PROKARYOTIC
Prokaryotic Protein Secretion (Bacteria)
o Bacterial cells need to secrete proteins in order to degrade large molecules outside the cell into smaller components that can then be taken up by the cell
o There are two types of bacteria; gram positive and gram negative, shown in the figure below.
o Gram positive bacteria have a much thicker cell wall formed of many layers of peptidoglycan and gram negative bacteria have a thin layer of peptidoglycan in their cell wall but have an outer membrane . as the cell wall is porous, transport is simple in gram positive bacteria whereas gram negative proteins must pass through the cytoplasmic membrane, the cell wall then the second membrane, making secretion of proteins a much more complex process.
o As opposed to eukaryotic cells, prokaryotic cells lack organelles such as the ER and Golgi, so secretion of proteins outside the cell membrane are performed through porins and translocons distributed throughout the cell membrane
Gram Negative Bacteria
o Gram negative bacteria have two ways of secretion - a non-periplasmic route where the secreted protein passes through a nano machine which passes through both membranes of the cell directly into the extracellular space (secretion types 1, 3, 4 and 6).
o Proteins can also be secreted via a periplasmic intermediate in a two step process, where firstly the protein is secreted from the cytoplasmic membrane into the periplasmic space (through the sec and tat translocon) then through the second membrane into the extracellular space (secretion types 2 and 5).
o The Sec translocon
o The sec translocon is important in extracellular secretion in bacteria, it is found in all three kingdoms of life and is used in transport proteins across organelles in eukaryotes.
o Sec dependent secretion in bacteria requires the secreted protein to be unfolded in order to cross the cytoplasmic membrane, a signal peptide and ATP in order to be transported.
o After they have passed the membrane, the signal peptide is cleaved and the protein is able to fold.
The sec mechanism
o After translation in the cytoplasm, the pro-protein is captured by secB which recognises the protein by the signal peptide at the N-terminus of the protein. This keeps the protein in an unfolded state to ease transport through the membrane.
o secB delivers the pro-protein to secA within the cytoplasmic membrane. SecA is bound to secYEFG, which creates a pore in the membrane.
o secA is an ATPase, providing the motive force to push the protein through the YEFG channel. When SecA binds to ATP, 20 amino acid residues are pushed through SecYEFG channel
o Hydrolysis of ATP causes secA to release from the protein and reform its original position.
ATP is bound again pushing more of the protein to be secreted through the channel.
o This cycle is continued until the protein is fully pushed through the pore. In some cases, secretion of the protein can be done while the protein is still being translated, which can provide the energy itself to begin secretion
o After the whole protein has passed through the channel, signal peptidases snip off the signal peptide from the protein, creating the mature protein.
o This is the inactive form so requires chaperones to complete tertiary folding
o The tat translocon
o The tat translocon is not as widespread as the sec translocon, it is found in bacterial, archaeal and thylakoid inner membranes and is used to secrete folded proteins (unlike the sec translocon which can only secrete unfolded proteins)
the tat mechanism
o tatA and tatC are found in the membrane and use a proton motive force as an energy source to pull the secretory protein through the membrane, rather than ATP.
o Firstly the tatB/C complex binds to the protein and recruits tatA.
o TatA weakens the membrane and induces a conformational change in the tatB/C complex
o The change in conformation pulls the protein through the membrane which forms a hole in the membrane.
o A proton flux is stimulated through the hole to seal the membrane back together after the formation of the hole from the bypassing protein
o the protein is then released from the tatB/C complex on the exterior side of the membrane, into the periplasmic space.
Secretion Types
o Some proteins stay in the periplasmic space, only secreted from one membrane, but others need to be completely exported so require another transduction pathway to reach the extracellular space.
o A chaperone usher formed of two components; one periplasmic chaperone which assists folding and prevents other interactions that may interfere pre folding, and an outer membrane usher, which acts as a platform for assembly.
o Type 5 secretion involves autotransporters in the sec translocon where the n terminal provides a passenger domain and c termini forms a barrel to thread the protein through and is folded in the process- such as degradative and cytotoxin proteins. There are many types of secretion depending on the path of the protein and the type of protein
Type1 secretion
o An ATPase on the inner membrane recognises the signal peptide of the protein to be secreted.
o An adapter protein is fused to the ATPase creating a channel for the protein to be secreted from.
o Proteins secreted from this system tend to be acidic and glycine rich, and show homology to adhesion proteins, indication this is how bacteria secrete their adhesion proteins.
Type2/5 secretion
o Both type 2 and type 5 secretion are on the outer membrane of gram negative bacteria and can transport proteins secreted to the periplasmic space via either the sec or tat translocon.
o Type 2 secretion
o has a periplasmic pseudopilus, an inner membrane platform and a cytoplasmic ATPase.
o It is thought that the type 2 secretion pathway recognises proteins to be secreted by a structural motif rather than a signal peptide.
o Secretory proteins from type 2 secretion include those such as hydrolases and exotoxins.
Type 5 secretion
o machinery is composed of autotransporters composed from polypeptides from the substrate proteins.
o The secreted proteins can either remain associated with the autotransporter on the outer membrane or are cleaved and released into the extracellular
Type 3, 4 and 6 secretion
o These secretion systems secrete proteins directly from the cytoplasm into a host cell.
o Type 2 secretion
o machinery consists of multiple ring like structures snapping both the inner and outer membrane and a needle facing into the extracellular matrix where the proteins exit where they firstly travel through the needle that open up a pore in the host.
o These types of secretory proteins have a non-cleavable N terminal secretion signal and are targeted to this secretory machinery in an unfolded state.
o Type 4 secretion
o pathway is similar however the proteins targeted to this channel have secretion signals composed of hydrophobic and positively charged residues located at the C termini.
Membrane vesicles
o Both gram negative and positive bacteria form vesicles that are pinched from the cell surface and cargo is taken into the extracellular space.
o The secreted proteins from vesicles are important in nutrient acquisition, iron hunting antibiotic resistance and biofilm formation.
o They also allow for pathogenesis in delivering virulence factors over long distances and modulating a host's immune system.
o There is evidence that proteins secreted from membrane vesicles are responsible for antibiotic resistance. Bacterial cells exposed to βlactam antibiotics such as penicillin increase vesicle formation containing βlactamases, degrading the treatment and can also increase the tolerance to the antibiotic in other bacterial cells, making bacterial vesicles an ideal therapeutic target
Classic antibiotics prevent the growth via cell wall biogenesis, DNA replication, transcription and protein synthesis.
o As the rate of antibiotic resistance is increasing, secretion systems are novel treatment that don't directly inhibit the growth of bacteria so don't provoke selection for mutations causing resistance.
o These systems are also highly conserved between bacteria so can be used widely.
o Secreted proteins play a major role in immune evasion, targeting these can help clear pathogens by the hosts immune system, rather than relying on typical antibiotics which may have no effect or become resistant. 16