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SNARE-fusion mediated inserƟon of membrane
proteins into naƟve and arƟficial membranes
1
1
1,2
Gustav Nordlund , Peter Brzezinski and Christoph von Ballmoos
1
Department of Biochemistry & Biophysics, Stockholm University, Sweden. 2Department of Chemistry & Biochemiststry, University of Bern, Switzerland
F1F0 ATP synthase
Membrane proteins carry out func ons such as nutrient uptake, ATP synthesis or
transmembrane signal transduc on. An increasing number of reports indicate that
cellular processes are underpinned by regulated interac ons between these proteins.
Consequently, func onal studies of these networks at a molecular level require
co-reconsƟtuƟon of the interacƟng components. Here, we report a SNARE-protein
based method for incorporaƟon of mulƟple membrane proteins into membranes, and
for delivery of large water-soluble substrates into closed membrane vesicles. The
approach is used for in vitro reconstruc on of a fully funcƟonal bacterial respiratory
chain from purified components. Furthermore, the method is used for func onal
incorporaƟon of the enƟre F1F0-ATP synthase complex into naƟve bacterial
membranes from which this component had been gene cally removed. The novel
methodology offers a tool to inves gate complex interac on networks between
membrane-bound proteins at a molecular level, which is expected to generate
func onal insights into key cellular func ons.
H+
N
KCN
Cartoon illustra ng the basic experimental setup. The two liposomes popula ons were allowed to fuse for 20 min and bo3 oxidase turnover was started with the addi on of DTT/Q1.
b
control (no syb)
1.2
Q
N
O
F1F0 ATP synthase
succinate
Q10H2
fumarate
Q10
O2
H+
nd
H2O
H+
ou
H+
H+
gr
50
100
150
200
Time (s)
c
fumarate
reductase
0.5
1
nmol ATP min-1
ΔΨ
ADP + Pi ATP
0.8
0.6
0.4
0.2
0
1
0.8
0.6
0.4
no syb
0.2
0
20
40
Fusion time (min)
60
a
b
50
ADP + Pi
ATP
H+
H+
H+
O2
e-
SNAP-25/syntaxin
H2O
e-
PMS
cyt. c
esynaptobrevin
ascorbate
source: http://boris.unibe.ch/65912/ | downloaded: 17.6.2017
-1
ATP synthesis (nmol ATP min )
10
8
syb + SNAP-25/syntaxin
6
4
no syb
2
0
0
20
Delivery of cytochrome c into closed vesicles.
a. Cartoon illustra ng the experimental setup.
Liposomes containing soluble cyt. c and synaptobrevin were mixed with proteoliposomes
containing CytcO oxidase, ATP synthase and
SNAP-25/syntaxin. The reac on was started by
the addi on of ascorbate and PMS as electron
donor and mediator, respec vely.
40
Fusion time (min)
60
b. Time course displaying the s mulated ATP
synthesis ac vity a er fusion of the two liposome popula on in the presence (filled circles)
and absence (open circles) of synaptobrevin.
Note: The low, but constant ac vity found in
the sample without synaptobrevin is due to
direct reduc on of bo3 oxidase by ascorbate/PMS.
H+
QH
H+
2
Q
H+
O2
H+
HO
2
H+
ΔΨ
NADH dehydrogenase
ADP + Pi
E. coli membrane proteins
H+
ATP
ATP synthesis rel. to BL21 (%)
NADH
NAD + H+
F1F0 ATP synthase
60
ATP synthase and E. coli membranes
Complex IV, ATP synthase and cytc
cytochrome c
oxidase
20
40
Fusion time (min)
Recons tu on of an intact bacterial respiratory chain.
a. Cartoon of the experimental setup. A er fusion, the respiratory chain was ini ated by addi on of
succinate, the substrate of complex II, reducing ubiquinone Q10 and thus energizing bo3 oxidase.
b. Time course of the experiment described in a. ATP synthesis rated were determined at the indicated me points in the presence (closed circles) or absence of synaptobrevin (open circles).
F1F0 ATP synthase
b
no syb
Respiratory enzyme activity
BL21
DK8
40
DK8 vs BL21 (%)
a. Raw data obtained from luminometer measurements obtained from experiment described in Introduc on Figure. Indicated are the addi ons
during the experiment.
b. ATP synthesis rates measured a er different addi ons as indicated in a(red). Controls without synaptobrevin are also shown (blue).
c. Time course of fusion during the experiment described in a. ATP synthesis rated were determined
at the indicated me points in the presence
(closed circles) or absence of synaptobrevin (open
circles).
syb + SNAP-25/syntaxin
0
a
syb + SNAP-25/syntaxin
1
0
1.2
-1
ATP synthesis (nmol ATP min )
0
Q1
DTT
a
ck
0
Ba
0
e-
synaptobrevin (syb)
bo3 oxidase
50
e-
ΔΨ
-1
ATP synthesis (nmol ATP × min )
ATP
100
H+
H2O
Q
1
150
O2
bo3 oxidase
H
200
ATP
Complexes II, IV and ATP synthase
syb + SNAP-25/syntaxin
KC
ubiqinone Q1
Order of addition
3
Luminescence (a.u. x 10 )
b
ADP + Pi
SNAP-25/syntaxin
Complex IV and ATP synthase
a
H+
30
20
NADH
Q1/DTT
10
0
DK8 +
ATPase
liposomes
DK8
140
120
100
80
60
40
20
0
NADH
Complex I
DK8 +
ATPase lip.
(no syb)
NADH
Q1/DTT
Complex IV
ATPase
liposomes
Delivery of ATP synthase into inverted membranes of ATP synthase lacking E. coli strain
a. Cartoon illustra ng the experimental setup. Inverted membrane vesicles of E. coli DK8 doped with
synaptobrevin were mixed with liposomes containing ATP synthase and SNAP-25/syntaxin and allowed to fuse.
b. Respiratory driven ATP synthesis a er fusion as described in a. The results are given compared to
a similar prepara on of strain BL21, normalized to the total membrane protein concentra on.
Shown are the rela ve ac vi es a er addi on of NADH (blue) and DTT/Q1 (red). Rela ve respiratory
enzyme ac vi es in inverted membranes of strains BL21 (light grey) and DK8 (dark grey) measured
as NADH oxida on for complex I and O2 consump on for complex IV (driven by either NADH oxidaon or DTT/Q1) are also shown (inset).
Reference:
Nordlund G., Brzezinski P., and von Ballmoos C (2014).
SNARE-fusion mediated inser on of membrane proteins into na ve and ar ficial membranes.
Nat. Commun. 5:4303 doi: 10.1038/ncomms5303