Bacterial Protein Translocation & Pathogenesis

David R. Sherman

HSB G-153

221-5381

dsherman@u.washington.edu

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)

Protein destinations

Approx 10% of proteins cross at least the inner membrane.

Approx 30% of proteins are membrane associated.

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.

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?

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

SecYEG topology

Homologous to eukaryotic Sec61p complex.9:494-500, 2001

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.

SRP targeting

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.

SecB protein targeting

Nat Struct Biol. 2001 8(6):492-8.

Sec interactions

9:494-500, 2001

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)

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 -helical domain

C (cleavage) domain.

TATFIND 1.2

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.

Classification is based on the sequence/structure of the transport machinery and their catalyzed reactions.

These systems are usually associated with virulence.

Assembly of type I pili

Allow for attachment during the initial stages of infection.

Assembled in two stages:Sec-dependentPap C/D-dependent

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.

Type II secretion -- the “general” secretory pathway

Occurs in 2 steps -- 1st is Sec-dependent; 2nd requires 10 proteins and ATP. Secretion signal?

Many examples:**cholera toxin**alkaline phosphataseproteaseselastaseType IV pili

Type IV pili (a type II machine)

Type III secretion

Triggered by contact w/ host cells.Sec-independent, similar to flagellar assembly.Assembly of the needle occurs at the tip.

Needle

Type IV secretion

Very versatile; Sec- and ATP-dependent.

Autotransporters: Neisseria IgA protease

Synthesized as a pre-proenzyme.C-term -barrel inserts in OM, pulls N-term through.N-term auto-cleaves, promoting release.

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.

So what’s in common?

All secretion systems must:

• assemble themselves.

• recognize the appropriate substrates.

• maintain proper folding state.

• determine their final locations.

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 Jan;35(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 Feb;185(4):1478-83.

Protein secretion and the pathogenesis of bacterial infections.Lee VT, Schneewind O. Genes Dev. 2001 Jul 15;15(14):1725-52.

Getting out: protein traffic in prokaryotes.Pugsley AP, Francetic O, Driessen AJ, de Lorenzo V. Mol Microbiol. 2004 Apr;52(1):3-11.

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Guinn et al, Mol Microbiol, 2004, 51(2):359-370.

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Guinn et al, Mol Microbiol, 2004, 51(2):359-370.

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Guinn et al, Mol Microbiol, 2004, 51(2):359-370.

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Guinn et al, Mol Microbiol, 2004, 51(2):359-370.

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Guinn et al, Mol Microbiol, 2004, 51(2):359-370.

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