Lecture 13 Protein Secretion Flashcards
Protein Secretion
Approximately 20% of the polypeptides synthesized by bacteria are located partially or completely outside of the cytoplasm.
How do they get there?
Known that in eukaryotic cells targeting of proteins to organelles such as the ER was mediated by recognition of a signal peptide by particular proteins
28 years ago (1989) it was discovered that bacteria had homologues to these proteins in the ER
One of these homologues was called Ffh in E. coli (homolog to the p54 subunit of the SRP), has a hydrophobic binding groove
This SRP and signal peptide complex are recognised by the bacterial homologue of the alpha subunit of the SRP receptor, FtsY. In bacteria, this serves the function of both the alpha and beta subunit of the SRP receptor
Both Ffh and FtsY contain GTP binding sites and when combined form a functional GTPase - formed the hydrophobic groove
This mechanism of signal peptide recognition by E. coli is proposed to be co-translational:
Co translational transport - polypeptide is translocated as soon as it leaves the ribosome (fed through the channel) doesn’t need that much energy as the energy of the ribosome pushes it through the open channel.
Ribosome making protein, Ffh bind Gth also binds the signal peptide in a groove. FtsY is a membrane protein that will interact with FfH, they come together to form a GTPase, which forms a structural change which enables the signal protein to go through the membrane.
Summary: Ffh and Ftsy come together to give some energy for the translocation of a protein through a membrane complex SecYEG.
SecY is the main component of the channel of the translocase, it is a heterotrimeric complex. SecY is a homolog of Sec61 in eukaryotes. Forms a circular pore.
SecYEG is highly conserved very important protein.
SecE functions as the ‘hinge’ that opens and closes the pore
There is no SecG homolog in eukaryotes. Thought to interact with SecA in bacteria
What is SecA?
Involved in post-translational targeting. It is a GTPase that pushes the substrate polypeptide through the SecY channel.
i.e. the protein has already been made, then transported
SecB holds the substrate in a translocation competent state with subsequent signal sequence recognition by SecA (mediates the interaction of the complex towards SecYG)
In both co- and post-translational secretion through the SecYEG translocase, the signal peptide is cleaved by a signal peptidase.
Sometimes the presence of a hydrophobic domain in the tranlocase pore can cause lateral translocation into the membrane by the chaperone YidC.
SUMMARY: Sec dependent secretion:
SRP mediated co translational
Sec mediated post translational
Proteins that do not embed in the membrane pass into the periplasm, from there bacteria secrete them outside. Can be either:
Sec independent: TypeIV, TypeIII, Type I SS
1 step: Go straight through from cytoplasm to outer membrane and beyond
Sec dependent: TypeV, TypeII SS
2 step: Go through SecYEG into periplasm then into outer membrane
TypeV is called an AUTOTRANSPORTER
TypeII is the most important G-ve SS. Also called a GSP: general secretory pathway. Type II SS push the protein through the channel after it has gone through SecYEG to exit the outer membrane
The Protein Secretion Systems
Type I:
Trimeric complex, no periplasmic intermediate. Sec independent.
Comprised of modules: the ATP Binding cassette (ABC), membrane fusion protein (MFP, in the periplasm part), and outer membrane protein (OMP).
Typically used in secreting proteins but also in drug efflux pumps. Secrete many classical toxins such as hemolysin. Prominent in G-ve bacteria.
An outer membrane protein like TolC can pair up with many MFPs and many differnt ABCs.
Type II:
Often called the general secretory pathway (GSP). Once in the periplasm, a complex macromolecular structure employing a piston like mechanism for secretion. Piston is built of Pilin
A ‘secretin’ in the outer membrane which is gated.
Cholera toxin is secreted by the T2SS. Will have an amino terminal hydrophobic groove (signal peptide) that is picked up by Fth and FtsY.
Type III:
SS predominant in pathogens not commensals. Another macromolecular structure, often referred to as an injectisome.
SPI (salmonella pathogenicity island) is a TIII SS. 50 protein structure.
Ancestrally related to flagella
Involved in the virulence of many intracellular Gram negative bacteria
Type IV:
Another macromolecular structure. As with the T3SS’s the genes encoding them are often on pathogenicity islands
Ancestrally related to conjugation apparatus - and is still involved in the transfer and uptake of DNA. Can also pull proteins from the periplasm (put there by SecYEG) and pass them through the channel.
Involved in the virulence of many intracellular Gram negative bacteria
N.b. the pertussis toxin goes through a T4SS through a sec dependent mechanism.
Type V:
Much simpler in concept. Often referred to as autotransporters. Have passenger and transpoter domains
Important for localising proteins to the outer membrane
These do have a periplasmic intermediate
Secreted effectors and virulence
Salmonella’s effectors mediate uptake through macrophages via a T3SS
1,Salmonellacells attach to the intestinal epithelium by means of adhesins, such as those encoded within SPI-3 and SPI-4 (salmonella pathogenicity islands 3 and 4)
2 and 3, Invasion of bacteria follows, and engulfment by M cells is mediated by virulence factors encoded within SPI-1 (T3SS) and SPI-5.
Salmonella does this by secreting the effector SipA into the host which causes actin rearrangment leading to salmonella uptake
5, Once taken up by phagocytes inside the cytoplasm,Salmonella replicates within the SCV. Factors encoded within SPI-2 and the pSLT plasmid are essential for survival in the phagosome (SifA prevents maturation of the phagolysosome).
7, Bacteria are internalized within phagocytes and located again within an SCV, where SPI-3, in addition to SPI-2 and the pSLT plasmid, play an important role
T3SS and T4SS:
Translocon at the tip of the injection apparatus forms a pore in host cell membranes and injects the effectors directly, to mediate manipulation of the immune response (e.g. siderophores) Approx 13 SPIs discovered, activated at different stages of host cell invasion.
*PhoP/Q represses SPI 1!
Virulent salmonella also have a plasmid that encodes for factors that are important for surviving inside the macrophage. A spiC (effectors) mutant of Salmonella shows reduced survival in macrophages due to interference with vesicular trafficking and lyzosome fusion.
