Genesis of extracytoplasmic structures and protein secretion in prokaryotic organisms Flashcards
Organelles and compartments in prokaryonts
Membrane compartments
a) forming de novo from plasma membrane
- Chromatophore
- Magnetosomes
- Thylakoids
b) inherited during cell cycle
- Anammoxosome
Organelles and compartments in prokaryonts
Membrane compartments
a) forming de novo from plasma membrane
- Chromatophore
- Magnetosomes
- Thylakoids
b) inherited during cell cycle
- Anammoxosome
The Translocase of the prokaryotic inner membrane (plasma membrane) is evolutionary related to the Translocon of the eukaryotic ER-membrane
Components of the bacterial transport system:
- SecA
- SecDFYajC
Components of the bacterial transport system with homology to components of the translocon in the ER membrane:
- FtsY
- SecYEG
- YidC
- lepB
FtsY is homolog to SRP-Ralpha, SecYEG is homolog to Sec61c, lepB is homolog to SPC18; YidC has homolog to EMC3.
Arche translocates have no SecA. Only bacteria and Euryarcheota have SecDF.
Acidic lipids esp. cardiolipin are crucial for effective translocation.
Co-translational targeting in bacteria
- minimal system consisting of SRP (4.5S RNA + FFH (NG+M domain) and Docking Protein (FtsY)
- essential for synthesis of membrane proteins of the PM
- existence of membrane-bound ribosomes is contentious
- actual translocation (e.g. of larger periplasmic domains) requires always (?) SecA
Post translational targeting in bacteria
SecA may recognize the cleavable signal sequence but also so-called “mature domains targeting signals (MTS)” present in several substrates which are also hydrophobic and at least support the effectivity of targeting and may be even essential for translocation.
The function of SecA during translocation
- secretion of soluble proteins is post translational sense lato
- SecA and ATP are essential
- PMF is supportive but not essential
- SecA most likely works according to the “push and Slide” model
Function of SecDF
SecDF and PMF are required for the post-initiation mode of translocation, which can occur in the absence of ATP and SecA. This function depends on the ability of the periplasmic P! domain to interact with a substrate and to undergo a structural transition between the I and F forms. The F state of SecDF may place the titled P1 head above the translocon pore, enabling it to capture an emerging pre protein. The pre protein -bearing F form could then return to the configuration, preventing the backward movement of the substrate and driving its forward movement. The release of bound pre protein from SecDF and the subsequent I to F conversion may be coupled to proton flow. These action cycles will eventually lead to the completion of translocation, in which the substrate is released from the translocon.
YidC - a protein that facilitates integration of membrane anchors into the lipid
- functions usually in cooperation with SecYEG during cotranslational integration and folding of membrane proteins; targeting may be SRP dependent; Also post translational activity ???
- may also function alone e.g. during integration of M13 procoat, Pf3 coat protein and some host proteins
- binds as a dimer to the ribosome
“Spontaneous insertion” of membrane proteins?
- means integration into lipid membranes without the direct help of other proteins
- “spontaneous insertion” may require certain lipids, a membrane potential or other chemical gradients across the membrane
Most process claimed to be “spontaneous insertion” of proteins during their biosynthesis finally turned out to be membrane protein dependent. The only candidate presently left is a domain of the potassium sensor KdpD from E. coli.
Some proteins show spontaneous insertion and translocation of proteins into or across biological membranes, resp., as part of their final function (e.g. several pore-forming toxins, some rare transcription factors and some viral proteins)
Special regulation on a nanometer scale - localized secretion mediated by special signal sequences
Secretion of M6 in Staphylococcus pyogenes occurs at the septum while secretion of Port is mainly occurring at the old pole.
Specific systems of transmembrane protein transport in prokaryota
a) Systems that are specific for proteobacteria and related organisms with outer membranes
- two step systems (usually SecYEG-dependent for PM and specific mechanisms for crossing the outer membranes)
- one step system crossing both membranes at once
b) Systems common to most prokaryotes
TypeVc Autotransporter adhesins
transport as for Va, but no cleavage of the extracellular domain; some protein up to 5000 aa large
Type II secretion (T2SS)
Main export pathway for extracellular hydrolytic enzymes in gram-.negative bacteria and for some AB5-type toxins.
Type IV-pili have a similar mechanism
Type IV secretion (T4SS)
- for DNA transfer (conjugation) in all prokaryotes (Inch archea)
- for protein secretion mainly in negibacteria
A bacterium may contain more than one kind of T4SS.
Structure of the Type III Apparatus (injectisome)
- Needle
- Inner rod
- Export gate
- Spokes
- Hub/ATPase
- Stalk
- Pods
- Export apparatus
- IR
- OR/neck
Type III secretion requires unfolding of the substrate
transport is ATP and PMF-dependent
Type VI secretion
T6SS complex remains stable until an unknown signal results in the contraction of the outer sheath and the propulsion of the inner tube into a neighboring cell (prokaryotic or eukaryotic). The effector proteins can exhibit their toxic activity. Concurrently, the ATPase ClpV disassembles the outer sheath so that the components can be recycled.
Type I secretion
Uses ABC-transporter in PM; Substrate: usually toxins (repeat-Type) e.g. hemolysin (E. coli), adenylatcyclase (B. pertussis), proteases (P. aeroginosa), leucotoxin (Pasteurella haemolytica) bacteriocins (Firmicutes)
- in proteobacteria, an uncleaved C-terminal leader is employed
- in Firmicutes and relates a cleavable N-terminal leader is used; the peptidase domain is also present in ABC from negibacteria, but lost there its proteolytic function
Tat pathway
- common in prokaryotes (80 %) in all plastids, also found in some mitochondria of protists
- signal similar to signal sequence, has RR-motive direct in front of the hydrophobic part
- processing by lepB
- can transport folded substrates, often with co-factors (2 kD — > 100 kD)
- minimal system Tat A + Tat C; in negibacteria also TatB
Model for membrane passage of Tat substrates
Upon interaction with the TatBC complex, Tat substrates bind with its signal to TatBC, followed by a recruitment of TatA. The amount of TatA depends on the size of the substrate. A major conformational change in TatBC which pulls the Tat substrate through the membrane which is weakened by TatA complex. In the moment of release of the Tat substrate, a very short-lived hole may be formed which could account for the large proton flux which accompanies Tat transport. The membrane is sealed and the substrate is laterally released, which makes the substrate accessible to signal sequence peptidases.
Type VII (ESX) / Type VII like system of posibacteria
- translocates folded substrates as homo or heterodimers
- transport most likely ATP driven (ATPases as subunits)
- signals for secretion, if known, are C-terminal
SpoIIIA-SpoIIQ System
- present in endospore forming Firmicutes
- consist of about 10 proteins (SpoIIAA-AH, SpoIIQ, GerM)
- some subunits have similarity to T2SS, others to T3SS subunits
- function still open (anchor for pili, transporter for proteins and/or small molecules?)
Cooperation of different transport systems during protein biogenesis
Translocase and TAT-machinery may cooperate during biogenesis of membrane proteins
Periplasmic space
- 10 % of the cell volume in E. coli
- highly viscous
- oxidizing
- involved in break-down of polymers
- environmental sensing by receptors in the PM (e.g. two component systems)
- contains peptidoglycan layer
Folding and modification in the periplasmic space
- periplasm provides a protected folding environment with some similarity to ER in eukaryotes
- Processing of the signal peptide e.g. by lepB (in GEP and in the TAT-pathway)
- to spie exend folding, quality control and degradation by ATP-independent chaperones and proteases in the periplasm or by ATP-dependent protease in the inner membrane (e.g. FisH)
- formation of disulfide bridges
- PPI-acitivity
- stress response system (e.g. CpxR-CpxA), activated by abnormal folded proteins in the periplasm or in the inner membrane
- several special modifications
a) Chaperone-assisted folding in the periplasm
- lack of ATP requires special types of chaperones; examples:
-> Spy (acting on several substrates)
-> FimC for Type I pili formation only; forms a kinetic assembly trap
in contrast to most other chaperones FimC accelerates folding (by factor 100)
-> folding catalyst like PPI or PDI
C) Degradation of miss-folded proteins
DegP-like systems in the periplasm
- HtrA/DegP/DegQ/DegS family - ATP independent regulated proteases, some of them (like DegP) are also acting as chaperone
- protease function may be stress induced (e.g. by heat)
- high protease activity may lead to stress response
- evolutionary conserved; also in gram+ bacteria outside the PM, in the inter membrane space of mitochondria, in plastids and as secreted protein in the ECM of Mammalia
Oligomeric states - DegP
different Oligomers - DegP6, DegP12, DegP15, DegP18, DegP24
DegP6 is a resting inactive form, able to bind unfolded proteins in open conformation and then transforms into greater Oligomers
DegP12/24 work as chaperone and stabilize proteins (e.g. monomers of OMPs) if they are able to fold fast - or function as self-compartmentalizing proteases acting only on unfolded proteins
d) N-linked glycosylation in Campylobacter jejune - acquired by horizontal gene transfer?
- proposed mechanism; only heptane sugars are transferred
- the only enzyme needed is the STT3-homolog PgIB
- similar mechanism in archea; in bacteria restricted to C. jejune and close relatives
Outer membrane
- highly asymmetric; inner leaflet (glycerol) phospholipids,. outer leaflet mainly LPS; contains lipoproteins and integral membrane proteins anchored via beta-barrel (outer membrane proteins - OMP)
- mistargeted glycerophospholipids are removed from the outer leaflet
- several interactions between LPS (between hydrophobic tails; between charged regions via cations; between sugars); form a barrier with an outer polar zone and an inner hydrophobic part -> protection against environment (e.g. hydrophobic toxins like bile salT)
-anchoring of peptidoglycan via Brauns lipoprotein (Lpp)
Biogenesis of outer membrane - a) Lipoproteins
- lipoproteins of E.coli face periplasm; Spirochetes have also LP that face the extracellular space
- 10 % of E.coli Lipoprotein in PM; 90 % in OM
- protein transport occurs via the GEP; processing of SP often by special peptidase
b) Integral membrane proteins - Biogenesis of OMPs
- beta-barrel structure
- assisted folding (skp, DegP (chaperone); surA (chaperone, PPI); DsbA/C (disulfide bonds); only surA is essential in vitro)
- specific insertion into OM via BAM-complex (BamA (omp85) and lipoproteins); supported by LPS
Structure and function of the BAM complex
- BamA assisted
- BamA budding
- BamA swing/elongation
- BamA lumen-catalyzed
c) Biogenesis of LPS
- lipid A is synthesized and modified at the cytoplasmic face of the inner membrane and than delivered to the OM
- in species that have an O-antigen the latter is delivered from cytoplasm via different routes to periplasm and ligated to the LPS at the inner leaflet of the OM before its final flip to the outer leaflet
Quality control - unfolded protein response
- The sigmaE-pathway is activated by miss localized/unfolded OMP
- DegS is stress-activated and rate limited regulatory protease
Folding and quality control of secretory proteins in gram-positive bacteria
- no genuine periplasmic space! -> most secretory proteins in firmicutes are selected for characteristics needed for rapid folding
- factors accelerating folding are:
-> PPIs
-> disulfide-isomerases
-> metal ions - factors for quality control are several proteases near the membrane and in the cell wall
Stress leads to signaling via CssS and upregulates extracellular proteases involved in quality control
Retrograde transport of macromolecules in bacteria
- no retrograde transport of unfolded proteins similar to ERAD known
- retrograde transport of macromolecular toxins and during the entry of phages known
- sometimes these pathogenic pathway employ machineries which serve the uptake of molecular larger than 0,8 kD like vitamin B12 or iron-loaded siderophores
Membrane blebbing
- “Negibacteria” sometimes are able to shed off vesicles from the OM, several functions have been assigned to these vesicles
- gram positive so far only one report (part of cell death process???)
- mitochondria (decending from gram positives) are also able to pinch off vesicles into the cytoplasm
- archea (e.g. Sulfolobus species) can pinch-off vesicles from the plasma membrane
