Lecture 13 - Bacterial Protein Transport Flashcards
Transport systems across inner membrane
1)
2)
1) Sec translocase
2) Tat
Most complex secretion system
G- outer membrane transport
Sec translocase different pathways
1)
2)
1) Classical - SecB chaperone
2) Alternative - SRP chaperone
Sec translocase classical pathway 1) 2) 3) 4)
1) Peptide translated in ribosome
2) N-terminal signal sequence bound by SecB chaperone
3) SecA recognises chaperone, hydrolyses ATP to pump peptide through SecYEG trimer
4) Signal peptidase removes signal sequence once peptide has been extruded through SecYEG
Sec translocase alternative pathway 1) 2) 3) 4)
1) Peptide tarnslated in ribosome
2) Signal sequence recognised by signal recognition particle (SRP)
3) SRP interacts with FTSy.
4) FTSy binds to SecA, secA hydrolyses ATP to pump peptide through SecYEG trimer
Function of YidC
YidC is involved in placing membrane-bound proteins in the membrane
Which protein is involved in placement of membrane-bound proteins?
YidC
Which proteins make up trimer involved in Sec tranlocation?
SecY, SecE, SecG
Sec signal sequence
N-terminal signal sequence
~20 amino acids
In what fashion does Sec translocation occur?
Stepwise, in groups of 20-30 amino acids at a time
Tat pathway 1) 2) 3) 4)
1) Signal sequence binds to TatBC in cell membrane
2) TatBC complexes with TatA
3) TatA is a pore-forming protein, brings a proton into the cell in exchange for extruding bound protein
4) Protein moves through TatABC translocon in a folded state
Pore-forming peptide in Tat pathway
TatA
Signal-recognising peptide in Tat pathway
TatBC
Difference between Tat and Sec
Tat transports natively folded proteins, cofactors can be attached
Sec can only transport unfolded proteins, hence the need for a chaperone molecule
Tat signal sequence
N terminal sequence
Twin-arginine motif
Types of outer membrane secretion systems 1) 2) 3) 4) 5) 6) 7) 8)
1) Autotransporter (type V)
2) Two partner secretion
3) Chaperone/usher pathway
4) Type I secretion system
5) Type II secretion system
6) Type III secretion system
7) Type IV secretion system
8) Type VI secretion system
Do autotransporters use Sec translocase?
Yes
Autotransporter pathway
1)
2)
3)
1) Sec translocase brings autotransporter peptide into periplasmic space by recognising signal sequence
2) Beta domain forms beta barrel in outer membrane with assistance of BAM protein
3) Alpha domain is secreted through beta barrel
Domains of a generic autotransporter
1)
2)
3)
1) Signal sequence
2) Alpha domain/Passenger domain/Functional domain - Will ultimately act as virulence factor. Can be toxin, protease, adhesin, invasin, etc
3) Beta domain - Forms beta barrel in membrane
On which terminal is a beta domain of an autotransporter?
Carboxy-terminal
Two partner secretion system
Similar concept to autotransporter
Difference is that beta barrel protein is encoded by a different gene
Examples of proteins secreted through twin partner secretion system
1)
2)
1) Haemolysin from Serratia marcescens
2) Filamentous haemagglutinin in Bordatella pertussis
Which system is used to secrete haemolysin and filamentous haemagglutinin?
Twin partner secretion system
Function of filamentous haemagglutinin
Lets B. pertissis bind to beta-1 integrins on lung epithelial cells
Mimics RGD motif on fibronectin
Proteins transported using Usher/Chaperone system
Pap pili
Type 1 pili
Usher/Chaperone system 1) 2) 3) 4)
1) Peptide translocated using Sec
2) Peptide binds to chaperone
3) Chaperone recognised by usher
4) Usher recognises chaperone, transports peptide
Function of chaperone donor strand
Inserts into binding groove of transported peptide, prevents it binding to anything
Makes peptide only bind to another subunit of pilus
Part of chaperone that prevents premature pilin binding
Donor strand
Do type I secretion systems use Sec?
No
Sec independent
What do type I secretion systems normally secrete?
Toxins, lipases, proteases
EG: Alpha haemolysin produced by some E coli
Type I secretion system signal peptide
C-terminal
~60 amino acids in length
Parts of type I secretion system
1)
2)
3)
1) ATP binding cassette
2) Membrane fusion protein, anchored in cytoplasmic membrane, spans periplasmic space
3) Beta barrel domain
Type I secretion system
1)
2)
3)
1) ATP binds to ATP binding cassette
2) Channel of beta barrel opens, membrane fusion protein is pulled down, binds to ATP binding cassette
3) Protein is translocated
Another term for type II secretion system
Main terminal branch of general secretory pathway
System involved in type 1/Pap pili secretion
Chaperone/Usher
System involved in type 4 pili secretion
Type II secretion system
Example of a type 4 pilus
Bfp of EPEC
Number of proteins involved in a type II secretion system
12 - 16 proteins
What is the name for the collection of accessory proteins in a type II secretion system?
Secreton
Outer membrane pore in type II secretion system
GspD
What is GspD?
Outer membrane pore in a type II secretion system
Secretin family of proteins
1)
2)
3)
1) Form a very stable ring-shaped membrane pores
2) Channel is 5-10nM in diameter
3) EG: GspD
Function of GspC
Links inner membrane complex to outer membrane pore in a type II secretion system
Function of GspS
Pilotin
Makes sure that GspD inserts into outer membrane
When absent, GspD inserts into both inner and outer membranes
What makes sure that GspD inserts into outer membrane?
GspS
What links the inner and outer membrane complexes of a type II secretion system?
GspC
Example of factor secreted by type II secretion system
Cholera toxin
How is cholera toxin secreted?
By a type II secretion system
A and B subunits are secreted through Sec separately, combine in periplasm
Type II secretion system signal peptide location
N-terminal
