Protein Secretion: a transport problem

1
Protein Secretion a transport problem
E. coli
Gram-negative
2
Terminal Secretion Systems of Gram-negatives
Type V secretion
Daniela Büttner and Ulla Bonas Trends in
Microbiology, Volume 10, Issue 4 , 2002, Pages
186-192
3
Tat (Twin arginine) transporter
A_A

• export of folded proteins across CM
• periplasmic proteins (often w. Redox cofactors
  i.e. TAMO-red.)
• several IM proteins
• new proteins later exported via Type II system
• energy electrochemical gradient (no ATP)
• signal peptide N-terminal signal
• SRRxFLK 'twin-arginine' motif
• cleaved off during transport (Signal peptidase
  1 LepB)

Palmer T., Sargent F, Berks BC, Trends Microbiol.
2005 Apr13(4)175-80.
4
Tat (Twin arginine) transportera proofreading
mechanism ?
Palmer T., Sargent F, Berks BC, Trends Microbiol.
2005 Apr13(4)175-80.
5
Tat (Twin arginine) transporter
Palmer T., Sargent F, Berks BC, Trends Microbiol.
2005 Apr13(4)175-80.
6
Tat transporter E. coli substrates
Palmer T., Sargent F, Berks BC, Trends Microbiol.
2005 Apr13(4)175-80.
7
Terminal Secretion Systems of Gram-negatives
Type V secretion
Daniela Büttner and Ulla Bonas Trends in
Microbiology, Volume 10, Issue 4 , 2002, Pages
186-192
8
The General Secretion Pathway (across IM
resembles ER-export in eukaryotes)
1) Association of peptide with secretion
system SecB or SRP bind leader (N-terminus,
20-30 aa hydrophobic core) 2) Translocation ATP
is main source of energy for translocation 3)
Type I signal peptidase cleaves off signal
sequence at a specific site. .
N
SP
SecYEG
FtsY
N
ATP
SecA
ADP
N
SRP
N
N
SecB
nascent polypeptide
nascent polypeptide
Ribosome
9
The General Secretion Pathway (Sec)
4) release and folding a) mature protein
released (peripl./extracell.) b) integrated into
the cytoplasmic membrane
8A structure of SecYEG
Breyton et al., Nature 2002
side view
N
a)
SP
b)
SecYEG
SecA
ATP
16 x 24 A channel
ADP
precursor protein
top view
a)
b)
10
Type V secretion (Autotransporter)
Type V secretion
11
Autotransporter
12
Model for Autotransporter function
protease (if secreted)
OM
peripl.
chaperone
N
SP
SecYEG
IM
SecA
ATP
N
SecB
ADP
precursor protein
13
Autotransporter
14
Terminal Secretion Systems of Gram-negatives
Type V secretion
Daniela Büttner and Ulla Bonas Trends in
Microbiology, Volume 10, Issue 4 , 2002, Pages
186-192
15
Chaperone-Usher pathway
donor strand complementation
Type 1 fimbriae (uropath. E. coli)
lectin domain (N)
pilin domain (C)
FimH
FimG
FimF
Ig-fold
FimA
FimH
FimG
FimF
FimD
FimD
OM
FimD (usher)
FimC
FimC (chaperone)
Sec
Sec
IM
Sec
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Type II secretion
Type V secretion
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secretion of cholera toxin via the type II
secretion pathway
4.
3.
2.
H
1.
sec
and proton motive force
Type II secretion shares many functional features
with the Type IV fimbriae assembly.
18
secretion of cholera toxin via the type II
secretion pathway
2. processing - S-S bridges via DsbA -
assembly of AB5 complex (cleavage of pilus
subunits G,H,I,J,K by prepilin-peptidase O)
sec
peripl.
IM
cytopl.
19
secretion of cholera toxin via the type II
secretion pathway
4.
3.
2.
H
1.
sec
and proton motive force
Type II secretion shares many functional features
with the Type IV fimbriae assembly.
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Experimental evidence for model B of the type II
secretion complex
E. coli expressing the pap-Operon of Klebsiella
oxytoca
anti-PulG immunogold
Type 1 fimbriae
pulG overexpression leads to bundle-like pilus
formation
Sauvonnet N, Vignon G, Pugsley AP, Gounon P. EMBO
J. 2000 May 1519(10)2221-8
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Wide variety of structures among Type II secreted
proteins
secretion signal no sequence similarity in
Choleratoxin B B5 recognized....via 3D
structure??
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virulence factors secreted via the Type II pathway
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Filamentous phages use parts of the Type II
pathway

• Cholera toxin phage
• filamentous
• requires only D (pore)

does EpsD shuffle between Type II complex and
phage assembly complex?
24
Type IV secretion
Type V secretion
25
Type IV secretion
Bartonella spp.
26
2 families of Type IV secretion systems
essential for T-complex transport
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Agrobacterium Type IV secretion
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Type IV secretion mechanism
Protein Function
substrate presentation VirD4 ATPase VirE1 chaperon
e Translocation energy VirB4 ATPase VirB11 ATPase
Protein channel VirB6,7,8,9,10 Attachment VirB2
Pilus VirB5 minor Pilus subunit VirB1 Adhesin
VirB2
VirB9
VirB10
VirB4
VirB11
(A. tumefaciens nomenclature)
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Type IV secretion of pertussis toxin (PT)
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Type I secretion
Type V secretion
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Topology of Type I protein secretion systems
Gram-negative
Gram-positive
Eukaryotes
yeast a-factor human Tap1/2 (ER), CFTR, MDR
exporter
32
Properties of Type I protein secretion systems
Gram-neg.
OMP even needed in spheroplasts binds MFP only
in presence of substrate sometimes shared
between systems MFP membrane fusion
protein N-term. in IM large domain in
PP ABC ATP binding cassette 200 aa conserved
motive (incl. 2 Walker-boxes ATP
binding) ATPase in presence of Substrate
recognizes 60 aa C-terminal secr. Signal general
composition 2 ABC 2 hydrophobic
domains Secretion in 1 step
33
Secretion by Type I protein secretion systems
OMP (TolC)
Membrane fusion protein
ABC protein
note TolC can interact with several different
Type I systems
34
Type I secretion RTX toxins and more
35
Organization of Type I protein secretion systems
Metalloprotease family
Toxin family
Hemoprotein
Colicin V
36
Type III secretion
Type V secretion
37
Type III secretion
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Flagella ??TypeIII secretion
G. Cornelis, Nat. Rev. Microbiol. VOLUME 4
NOVEMBER 2006 811
39
Assembly of Type III secretion systems

• export PrgH, PrgK, InvG (InvH) base structure

2. together with export machinery (composed
of InvA, InvC, SpaP, SpaQ, SpaR, and SpaS)
functional TTS machinery
3. exclusive secretion of needle protein and
inner rod ? specific length reached substrate
specificity switch ? effector proteins secretion
? specificity switch is governed by InvJ
40
or ?yscP (Yersinia)
TTSS how is assembly controlled ?
anchor assembly kinetics model
ruler model
PrgI
PrgJ
InvJ
41
Type III secretion mechanism
secretion signal N-terminal 20-60 aa (not
cleaved!!) can be transplanted sequence not
conserved 2 parts
1
11-17
60-80
N-
-C
secr.
transl.
effector domain
chaperone
N-termini of Yersinia effectors
42
Effector proteins transported via Type III
secretion systems
43
Conclusions
Type V secretion
Daniela Büttner and Ulla Bonas Trends in
Microbiology, Volume 10, Issue 4 , 2002, Pages
186-192