Dia 1

1
Acknowledgements
Chemical Biology and Organic Chemistry, Utrecht
University Eefjan Breukink, Vincent van Dam,
Robert Sijbrandi, Tamimount Mohammadi, Richard
Huijbregts, Matthijs Kol, Toon de
Kroon Swammerdam Institute for Life Sciences,
University of Amsterdam Tanneke den
Blaauwen Center for Protein Engineering,
University of Liège Martine Nguyen-Distèche Inst
itute for molecular and cellular biochemistry and
biophysics, University Paris-Sud Dominique
Mengin-Lecreulx
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Bacterial cell enveloppe
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Phospholipid translocation E. coli IM

• PL synthesis cis-side
• Translocation
• fast, bidirectional
• low headgroup specificity
• PMF/ATP not required
• not obligatory coupled to synthesis
• protein mediated (specific/ more general)

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Flop plotted against incubation time
KALP31 12000
C6NBD-PG
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Concentration dependence
KALP31
C6NBD-PG
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Concentration dependence
KALP31
C6NBD-PG
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The integral part of (some of) the membrane
proteins in the inner membrane catalyze
transbilayer movement of phospholipids
Kol et al. (2004) Biochemistry
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Relative rate constant of NBD-PG flop as a
function of the lipid matrix the inhibitory
effect of cholesterol
WALP23
KALP23
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Bacterial cell wall synthesis a central role
for the membrane embedded precursor Lipid II
Lipid II
out
in
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Structure of Lipid II
G
GlcNAc
D-Ala-D-Ala
M
A
E
MurNAc
A
A
K
Pi
Pi
pentapeptide
Pyrophosphate
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Undecaprenyl chain
2
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MD simulation of Lipid II in a bilayer
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MD simulation of Lipid II in a bilayer
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In-vitro synthesis of Lipid II
Breukink et al. (2003) JBC
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Does Lipid II spontaneously flip/flop across the
membrane?
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NBD-fluorescence (a.u.)
1
0
0
25
50
100
75
time (s)
Van Dam et al. (2008) Mol. Microbiol.
DOPE/DOPG/NBD-Lipid II 75250.2
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Does Lipid II spontaneously flip/flop across the
membrane?
2
dithionite
triton X-100
50
NBD-fluorescence (a.u.)
1
If there is transport, it is not a fast process!
0
0
25
50
100
75
time (s)
Van Dam et al. (2008) Mol. Microbiol.
DOPE/DOPG/NBD-Lipid II 75250.2
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Lipid II does not spontaneously flip/flop across
the membrane?
After removal of dithionite ?Incubation at 30ºC
?
DOPE/DOPG/NBD-Lipid II 75250.2
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• Lipid II translocation is not induced by TMs
  that induce PL translocation
• Lipid II synthesis and translocation are not
  obligatory coupled

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Lipid II
out
Lipid I
MraY
MurG
Lipid II
M
M
G
in
G
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A Lipid II translocation assay based on FRET
between Lipid II and vancomycin
out
in
Pi
Plasma membrane
Pi
M
Fluorescent lipid II
G
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A Lipid II translocation assay based on FRET
between Lipid II and vancomycin
G
M
out
Pi
Pi
in
Pi
Plasma membrane
Pi
M
Fluorescent lipid II
G
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A Lipid II translocation assay based on FRET
between Lipid II and vancomycin
G
M
out
Pi
Pi
in
Pi
Plasma membrane
Pi
M
Fluorescent lipid II
G
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A Lipid II translocation assay based on FRET
between Lipid II and vancomycin
FRET
G
M
out
Pi
Pi
in
Pi
Plasma membrane
Pi
M
Fluorescent lipid II
G
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Interaction between fluorescently labeled
vancomycin and Lipid II results in FRET!
Model membrane vesicles with NBD-Lipid II
incorporated, excitation at 481 nm
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In vitro Lipid II translocationFRET assay
RSO

• General procedure
• Right side out (RSO) vesicles E. coli W3899
• NBD-UDP-MurNAc-pentapeptide UDP-GlcNAc
• Two freeze-thaw cycles
• Lipid II synthesis (on ice)
• Fluorescence spectra in the
• presence of vanco-TMR (15 C)

Freeze-thaw
Synthesis
out
in
Pi
Membrane
Pi
M
Fluorescent lipid II
G
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The appearance of FRET demonstrates that Lipid II
translocation occurred
Spectra at 0, 5, 10, 20 and 30 min at 15 C with
vanco-TMR
Transport is not dependent on ATP PMF not
affected by trypsin/protease K
treatment Transport is affected by protein
modifying agents
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Photocross-link approach to identify the
proteins involved in Lipid II translocation
G
M
out
Pi
Pi
in
Pi
Plasma membrane
Pi
M
Photoactivatable lipid II
G
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Right Side Out E. coli vesicles (RSOs)
RSO
UDP-GlcNAc

Lipid II synthesis/ translocation
RSO
UV irradiation (30 s)
SDS PAGE/ Autoradiography
RSO
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14C methylated protein marker
Lipid II synthesis, UV light
Lipid II synthesis, no UV
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A specific set of proteins is crosslinked
Crosslinked proteins -autoradiogram
Total of proteins - coomassie
14C methylated protein marker
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FtsW
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MurG co-depletes with FtsW
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FtsW depletion results in decreased Lipid II
translocation
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FtsW overproduction results in increased Lipid
II translocation
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The same is true for E. coli FtsW
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Working model
Lipid II
out
PBP
FtsW
Lipid II
M
G
in
MurG
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The presence of FtsW makes Lipid II accesible to
dithionite
reconstitution experiments