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Secretion
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- 1Why study bacterial secreion?- Fundamental info of protein targeting by studying experimentally tractable system - easy to genetically manipulate, grow
- Important for bacterial pathogenesis as many virulence factors are secreted - so secretion systems are potential chemotherapeutic targets
- Info can be exploited for biotechnology - useful for commercial protein production - 2What is the difference between gram negative and gram positive bacteria?Different envelope architectures - both have cytoplasmic inner membrane and a pepridoglycan layer - gram -ve also have a true outer membrane
(see book for pics) - 3Define export and secretionExport is movement across the inner membrane'; secretion is movement to the extracellular medium/ environment
In gram -ve, protein is exported from cytoplasm across the inner membrane - to the periplasm - in secretion from gram -ve, protein is target to extracellular environment - 4How were secretion systems identified?A lot of work using conditionally lethal mutants - most work done of E.Coli K12 and genes on export machinery are called sec genes
Gene and protein fusion technologies have (using lac z), Pho a (alkaline phosphatase), BlaM (beta-lactamase - a periplasmic enzyme) and GFPs as reporters has been using in identifying genes - 5How does the sec pathway work?Proteins destined for export - synthesised as pre-proteins - n-terminal extension of around 14-20 AAs (signal sequence) - directs to sec - signal removed by membrane-bound peptidase during translocation of the exported protein through the cytoplasmic mem - takes place on periplasmic face of the inner membrane
As protein synthesised, signal sequence and parts of the mature protein interact with cytoplasmic tetrameric SecB protein - acts as a molecular chaperone - largely after translation has occurred co- and post
Complex is then taken to membrane-associate SecA (receptor) ATPase - interacts with the SecYEG translocon
Cycles of ATP hydrolysis of Sec A are used to pump the protein through the translocon Chanel as it is exported to the periplasm
- exported protein needs to fold - may be assisted by activity of various Dsb proteins in the periplasm (in Dsb mutants periplasmic folding impaired) - 6What are some feature of signal sequences?Don not have consensus primary sequence - but have 3 domains - positively charged amino terminal region, hydrophobic core consisting of 5 to 15 H phobic AAs followed by signal peptidase cleavage site - usually AlaXAla - around 14-20 residues from the met start
Proteins with a lipid modification are processed by a specific, alternative signal peptidase - 7What other secretion pathways are there?Not all exported proteins use the Sec B/Sec A route - some interact with bacterial homologues of the euk signal recognition particle SRP/STP receptor
Also have TAT pathway - pH dependent - allows export of some proteins with a twin arginine in signal peptide- proteins targeted via TAT pathway are translocated in a fully-folded state and include some periplasmic proteins containing redox co-factors e.g. Fes, molyndopterin
TAT has a proofreading mechanism - proofreading chaperone binds to the exposed twin-arginine peptide signal of pre-protein - blocks signals and stops recognition by TAT BC which recognises TAT a and translocates it - remains bound until co-factor is loaded -prevents any attempted export whilst co-factor is bound - then released - 8How are membrane proteins targeted?Pre proteins can also be target using the ribosome-bound signal recognition particle as soon as they emerge from the ribosome exit tunnel - mainly for proteins very hydrophobic e.g. membrane proteins that have H phobic signal sequences - co-translational translocation - then taken to Sec YEG - FysY is receptor - sec A only used for very hydrophilic proteins
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- (Lecture notes ) Biology Fichas sobre Secretion , creado por Jenna Robinson el 12/05/2017.
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- Jenna Robinson
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