type
3	I-protein_type
-	I-protein_type
3	I-protein_type
proteins	I-protein_type
.	O

The	O
monomeric	B-oligomeric_state
G	B-protein_type
protein	I-protein_type
Rab11	B-protein
binds	O
mammalian	B-taxonomy_domain
PI4KB	B-protein
through	O
the	O
helical	B-structure_element
domain	I-structure_element
of	O
the	O
kinase	B-protein_type
.	O

Although	O
Rab11	B-protein
does	O
not	O
appear	O
to	O
be	O
required	O
for	O
recruitment	O
of	O
PI4KB	B-protein
to	O
the	O
Golgi	O
,	O
PI4KB	B-protein
is	O
required	O
for	O
Golgi	O
recruitment	O
of	O
Rab11	B-protein
.	O

Arf1	B-protein
,	O
the	O
other	O
small	B-protein_type
GTP	I-protein_type
binding	I-protein_type
protein	I-protein_type
,	O
is	O
known	O
to	O
influence	O
the	O
activity	O
and	O
localization	O
of	O
PI4KB	B-protein
,	O
but	O
it	O
does	O
not	O
appear	O
to	O
interact	O
directly	O
with	O
PI4KB	B-protein
(	O
our	O
unpublished	O
data	O
).	O

The	O
yeast	B-taxonomy_domain
homologue	O
of	O
NCS1	B-protein
called	O
frequenin	B-protein
has	O
been	O
shown	O
to	O
interact	O
with	O
Pik1p	B-protein
,	O
the	O
yeast	B-taxonomy_domain
orthologue	O
of	O
PI4KB	B-protein
and	O
regulate	O
its	O
activity	O
and	O
perhaps	O
its	O
membrane	O
association	O
,	O
but	O
the	O
role	O
of	O
NCS	B-protein
-	I-protein
1	I-protein
in	O
PI4KB	B-protein
recruitment	O
in	O
mammalian	B-taxonomy_domain
cells	O
is	O
unclear	O
.	O

NCS	B-protein
-	I-protein
1	I-protein
is	O
an	O
N	O
-	O
terminally	O
myristoylated	B-protein_state
protein	O
that	O
participates	O
in	O
exocytosis	O
.	O

It	O
is	O
expressed	O
only	O
in	O
certain	O
cell	O
types	O
,	O
suggesting	O
that	O
if	O
it	O
contributes	O
to	O
PI4KB	B-protein
membrane	O
recruitment	O
,	O
it	O
does	O
so	O
in	O
a	O
tissues	O
specific	O
manner	O
.	O

The	O
interaction	O
of	O
PI4KB	B-protein
with	O
14	B-protein_type
-	I-protein_type
3	I-protein_type
-	I-protein_type
3	I-protein_type
proteins	I-protein_type
,	O
promoted	O
by	O
phosphorylation	B-ptm
of	O
PI4KB	B-protein
by	O
protein	B-protein
kinase	I-protein
D	I-protein
,	O
influences	O
the	O
activity	O
of	O
PI4KB	B-protein
by	O
stabilizing	O
its	O
active	B-protein_state
conformation	O
.	O

However	O
,	O
14	B-protein_type
-	I-protein_type
3	I-protein_type
-	I-protein_type
3	I-protein_type
proteins	I-protein_type
do	O
not	O
appear	O
to	O
interfere	O
with	O
membrane	O
recruitment	O
of	O
this	O
kinase	B-protein_type
.	O

ACBD3	B-protein
is	O
a	O
Golgi	O
resident	O
protein	O
,	O
conserved	B-protein_state
among	O
vertebrates	B-taxonomy_domain
(	O
SI	O
Fig	O
.	O
7	O
),	O
that	O
interacts	O
directly	O
with	O
PI4KB	B-protein
(	O
see	O
also	O
SI	O
Fig	O
.	O
8	O
and	O
SI	O
Discussion	O
),	O
and	O
whose	O
genetic	O
inactivation	O
interferes	O
with	O
the	O
Golgi	O
localization	O
of	O
the	O
kinase	B-protein_type
.	O

For	O
these	O
reasons	O
we	O
focused	O
on	O
the	O
interaction	O
of	O
the	O
PI4KB	B-protein
enzyme	O
with	O
the	O
Golgi	O
resident	O
ACBD3	B-protein
protein	O
in	O
this	O
study	O
.	O

Here	O
we	O
present	O
the	O
mechanism	O
for	O
membrane	O
recruitment	O
of	O
PI4KB	B-protein
by	O
the	O
Golgi	O
resident	O
ACBD3	B-protein
protein	O
.	O

We	O
show	O
that	O
these	O
proteins	O
interact	O
directly	O
with	O
a	O
Kd	B-evidence
value	O
in	O
the	O
submicromolar	O
range	O
.	O

The	O
interaction	O
is	O
sufficient	O
to	O
recruit	O
PI4KB	B-protein
to	O
model	O
membranes	O
in	O
vitro	O
as	O
well	O
as	O
to	O
the	O
mitochondria	O
where	O
PI4KB	B-protein
is	O
never	O
naturally	O
found	O
.	O

To	O
understand	O
this	O
process	O
at	O
the	O
atomic	O
level	O
we	O
solved	B-experimental_method
the	O
solution	B-evidence
structure	I-evidence
of	O
ACBD3	B-complex_assembly
:	I-complex_assembly
PI4KB	I-complex_assembly
sub	O
complex	O
(	O
Fig	O
.	O
1A	O
)	O
and	O
found	O
that	O
the	O
PI4KB	B-protein
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
contains	O
a	O
short	B-structure_element
amphipatic	I-structure_element
helix	I-structure_element
(	O
residues	O
44	B-residue_range
–	I-residue_range
64	I-residue_range
)	O
that	O
binds	O
the	O
ACBD3	B-protein
Q	B-structure_element
domain	I-structure_element
.	O

The	O
Q	B-structure_element
domain	I-structure_element
adopts	O
a	O
helical	B-structure_element
hairpin	I-structure_element
fold	I-structure_element
that	O
is	O
further	O
stabilized	O
upon	O
binding	O
the	O
kinase	B-structure_element
helix	I-structure_element
(	O
Fig	O
.	O
2A	O
).	O

Our	O
data	O
strongly	O
suggest	O
that	O
formation	O
of	O
the	O
complex	O
does	O
not	O
directly	O
influence	O
the	O
catalytic	O
abilities	O
of	O
the	O
kinase	B-protein_type
but	O
experiments	O
with	O
model	O
membranes	O
revealed	O
that	O
ACBD3	B-protein
enhances	O
catalytic	O
activity	O
of	O
the	O
kinase	B-protein_type
by	O
a	O
recruitment	O
based	O
mechanism	O
;	O
it	O
recruits	O
the	O
kinase	B-protein_type
to	O
the	O
membrane	O
and	O
thus	O
increases	O
the	O
local	O
concentration	O
of	O
the	O
substrate	O
in	O
the	O
vicinity	O
of	O
the	O
kinase	B-protein_type
.	O

Based	O
on	O
our	O
and	O
previously	O
published	O
structures	B-evidence
we	O
built	O
a	O
pseudoatomic	B-evidence
model	I-evidence
of	O
PI4KB	B-protein
multi	O
-	O
protein	O
assembly	O
on	O
the	O
membrane	O
(	O
Fig	O
.	O
5	O
)	O
that	O
illustrates	O
how	O
the	O
enzyme	O
is	O
recruited	O
and	O
positioned	O
towards	O
its	O
lipidic	O
substrate	O
and	O
how	O
it	O
in	O
turn	O
recruits	O
Rab11	B-protein
.	O

+	B-taxonomy_domain
RNA	I-taxonomy_domain
viruses	I-taxonomy_domain
replicate	O
at	O
specific	O
PI4P	B-chemical
-	O
enriched	O
membranous	O
compartments	O
.	O

These	O
are	O
called	O
replication	O
factories	O
(	O
because	O
they	O
enhance	O
viral	B-taxonomy_domain
replication	O
)	O
or	O
membranous	O
webs	O
(	O
because	O
of	O
their	O
appearance	O
under	O
the	O
electron	O
microscope	O
).	O

To	O
generate	O
replication	O
factories	O
,	O
viruses	B-taxonomy_domain
hijack	O
several	O
host	O
factors	O
including	O
the	O
PI4K	B-protein_type
kinases	B-protein_type
to	O
secure	O
high	O
content	O
of	O
the	O
PI4P	B-chemical
lipid	B-chemical
.	O

Non	B-protein_type
-	I-protein_type
structural	I-protein_type
3A	I-protein_type
proteins	I-protein_type
from	O
many	O
picornaviruses	B-taxonomy_domain
from	O
the	O
Enterovirus	B-taxonomy_domain
(	O
e	O
.	O
g	O
.	O
poliovirus	B-species
,	O
coxsackievirus	B-species
-	I-species
B3	I-species
,	O
rhinovirus	B-species
-	I-species
14	I-species
)	O
and	O
Kobuvirus	B-taxonomy_domain
(	O
e	O
.	O
g	O
.	O
Aichi	B-species
virus	I-species
-	I-species
1	I-species
)	O
genera	O
directly	O
interact	O
with	O
ACBD3	B-protein
.	O

Our	O
data	O
suggest	O
that	O
they	O
could	O
do	O
this	O
via	O
3A	B-complex_assembly
:	I-complex_assembly
ACBD3	I-complex_assembly
:	I-complex_assembly
PI4KB	I-complex_assembly
complex	O
formation	O
.	O

The	O
structure	B-evidence
of	O
the	O
ACBD3	B-protein
Q	B-structure_element
domain	I-structure_element
and	O
the	O
kinase	B-structure_element
helix	I-structure_element
described	O
here	O
provides	O
a	O
novel	O
opportunity	O
for	O
further	O
research	O
on	O
the	O
role	O
of	O
ACBD3	B-protein
,	O
PI4KB	B-protein
,	O
and	O
the	O
ACBD3	B-complex_assembly
:	I-complex_assembly
PI4KB	I-complex_assembly
interaction	O
in	O
picornaviral	B-taxonomy_domain
replication	O
.	O

This	O
could	O
eventually	O
have	O
implications	O
for	O
therapeutic	O
intervention	O
to	O
combat	O
picornaviruses	B-taxonomy_domain
-	O
mediated	O
diseases	O
ranging	O
from	O
polio	O
to	O
the	O
common	O
cold	O
.	O

Biochemical	B-experimental_method
characterization	I-experimental_method
of	O
the	O
ACBD3	B-complex_assembly
:	I-complex_assembly
PI4KB	I-complex_assembly
complex	O
.	O

(	O
A	O
)	O
Schematic	O
representation	O
of	O
the	O
ACBD3	B-protein
and	O
PI4KB	B-protein
constructs	O
used	O
for	O
the	O
experiments	O
.	O

ACBD3	B-protein
contains	O
the	O
acyl	B-structure_element
-	I-structure_element
CoA	I-structure_element
binding	I-structure_element
domain	I-structure_element
(	O
ACBD	B-structure_element
),	O
charged	B-structure_element
amino	I-structure_element
acids	I-structure_element
region	I-structure_element
(	O
CAR	B-structure_element
),	O
glutamine	B-structure_element
rich	I-structure_element
region	I-structure_element
(	O
Q	B-structure_element
),	O
and	O
Golgi	B-structure_element
dynamics	I-structure_element
domain	I-structure_element
(	O
GOLD	B-structure_element
).	O

PI4KB	B-protein
is	O
composed	O
of	O
the	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
,	O
helical	B-structure_element
domain	I-structure_element
,	O
and	O
kinase	B-structure_element
domain	I-structure_element
which	O
can	O
be	O
divided	O
into	O
N	B-structure_element
-	I-structure_element
and	I-structure_element
C	I-structure_element
-	I-structure_element
terminal	I-structure_element
lobes	I-structure_element
.	O

(	O
B	O
)	O
In	B-experimental_method
vitro	I-experimental_method
pull	I-experimental_method
-	I-experimental_method
down	I-experimental_method
assay	I-experimental_method
.	O

Pull	B-experimental_method
-	I-experimental_method
down	I-experimental_method
assays	I-experimental_method
were	O
performed	O
using	O
NiNTA	O
-	O
immobilized	O
N	O
-	O
terminal	O
His6GB1	B-protein_state
-	I-protein_state
tagged	I-protein_state
proteins	O
as	O
indicated	O
and	O
untagged	B-protein_state
full	B-protein_state
-	I-protein_state
length	I-protein_state
PI4KB	B-protein
or	O
ACBD3	B-protein
.	O

The	O
inputs	O
and	O
bound	O
proteins	O
were	O
analyzed	O
on	O
SDS	B-experimental_method
gels	I-experimental_method
stained	O
with	O
Coomassie	O
Blue	O
.	O

Please	O
,	O
see	O
SI	O
Fig	O
.	O
9	O
for	O
original	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
gels	O
.	O
(	O
C	O
)	O
Analytical	B-experimental_method
Ultracentrifugation	I-experimental_method
.	O

AUC	B-experimental_method
analysis	O
of	O
the	O
ACBD3	B-complex_assembly
:	I-complex_assembly
PI4KB	I-complex_assembly
full	B-protein_state
-	I-protein_state
length	I-protein_state
complex	O
at	O
the	O
concentration	O
of	O
5	O
μM	O
(	O
both	O
proteins	O
,	O
left	O
panel	O
)	O
and	O
ACBD3	B-complex_assembly
Q	I-complex_assembly
domain	I-complex_assembly
:	I-complex_assembly
PI4KB	I-complex_assembly
N	I-complex_assembly
terminal	I-complex_assembly
region	I-complex_assembly
complex	O
at	O
the	O
concentration	O
of	O
35	O
μM	O
(	O
both	O
proteins	O
,	O
right	O
panel	O
).	O
(	O
D	O
)	O
Surface	B-experimental_method
plasmon	I-experimental_method
resonance	I-experimental_method
.	O

SPR	B-experimental_method
analysis	O
of	O
the	O
PI4KB	B-protein
binding	O
to	O
immobilized	O
ACBD3	B-protein
.	O

Sensorgrams	B-evidence
for	O
four	O
concentrations	O
of	O
PI4KB	B-protein
are	O
shown	O
.	O

Structural	B-experimental_method
analysis	I-experimental_method
of	O
the	O
ACBD3	B-complex_assembly
:	I-complex_assembly
PI4KB	I-complex_assembly
complex	O
.	O

(	O
A	O
)	O
Overall	O
structure	B-evidence
of	O
the	O
ACBD3	B-protein
Q	B-structure_element
domain	I-structure_element
by	O
itself	O
and	O
in	B-protein_state
complex	I-protein_state
with	I-protein_state
the	O
PI4KB	B-protein
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
.	O

Superposition	B-experimental_method
of	O
the	O
30	O
converged	O
structures	B-evidence
obtained	O
for	O
the	O
Q	B-structure_element
domain	I-structure_element
(	O
top	O
)	O
and	O
the	O
45	O
converged	O
structures	B-evidence
obtained	O
for	O
the	O
complex	O
(	O
bottom	O
),	O
with	O
only	O
the	O
folded	B-protein_state
part	O
of	O
PI4KB	B-protein
shown	O
(	O
see	O
SI	O
Fig	O
.	O
2	O
for	O
the	O
complete	O
view	O
).	O
(	O
B	O
)	O
Detailed	O
view	O
of	O
the	O
complex	O
.	O

The	O
interaction	O
is	O
facilitated	O
by	O
only	O
two	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
(	O
ACBD3	B-protein
Tyr261	B-residue_name_number
:	O
PI4KB	B-protein
His63	B-residue_name_number
and	O
ACBD3	B-protein
Tyr288	B-residue_name_number
:	O
PI4KB	B-protein
Asp44	B-residue_name_number
),	O
while	O
the	O
hydrophobic	B-site
surface	I-site
of	O
the	O
kinase	B-structure_element
helix	I-structure_element
nests	O
in	O
the	O
ACBD3	B-protein
Q	B-structure_element
domain	I-structure_element
.	O

ACBD3	B-protein
is	O
shown	O
in	O
magenta	O
and	O
PI4KB	B-protein
in	O
orange	O
.	O

(	O
C	O
)	O
Top	O
view	O
of	O
the	O
kinase	B-structure_element
helix	I-structure_element
.	O

The	O
kinase	B-structure_element
helix	I-structure_element
is	O
amphipathic	B-protein_state
and	O
its	O
hydrophobic	B-site
surface	I-site
overlaps	O
with	O
the	O
ACBD3	B-protein
binding	B-site
surface	I-site
(	O
shown	O
in	O
magenta	O
).	O

Strong	O
and	O
weak	O
hydrophobes	O
are	O
in	O
green	O
and	O
cyan	O
respectively	O
,	O
basic	O
residues	O
in	O
blue	O
,	O
acidic	O
residues	O
in	O
red	O
and	O
nonpolar	O
hydrophilic	O
residues	O
in	O
orange	O
.	O
(	O
D	O
)	O
Pull	B-experimental_method
-	I-experimental_method
down	I-experimental_method
assay	I-experimental_method
with	O
a	O
NiNTA	O
-	O
immobilized	O
N	O
-	O
terminally	O
His6GB1	B-protein_state
-	I-protein_state
tagged	I-protein_state
PI4KB	B-protein
kinase	B-protein_type
and	O
untagged	B-protein_state
ACBD3	B-protein
protein	O
.	O

Wild	B-protein_state
type	I-protein_state
proteins	O
and	O
selected	O
point	O
mutants	B-protein_state
of	O
both	O
PI4KB	B-protein
and	O
ACBD3	B-protein
were	O
used	O
.	O

Please	O
,	O
see	O
SI	O
Fig	O
.	O
9	O
for	O
original	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
gels	O
.	O

ACBD3	B-protein
is	O
sufficient	O
to	O
recruit	O
the	O
PI4KB	B-protein
kinase	B-protein_type
to	O
membranes	O
.	O

(	O
A	O
)	O
GUVs	B-experimental_method
recruitment	I-experimental_method
assay	I-experimental_method
.	O

Top	O
–	O
Virtually	O
no	O
membrane	O
bound	O
kinase	B-protein_type
was	O
observed	O
when	O
600	O
nM	O
PI4KB	B-protein
was	O
added	O
to	O
the	O
GUVs	B-experimental_method
.	O

Bottom	O
–	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
600	O
nM	O
GUV	B-protein_state
tethered	I-protein_state
ACBD3	B-protein
a	O
significant	O
signal	O
of	O
the	O
kinase	B-protein_type
is	O
detected	O
on	O
the	O
surface	O
of	O
GUVs	B-experimental_method
.	O

(	O
B	O
)	O
Golgi	B-experimental_method
displacement	I-experimental_method
experiment	I-experimental_method
.	O

Upper	O
panel	O
:	O
ACBD3	B-protein
Q	B-structure_element
domain	I-structure_element
fused	O
to	O
GFP	B-experimental_method
was	O
overexpressed	B-experimental_method
and	O
the	O
endogenous	O
PI4KB	B-protein
was	O
immunostained	B-experimental_method
.	O

Middle	O
panel	O
:	O
The	O
same	O
experiment	O
performed	O
with	O
GFP	B-experimental_method
alone	O
.	O

Lower	O
panel	O
:	O
The	O
same	O
experiment	O
performed	O
with	O
mutant	B-protein_state
Q	B-structure_element
domain	I-structure_element
(	O
F258A	B-mutant
,	O
H284A	B-mutant
,	O
Y288A	B-mutant
)	O
that	O
does	O
not	O
bind	O
the	O
PI4KB	B-protein
.	O
(	O
C	O
)	O
ACBD3	B-protein
Q	B-structure_element
domain	I-structure_element
overexpression	B-experimental_method
inhibits	O
ceramide	B-chemical
transport	O
to	O
Golgi	O
–	O
COS	O
-	O
7	O
cells	O
transfected	O
with	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
ACBD3	B-protein
Q	B-structure_element
domain	I-structure_element
-	O
FKBP	B-protein
-	O
mRFP	B-experimental_method
were	O
loaded	O
with	O
0	O
.	O
05	O
μM	O
Bodipy	B-chemical
FL	I-chemical
-	I-chemical
Ceramide	I-chemical
for	O
20	O
min	O
,	O
then	O
washed	O
and	O
depicted	O
after	O
20	O
min	O
.	O

Middle	O
panel	O
–	O
The	O
same	O
experiment	O
performed	O
with	O
mRFP	B-experimental_method
-	O
FKBP	B-protein
alone	O
.	O

Lower	O
panel	O
–	O
The	O
same	O
experiment	O
performed	O
with	O
mutant	B-protein_state
Q	B-structure_element
domain	I-structure_element
(	O
F258A	B-mutant
,	O
H284A	B-mutant
,	O
Y288A	B-mutant
)	O
that	O
does	O
not	O
bind	O
the	O
PI4KB	B-protein
.	O
(	O
D	O
)	O
Scheme	O
of	O
the	O
mitochondria	B-experimental_method
recruitment	I-experimental_method
experiment	I-experimental_method
.	O

–	O
The	O
AKAP1	B-protein
-	O
FRB	B-structure_element
-	O
CFP	B-experimental_method
construct	O
is	O
localized	B-evidence
at	O
the	O
outer	O
mitochondrial	O
membrane	O
,	O
while	O
the	O
GFP	B-experimental_method
-	O
PI4KB	B-protein
and	O
Q	B-structure_element
domain	I-structure_element
-	O
FKBP	B-protein
-	O
mRFP	B-experimental_method
constructs	O
are	O
localized	B-evidence
in	O
the	O
cytoplasm	O
where	O
they	O
can	O
form	O
a	O
complex	O
.	O

Upon	O
addition	O
of	O
rapamycin	B-chemical
the	O
Q	B-structure_element
domain	I-structure_element
-	O
FKBP	B-protein
-	O
mRFP	B-experimental_method
construct	O
translocates	O
to	O
the	O
mitochondria	O
and	O
takes	O
GFP	B-experimental_method
-	O
PI4KB	B-protein
with	O
it	O
.	O
(	O
E	O
)	O
Mitochondria	B-experimental_method
recruitment	I-experimental_method
experiment	I-experimental_method
.	O

Left	O
–	O
cells	O
transfected	O
with	O
AKAP1	B-protein
-	O
FRB	B-structure_element
-	O
CFP	B-experimental_method
,	O
GFP	B-experimental_method
-	O
PI4KB	B-protein
and	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
Q	B-structure_element
domain	I-structure_element
-	O
FKBP	B-protein
-	O
mRFP	B-experimental_method
constructs	O
before	O
and	O
five	O
minutes	O
after	O
addition	O
of	O
rapamycin	B-chemical
.	O

Right	O
–	O
The	O
same	O
experiment	O
performed	O
using	O
the	O
H264A	B-mutant
Q	B-structure_element
domain	I-structure_element
mutant	B-protein_state
.	O

ACBD3	B-protein
indirectly	O
increases	O
the	O
activity	O
of	O
PI4KB	B-protein
.	O

(	O
A	O
)	O
Micelles	B-experimental_method
-	I-experimental_method
based	I-experimental_method
kinase	I-experimental_method
assay	I-experimental_method
–	O
PI	B-chemical
in	O
TX100	O
micelles	O
was	O
used	O
in	O
a	O
luminescent	B-experimental_method
kinase	I-experimental_method
assay	I-experimental_method
and	O
the	O
production	O
of	O
PI4P	B-chemical
was	O
measured	O
.	O

Bar	O
graph	O
presents	O
the	O
mean	O
values	O
of	O
PI4P	B-chemical
generated	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
the	O
proteins	O
as	O
indicated	O
,	O
normalized	O
to	O
the	O
amount	O
of	O
PI4P	B-chemical
generated	O
by	O
PI4KB	B-protein
alone	O
.	O

Error	O
bars	O
are	O
standard	B-evidence
errors	I-evidence
of	I-evidence
the	I-evidence
mean	I-evidence
(	O
SEM	B-evidence
)	O
based	O
on	O
three	O
independent	O
experiments	O
.	O
(	O
B	O
)	O
GUV	B-experimental_method
-	I-experimental_method
based	I-experimental_method
phosphorylation	I-experimental_method
assay	I-experimental_method
–	O
GUVs	B-experimental_method
containing	O
10	O
%	O
PI	B-chemical
were	O
used	O
as	O
a	O
substrate	O
and	O
the	O
production	O
of	O
PI4P	B-chemical
was	O
measured	O
using	O
the	O
CFP	B-experimental_method
-	I-experimental_method
SidC	I-experimental_method
biosensor	I-experimental_method
.	O

(	O
C	O
)–	O
Quantification	O
of	O
the	O
GUV	B-experimental_method
phosphorylation	I-experimental_method
assay	I-experimental_method
–	O
Mean	B-evidence
membrane	I-evidence
fluorescence	I-evidence
intensity	I-evidence
of	O
the	O
PI4P	B-chemical
reporter	O
(	O
SidC	B-protein
-	O
label	O
)	O
under	O
different	O
protein	O
/	O
ATP	B-chemical
conditions	O
.	O

The	O
mean	B-evidence
membrane	I-evidence
intensity	I-evidence
value	O
is	O
relative	O
to	O
the	O
background	O
signal	O
and	O
the	O
difference	O
between	O
the	O
membrane	O
and	O
background	O
signal	O
in	O
the	O
reference	O
system	O
lacking	O
ATP	B-chemical
.	O

The	O
error	O
bars	O
stand	O
for	O
SEM	B-evidence
based	O
on	O
three	O
independent	O
experiments	O
(	O
also	O
SI	O
Fig	O
.	O
6	O
).	O

Pseudoatomic	B-evidence
model	I-evidence
of	O
the	O
PI4KB	B-protein
multiprotein	O
complex	O
assembly	O
.	O

PI4KB	B-protein
in	O
orange	O
,	O
Rab11	B-protein
in	O
purple	O
,	O
ACBD3	B-protein
in	O
blue	O
.	O

The	O
model	O
is	O
based	O
on	O
our	O
NMR	B-experimental_method
structure	B-evidence
and	O
a	O
previously	O
published	O
crystal	B-evidence
structure	I-evidence
of	O
PI4KB	B-complex_assembly
:	I-complex_assembly
Rab11	I-complex_assembly
complex	O
(	O
PDB	O
code	O
4D0L	O
),	O
ACBD	B-structure_element
and	O
GOLD	B-structure_element
domain	O
were	O
homology	B-experimental_method
modeled	I-experimental_method
based	O
on	O
high	O
sequence	O
identity	O
structures	B-evidence
produced	O
by	O
the	O
Phyre2	B-experimental_method
web	O
server	O
.	O

The	O
GOLD	B-structure_element
domain	O
is	O
tethered	O
to	O
the	O
membrane	O
by	O
GolginB1	B-protein
(	O
also	O
known	O
as	O
Giantin	B-protein
)	O
which	O
is	O
not	O
shown	O
for	O
clarity	O
.	O

Intrinsically	B-structure_element
disordered	I-structure_element
linkers	I-structure_element
are	O
modeled	O
in	O
an	O
arbitrary	O
but	O
physically	O
plausible	O
conformation	O
.	O