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PspA, IM30 (Vipp1) and LiaH, which all belong to the PspA/IM30 protein family, form high molecular weight oligomeric structures. For all proteins membrane binding and protection of the membrane structure and integrity has been shown or postulated. Here we discuss the possible membrane chaperoning activity of PspA, IM30 and LiaH and propose that larger oligomeric structures bind to stressed membrane regions, followed by oligomer disassembly and membrane stabilization by protein monomers or smaller/different oligomeric scaffolds.
The proteins PspA, IM30 (Vipp1), LiaH and YjfJ belong to the PspA/IM30 protein family. Structurally, all family members are predicted to be mainly α–helical,1-4 and PspA, IM30 and LiaH form high-molecular weight homo-oligomeric ring structures. Solely YjfJ is reported to not form such complexes,5 and, in fact, YjfJ is the most distant and least studied protein in this family. Therefore, it will not be further discussed here. The tendency of purified PspA, LiaH and IM30 to form ring structures or other higher order oligomers is extremely pronounced and monomers represent a minor fraction in solution.6-9 The observed homo-oligomeric rings are strikingly similar: PspA has been calculated to form 36-mers, and LiaH also forms ring structures of that size range.3,7 IM30 has the intrinsic propensity to form varying ring sizes containing (at least) 48–68 monomers.1,2 Nevertheless, besides a common phylogenic origin and obvious structural similarities, PspA, IM30 and LiaH have different physiological functions.
The phage shock protein (Psp) system represents a conserved stress response system of bacteria and archaea.10 In enterobacteria, Psp response proteins are encoded by the pspABCDE operon and monocistronic pspF and pspG genes. Thus far, the enterobacterial Psp system is the best characterized representative involving a member of the PspA/IM30 family.
In the absence of stress, PspA inhibits the transcription factor PspF.11 One model suggests that the Psp system is activated when PspA itself senses membrane defects or stress, resulting in liberation of PspF.12 As a result, transcription of the psp genes is enhanced and the PspA level increases, which triggers the formation of membrane-protecting PspA assemblies.13 In agreement with this hypothesis, PspA can stabilize stressed membranes in vitro.14 Alternatively, the main sensory role is attributed to the membrane components PspB and PspC.15-17 In this model, membrane stress results in recruitment of PspA to PspC, and a concomitant release of PspF activates the Psp response. Indeed, sensing of mistargeted secretins or membrane-destabilizing proteins requires PspB and PspC, which may function in conjunction with PspA.18,19 In non-enterobacteria, PspBC may be replaced by other sensory systems. Recent analyses of the PspA-PspF interaction suggest that the PspAF complex does not necessarily need to dissociate to modulate psp gene expression.20 In summary, the function of PspA appears to be dual: (i) membrane binding and membrane protection as well as (ii) upregulation of corresponding genes.
IM30 (Inner Membrane-associated protein of 30 kDa), alternatively named Vipp1 (Vesicle-Inducing Protein in Plastids), is conserved in all organisms harboring thylakoid membranes (TMs), i.e. cyanobacteria (except several Prochlorococcus marinus strains), algae and higher plants.21 It has been reported that IM30 can somehow functionally replace PspA in Escherichia coli.22,23 Thus, while membrane binding and membrane protection appears to be conserved, IM30 must have gained extra functions. Although many IM30-specific functions have been proposed in the past (for a recent review, see ref. 21), a recent report suggests that IM30 can trigger membrane fusion upon membrane binding, a process crucial for TM biogenesis and maintenance.24 A fusogenic activity would in fact explain many of the previously proposed IM30 activities.
The Lia system is highly conserved in Bacillus and Listeria species (low GC Gram-positive bacteria).25 Its expression is controlled by the LiaFSR three-component system.26 In the absence of stress conditions, the bifunctional histidine kinase LiaS is kept in its phosphatase state by the inhibitor protein LiaF, thereby repressing the activity of the transcriptional factor LiaR.27 Under stress conditions, LiaF presumably releases LiaS, which switches its activity from phosphatase to kinase state. Once activated by phosphorylation, LiaR induces transcription of the liaIH operon.26,27 LiaI is the membrane anchor of LiaH, which dynamically scans the membrane and recruits LiaH to the site of envelope damages.28 It is postulated that oligomeric LiaI/LiaH complexes stabilize a perturbed envelope at the sites of membrane damage.28
While all studied members of the PspA/IM30 family have acquired specific functions during the course of evolution, PspA3,5,12,29,30 and IM301,2,4,6,12,31-33 have been shown to directly bind to membrane surfaces and thereby to alter the structure and organization of membranes eventually resulting in membrane stabilization. LiaH is suggested to have the same activity, but experimental verification is still missing.28
PspA and IM30 preferentially bind to negatively charged membranes or to surface curvature-stressed membranes.6,12,24 Surface attachment of IM30 or PspA increases the packing density of individual lipids, i.e., the lipid bilayer becomes more ordered.6,12 Increasing the lipid packing density might be a vital repair strategy if stress-induced defects occur in lipid bilayers. Membrane stress resulting in membrane reorganization and eventually in membrane defects might be induced by heat or osmotic stress, by membrane remodeling agents, such as alcohols, as well as by external forces. Importantly, the major lipid species of bacterial as well as TMs are non-bilayer forming lipids (such as PE (Phosphatidylethanolamine) in E. coli and MGDG (Monogalactosyldiacylglycerol) in cyanobacterial/chloroplasts' TMs). While MGDG is crucial for TM organization,34 excess of MGDG severely decreases the membrane stability.35 In fact, under prolonged stress affecting membrane integrity or fluidity, the MGDG content is adjusted.36,37 Membrane binding of PspA or IM30 proteins might therefore simply spatiotemporally stabilize short-living protein-free (instable) lipid patches by insertion of the hydrophobic side-chains of a membrane-binding amphipathic helix between the lipid acyl-chains of the cytoplasmic lipid bilayer leaflet. Such membrane stabilizing mechanism is discussed for many membrane-active proteins or peptides that contain amphipathic helices, such as antimicrobial peptides38-40 or BAR (Bin/Amphiphysin/Rvs)-domain proteins.41,42 In case of negative curvature stress or other defects in lipid bilayer structures, binding of PspA or IM30 proteins to membrane surfaces might stabilize the bilayer structure by the discussed hydrophobic insertion mechanism.
The ability of selected proteins to bind and to protect stressed membranes is important for the fitness and survival of microorganisms and plants, which are constantly exposed to different stresses, like cold, heat, reactive oxygen species (generated, among other, by an intense photosynthetic activity), acid or organic solvents.43-46 Membrane binding and protection has already been described for oligomeric proteins belonging to the Small Heat Shock Protein (sHSP) family. sHSPs are low-molecular weight proteins (15–42kDa) that form dynamic 9- to 50-mers.47 However, the sHSPs active unit is most certainly a dimer.48 Hence, dimer formation requires dissociation of the oligomeric structure. Under heat stress conditions, the cyanobacterial sHSP HspA (also named Hsp17) delocalizes from the cytoplasm and binds to TMs to protect the membrane ultrastructure49 by increasing the lipid order and reducing membrane fluidity.50 Lo18, a sHSP from Oenococcus oeni, forms 16-mer spherical complexes.51 Under ethanol-induced membrane stress, 16-mer complexes disassemble into dimers that bind to membranes and stabilize them.52 Furthermore, expression of the O. oeni Lo18 in Lactococcus lactis improved acid stress tolerance of L. lactis,53 and heterologous expression of HSP17 from Caenorhabditis elegans enabled E. coli to grow at temperatures that are normally lethal for the wild-type strain.54
It is tempting to postulate a similar role for members of the PspA/IM30 family, that is, that the oligomers dissociate on membrane surfaces upon membrane binding to stabilize defined membrane patches. In fact, for PspA11,20 and IM306,24 dissociation of their large ring assemblies is indicated. IM30 LMWO (lower molecular weight oligomers) bind with an about 5-fold higher affinity to PG liposomes than IM30 rings,6 which argues for binding of monomers rather than high molecular mass oligomers to membrane surfaces and/or IM30 ring disassembly upon membrane binding.24 Increased membrane affinity of LMWO (lower molecular weight oligomers) IM30 is discussed to originate from exposure of amphiphilic helices, which are otherwise involved in formation of coiled-coil-type interactions within IM30 rings.21 Since LMWO (lower molecular weight oligomers) appear to not be stable in solution, formation of the ring structures might simply be vital for shielding the amphiphilic helices. Thereby, PspA or IM30 proteins remain soluble in the absence of stress conditions.
Binding of an IM30 ring to TMs requires a free lipid area of about 380 nm2 (total surface of the ring considered as a disc with an assumed ring diameter of 22 nm).2 In Synechocystis PCC 6803 TMs, the proportion of proteins (weight/weight) is 68%55 and in spinach TMs, the proteins area occupancy has been estimated to be 75%.56 Thus, binding of IM30 rings seems barely probable in vivo within the crowded environment of a TM, unless stressed membrane areas are protein depleted or an unknown mechanism creates such large protein-free lipid domains. In fact, crowding is not homogeneous in cyanobacterial and plant TMs that possess lateral heterogeneity with densely packed and more fluid areas.57,58 However, while a single IM30 ring covers an area of ~380 nm2, the center of a ring is open leaving a free area of ~80 nm2.2 Binding of smaller units (such as monomers) or flexibly rearranging scaffolds to damaged membranes could completely cover and protect damaged and/or stressed membranes by stabilizing the order of the lipid bilayer via filling the “gaps” between lipid head groups.
Functional characterization of PspA/IM30 proteins has been notoriously difficult. But combining the lessons learned from its best studied members – the PspA from enterobacteria, IM30 (Vipp1) from cyanobacteria and plants, and LiaH from Firmicutes– allowed extracting a first set of common features with respect to their oligomerization, membrane binding and putative membrane protecting function, as discussed here. We propose that membrane protection of members of the PspA/IM30 protein family is mediated by (1.) binding of oligomeric/multimeric assemblies or ring structures to stressed membrane areas followed (2.) by ring dissociation, which results in exposure of membrane-binding amphiphilic helices and finally in coverage of a stressed membrane area by monomers or flexible scaffolds (Fig. 1). Notably, membrane attachment of oligomeric proteins and subsequent local disassembly results in a high monomer concentration that is confined to a selected membrane area.
Clearly, besides the proposed membrane-protective activity of PspA/IM30 proteins, they have gained more specific functions in their special systems, and these activities are likely differently regulated. However, the here described insights can now serve as a starting point for more detailed functional and biochemical analyses that will ultimately unravel both general and organism-specific features of the ubiquitously distributed PspA/IM30 protein family.
No potential conflicts of interest were disclosed.
This work was supported by the University of Mainz; and the Ernst & Margarete Wagemann Foundation.