Bacterial Protein Translocation

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Bacterial Protein Translocation Pathogenesis

• David R. Sherman
• HSB G-153
• 221-5381
• dsherman_at_u.washington.edu

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Lecture outline

• Cellular addresses
• Getting stuck in the membrane YidC
• Crossing the inner membrane
• Sec-dependent
• SRP
• Sec B
• TAT-mediated
• Crossing the outer barrier - specialized
  secretion systems
• An example in gram () bacteria (paper
  discussion)

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Protein destinations
Approx 10 of proteins cross at least the inner
membrane. Approx 30 of proteins are membrane
associated.
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Machinery of bacterial protein translocation
Cytosolic membrane (Gram /-) YidC.
Sec machinery. Tat
translocation. Cell wall (Gram /-) very little
known. Outer membrane (Gram -) several
specialized systems. Much better studied
in Gram-negatives.
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Membrane insertion via YidC

• Multi-pass membrane protein.
• Needed for insertion of some (all?) membrane
  proteins.
• Can act alone or w/ Sec YEG.
• Evolutionary origin of secretion?

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Across the cytoplasmic membrane -- the Sec
machinery
General features Sec YEG heterotrimeric
pore-forming membrane proteins. SecA
membrane-associated ATPase. Substrates are
generally unfolded. Substrates have a signal
peptide usually N-terminal 1() basic AAs
followed by10-20 hydrophobic AAs
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SecYEG topology
9494-500, 2001
Homologous to eukaryotic Sec61p complex.
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SRP-mediated translocation
SRP homologous to eukaryotic SRP Ffh (54
homolog) 48kDa GTPase ffs (4.5S RNA) essential
for cell viability Recognizes ribosome-bound
nascent membrane proteins. Substrate recognition
is via signal sequence. SecA is NOT needed for
membrane association, but IS needed for
translocation.
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SRP targeting
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SecB-mediated translocation
SecB acidic, cytosolic chaperone. recognizes
mature, unfolded proteins. destination --
periplasm, outer membrane or beyond. Substrate
recognition is NOT via the signal
sequence. Binding motif 9 AAs
long. hydrophobic and basic. acidic AAs
strongly disfavored.
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SecB protein targeting
Nat Struct Biol. 2001 8(6)492-8.
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Sec interactions
9494-500, 2001
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Twin-arginine (Tat)-mediated protein translocation
Independent of the Sec system. TatA and TatC are
essential. Transports folded proteins. Not
found in eukaryotes some bacteria. of Tat
substrates per organism varies very widely
-- None (Clostridium tetani, Fusobacterium) 145
(Streptomyces coelicolour)
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Twin-arginine (Tat)-mediated protein translocation
Targeting signal (in the first 35 AAs) has 3
regions N-term is positively charged
(S/T)-R-R-x-F-L-K hydrophobic a-helical
domain C (cleavage) domain. TATFIND 1.2
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Specialized secretion pathways (Gram-negative
bacteria)
1. Type I pili. 2. Type I secretion. 3. Type
II secretion/general secretory pathway/type IV
pili. 4. Type III secretion (TTSS). 5. Type IV
secretion. 6. Type V/autotransporters. Classific
ation is based on the sequence/structure of the
transport machinery and their catalyzed
reactions. These systems are usually associated
with virulence.
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Assembly of type I pili
Allow for attachment during the initial stages of
infection. Assembled in two stages Sec-dependent
Pap C/D-dependent
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Type I secretion of repeat toxins HlyA
Lipid-modified toxin. 11-17 repeats of 9
AAs. Binds Ca Punches holes.
Sec-independent. Requires ABC-transporter
(HlyB). C-term signal sequence.
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Type II secretion -- the general secretory
pathway
Many examples cholera toxin alkaline
phosphatase proteases elastase Type IV pili
Occurs in 2 steps -- 1st is Sec-dependent 2nd
requires 10 proteins and ATP. Secretion signal?
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Type IV pili (a type II machine)
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Type III secretion
Needle
Triggered by contact w/ host cells. Sec-independen
t, similar to flagellar assembly. Assembly of the
needle occurs at the tip.
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Type IV secretion
Very versatile Sec- and ATP-dependent.
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Autotransporters Neisseria IgA protease
Synthesized as a pre-proenzyme. C-term b-barrel
inserts in OM, pulls N-term through. N-term
auto-cleaves, promoting release.
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Cell wall proteins of Gram ()s
Initially Sec-dependent. N-term signal
cleaved. C-term signal sorts to
CW. L-P-x-T-G. Amide linkage to peptidoglycan.
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So whats in common?

• All secretion systems must
• assemble themselves.
• recognize the appropriate substrates.
• maintain proper folding state.
• determine their final locations.

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Additional reading (not assigned)
The structural basis of protein targeting and
translocation in bacteria. Driessen AJ, Manting
EH, van der Does C. Nat Struct Biol. 2001
8(6)492-8. The Tat protein export
pathway. Berks BC, Sargent F, Palmer T. Mol
Microbiol. 2000 Jan35(2)260-74. Prokaryotic
utilization of the twin-arginine translocation
pathway a genomic survey. Dilks K, Rose RW,
Hartmann E, Pohlschroder M. J Bacteriol. 2003
Feb185(4)1478-83. Protein secretion and the
pathogenesis of bacterial infections. Lee VT,
Schneewind O. Genes Dev. 2001 Jul
1515(14)1725-52. Getting out protein traffic
in prokaryotes. Pugsley AP, Francetic O, Driessen
AJ, de Lorenzo V. Mol Microbiol. 2004
Apr52(1)3-11.
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Fig 1.
Guinn et al, Mol Microbiol, 2004, 51(2)359-370.
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Fig. 2
Guinn et al, Mol Microbiol, 2004, 51(2)359-370.
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Fig. 3
Guinn et al, Mol Microbiol, 2004, 51(2)359-370.
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Fig. 4
Guinn et al, Mol Microbiol, 2004, 51(2)359-370.
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Fig. 5
Guinn et al, Mol Microbiol, 2004, 51(2)359-370.
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Fig. 6
Guinn et al, Mol Microbiol, 2004, 51(2)359-370.
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Fig. 7
Guinn et al, Mol Microbiol, 2004, 51(2)359-370.