PspA adopts an ESCRT-III-like fold and remodels bacterial membranes.

PspA is the main effector of the phage shock protein (Psp) system and preserves the bacterial inner membrane integrity and function. Here, we present the 3.6 Å resolution cryoelectron microscopy (cryo-EM) structure of PspA assembled in helical rods. PspA monomers adopt a canonical ESCRT-III fold in an extended open conformation. PspA rods are capable of enclosing lipids and generating positive membrane curvature. Using cryo-EM, we visualized how PspA remodels membrane vesicles into µm-sized structures and how it mediates the formation of internalized vesicular structures. Hotspots of these activities are zones derived from PspA assemblies, serving as lipid transfer platforms and linking previously separated lipid structures. These membrane fusion and fission activities are in line with the described functional properties of bacterial PspA/IM30/LiaH proteins. Our structural and functional analyses reveal that bacterial PspA belongs to the evolutionary ancestry of ESCRT-III proteins involved in membrane remodeling.

Conserved structures of ESCRT-III superfamily members across domains of life.

Structural and evolutionary studies of cyanobacterial phage shock protein A (PspA) and inner membrane-associated protein of 30 kDa (IM30) have revealed that these proteins belong to the endosomal sorting complex required for transport-III (ESCRT-III) superfamily, which is conserved across all three domains of life. PspA and IM30 share secondary and tertiary structures with eukaryotic ESCRT-III proteins, whilst also oligomerizing via conserved interactions. Here, we examine the structures of bacterial ESCRT-III-like proteins and compare the monomeric and oligomerized forms with their eukaryotic counterparts. We discuss conserved interactions used for self-assembly and highlight key hinge regions that mediate oligomer ultrastructure versatility. Finally, we address the differences in nomenclature assigned to equivalent structural motifs in both the bacterial and eukaryotic fields and suggest a common nomenclature applicable across the ESCRT-III superfamily.

Mg2+ limitation leads to a decrease in chlorophyll, resulting in an unbalanced photosynthetic apparatus in the cyanobacterium Synechocytis sp. PCC6803.

Mg2+, the most abundant divalent cation in living cells, plays a pivotal role in numerous enzymatic reactions and is of particular importance for organisms performing oxygenic photosynthesis. Its significance extends beyond serving as the central ion of the chlorophyll molecule, as it also acts as a counterion during the light reaction to balance the proton gradient across the thylakoid membranes. In this study, we investigated the effects of Mg2+ limitation on the physiology of the well-known model microorganism Synechocystis sp. PCC6803. Our findings reveal that Mg2+ deficiency triggers both morphological and functional changes. As seen in other oxygenic photosynthetic organisms, Mg2+ deficiency led to a decrease in cellular chlorophyll concentration. Moreover, the PSI-to-PSII ratio decreased, impacting the photosynthetic efficiency of the cell. In line with this, Mg2+ deficiency led to a change in the proton gradient built up across the thylakoid membrane upon illumination.

Ultrasound-guided determination demonstrates influence of age, sex and type of sport on medial femoral condyle cartilage thickness in children and adolescents.

PURPOSE: To analyse the reliability of ultrasound-guided measurement of the cartilage thickness at the medial femoral condyle in athletically active children and adolescents before and after mechanical load in relation to age, sex and type of sport. METHODS: Three successive measurements were performed in 157 participants (median/min-max age: 13.1/6.0-18.0 years, 106 males) before and after mechanical load by squats at the same site of the medial femoral condyle by defined transducer positioning. Test-retest reliability was examined using Cronbach's α $\alpha $ calculation. Differences in cartilage thickness were analysed with respect to age, sex and type of practiced sports, respectively. RESULTS: Excellent reliability was achieved both before and after mechanical load by 30 squats with a median cartilage thickness of 1.9 mm (range: 0.5-4.8 mm) before and 1.9 mm (0.4-4.6 mm) after mechanical load. Male cartilages were thicker (p < 0.01) before (median: 2.0 mm) and after (2.0 mm) load when compared to female cartilage (before: 1.6 mm; after: 1.7 mm). Median cartilage thickness was about three times higher in karate athletes (before: 2.3 mm; after: 2.4 mm) than in sports shooters (0.7; 0.7 mm). Cartilage thickness in track and field athletes, handball players and soccer players were found to lay in-between. Sport type related thickness changes after mechanical load were not significant. CONCLUSION: Medial femoral condyle cartilage thickness in childhood correlates with age, sex and practiced type of sports. Ultrasound is a reliable and simple, pain-free approach to evaluate the cartilage thickness in children and adolescents. LEVEL OF EVIDENCE: Level III.

Unfolding Individual Domains of BmrA, a Bacterial ABC Transporter Involved in Multidrug Resistance.

The folding and stability of proteins are often studied via unfolding (and refolding) a protein with urea. Yet, in the case of membrane integral protein domains, which are shielded by a membrane or a membrane mimetic, urea generally does not induce unfolding. However, the unfolding of α-helical membrane proteins may be induced by the addition of sodium dodecyl sulfate (SDS). When protein unfolding is followed via monitoring changes in Trp fluorescence characteristics, the contributions of individual Trp residues often cannot be disentangled, and, consequently, the folding and stability of the individual domains of a multi-domain membrane protein cannot be studied. In this study, the unfolding of the homodimeric bacterial ATP-binding cassette (ABC) transporter Bacillus multidrug resistance ATP (BmrA), which comprises a transmembrane domain and a cytosolic nucleotide-binding domain, was investigated. To study the stability of individual BmrA domains in the context of the full-length protein, the individual domains were silenced by mutating the existent Trps. The SDS-induced unfolding of the corresponding constructs was compared to the (un)folding characteristics of the wild-type (wt) protein and isolated domains. The full-length variants BmrAW413Y and BmrAW104YW164A were able to mirror the changes observed with the isolated domains; thus, these variants allowed for the study of the unfolding and thermodynamic stability of mutated domains in the context of full-length BmrA.

Membrane destabilization and pore formation induced by the Synechocystis IM30 protein.

The inner membrane-associated protein of 30 kDa (IM30) is essential in chloroplasts and cyanobacteria. The spatio-temporal cellular localization of the protein appears to be highly dynamic and triggered by internal as well as external stimuli, mainly light intensity. The soluble fraction of the protein is localized in the cyanobacterial cytoplasm or the chloroplast stroma, respectively. Additionally, the protein attaches to the thylakoid membrane as well as to the chloroplast inner envelope or the cyanobacterial cytoplasmic membrane, respectively, especially under conditions of membrane stress. IM30 is involved in thylakoid membrane biogenesis and/or maintenance, where it either stabilizes membranes and/or triggers membrane-fusion processes. These apparently contradicting functions have to be tightly controlled and separated spatiotemporally in chloroplasts and cyanobacteria. IM30's fusogenic activity depends on Mg2+ binding to IM30; yet, it still is unclear how Mg2+-loaded IM30 interacts with membranes and promotes membrane fusion. Here, we show that the interaction of Mg2+ with IM30 results in increased binding of IM30 to native, as well as model, membranes. Via atomic force microscopy in liquid, IM30-induced bilayer defects were observed in solid-supported bilayers in the presence of Mg2+. These structures differ dramatically from the membrane-stabilizing carpet structures that were previously observed in the absence of Mg2+. Thus, Mg2+-induced alterations of the IM30 structure switch the IM30 activity from a membrane-stabilizing to a membrane-destabilizing function, a crucial step in membrane fusion.

Transmembrane signaling and cytoplasmic signal conversion by dimeric transmembrane helix 2 and a linker domain of the DcuS sensor kinase.

Transmembrane (TM) signaling is a key process of membrane-bound sensor kinases. The C4-dicarboxylate (fumarate) responsive sensor kinase DcuS of Escherichia coli is anchored by TM helices TM1 and TM2 in the membrane. Signal transmission across the membrane relies on the piston-type movement of the periplasmic part of TM2. To define the role of TM2 in TM signaling, we use oxidative Cys cross-linking to demonstrate that TM2 extends over the full distance of the membrane and forms a stable TM homodimer in both the inactive and fumarate-activated state of DcuS. An S186xxxGxxxG194 motif is required for the stability and function of the TM2 homodimer. The TM2 helix further extends on the periplasmic side into the α6-helix of the sensory PASP domain and on the cytoplasmic side into the α1-helix of PASC. PASC has to transmit the signal to the C-terminal kinase domain. A helical linker on the cytoplasmic side connecting TM2 with PASC contains an LxxxLxxxL sequence. The dimeric state of the linker was relieved during fumarate activation of DcuS, indicating structural rearrangements in the linker. Thus, DcuS contains a long α-helical structure reaching from the sensory PASP (α6) domain across the membrane to α1(PASC). Taken together, the results suggest piston-type TM signaling by the TM2 homodimer from PASP across the full TM region, whereas the fumarate-destabilized linker dimer converts the signal on the cytoplasmic side for PASC and kinase regulation.

Quantitative characterization of tetraspanin 8 homointeractions in the plasma membrane.

The spatial distribution of proteins in cell membranes is crucial for signal transduction, cell communication and membrane trafficking. Members of the Tetraspanin family organize functional protein clusters within the plasma membrane into so-called Tetraspanin-enriched microdomains (TEMs). Direct interactions between Tetraspanins are believed to be important for this organization. However, studies thus far have utilized mainly co-immunoprecipitation methods that cannot distinguish between direct and indirect, through common partners, interactions. Here we study Tetraspanin 8 homointeractions in living cells via quantitative fluorescence microscopy. We demonstrate that Tetraspanin 8 exists in a monomer-dimer equilibrium in the plasma membrane. Tetraspanin 8 dimerization is described by a high dissociation constant (Kd = 14 700 ± 1100 Tspan8/µm2), one of the highest dissociation constants measured for membrane proteins in live cells. We propose that this high dissociation constant, and thus the short lifetime of the Tetraspanin 8 dimer, is critical for Tetraspanin 8 functioning as a master regulator of cell signaling.

Myristic Acid Inhibits the Activity of the Bacterial ABC Transporter BmrA.

ATP-binding cassette (ABC) transporters are conserved in all kingdoms of life, where they transport substrates against a concentration gradient across membranes. Some ABC transporters are known to cause multidrug resistances in humans and are able to transport chemotherapeutics across cellular membranes. Similarly, BmrA, the ABC transporter of Bacillus subtilis, is involved in excretion of certain antibiotics out of bacterial cells. Screening of extract libraries isolated from fungi revealed that the C14 fatty acid myristic acid has an inhibitory effect on the BmrA ATPase as well as the transport activity. Thus, a natural membrane constituent inhibits the BmrA activity, a finding with physiological consequences as to the activity and regulation of ABC transporter activities in biological membranes.

Lipid Bilayer Interactions of Peptidic Supramolecular Polymers and Their Impact on Membrane Permeability and Stability.

The synthesis and physicochemical characterization of supramolecular polymers with tunable assembly profiles offer exciting opportunities, involving the development of new biomedical carriers. Because synthetic nanocarriers aim to transport substances across or toward cellular membranes, we evaluated the interactions of amphiphilic peptide-based supramolecular polymers with lipid bilayers. Here, we focused on nanorod-like supramolecular polymers, obtained from two C3-symmetric dendritic peptide amphiphiles with alternating Phe/His sequences, equipped with a peripheral tetraethylene glycol dendron (C3-PH) or charged ethylenediamine end groups (C3-PH+). Triggered by pH changes, these amphiphiles assemble reversibly. Our results show that the supramolecular polymers have an impact on the lipid order in model membranes. Changes in the lipid order were observed depending on the charge state of the amphiphilic building blocks, as well as the chemical composition and physical properties of the bilayer. Furthermore, we further performed cell viability assays with the C3-PH+ and C3-PH supramolecular polymers. For C3-PH, the cell viability and extent of proliferation were decreased and the membrane permeability was enhanced, indicating a strong interaction of the polymer with cellular membranes. The results have implications for the design of novel pH-switchable supramolecular drug carriers and delivery vehicles that can respond to an altered microenvironment of tumorous or inflamed tissue.

Proton Leakage Is Sensed by IM30 and Activates IM30-Triggered Membrane Fusion.

The inner membrane-associated protein of 30 kDa (IM30) is crucial for the development and maintenance of the thylakoid membrane system in chloroplasts and cyanobacteria. While its exact physiological function still is under debate, it has recently been suggested that IM30 has (at least) a dual function, and the protein is involved in stabilization of the thylakoid membrane as well as in Mg2+-dependent membrane fusion. IM30 binds to negatively charged membrane lipids, preferentially at stressed membrane regions where protons potentially leak out from the thylakoid lumen into the chloroplast stroma or the cyanobacterial cytoplasm, respectively. Here we show in vitro that IM30 membrane binding, as well as membrane fusion, is strongly increased in acidic environments. This enhanced activity involves a rearrangement of the protein structure. We suggest that this acid-induced transition is part of a mechanism that allows IM30 to (i) sense sites of proton leakage at the thylakoid membrane, to (ii) preferentially bind there, and to (iii) seal leaky membrane regions via membrane fusion processes.

Lipid Binding Controls Dimerization of the Coat Protein p24 Transmembrane Helix.

Coat protein (COP) I and COP II complexes are involved in the transport of proteins between the endoplasmic reticulum and the Golgi apparatus in eukaryotic cells. The formation of COP I/II complexes at membrane surfaces is an early step in vesicle formation and is mastered by p24, a type I transmembrane protein. Oligomerization of p24 monomers was suggested to be mediated and/or stabilized via interactions within the transmembrane domain, and the p24 transmembrane helix appears to selectively bind a single sphingomyelin C18:0 molecule. Furthermore, a potential cholesterol-binding sequence has also been predicted in the p24 transmembrane domain. Thus, sphingomyelin and/or cholesterol binding to the transmembrane domain might directly control the oligomeric state of p24 and, thus, COP vesicle formation. In this study, we show that sequence-specific dimerization of the p24 transmembrane helix is mediated by a LQ7 motif, with Gln187 being of special importance. Whereas cholesterol has no direct impact on p24 dimerization, binding of the sphingolipid can clearly control dimerization of p24 in rigid membrane regions. We suggest that specific binding of a sphingolipid to the p24 transmembrane helix affects p24 dimerization in membranes with increased cholesterol contents. A clearly defined p24 dimerization propensity likely is crucial for the p24 activity, which involves shuttling in between the endoplasmic reticulum and the Golgi membrane, in which cholesterol and SM C18:0 concentrations differ.

Mg2+ binding triggers rearrangement of the IM30 ring structure, resulting in augmented exposure of hydrophobic surfaces competent for membrane binding.

The "inner membrane-associated protein of 30 kDa" (IM30), also known as "vesicle-inducing protein in plastids 1" (Vipp1), is found in the majority of photosynthetic organisms that use oxygen as an energy source, and its occurrence appears to be coupled to the existence of thylakoid membranes in cyanobacteria and chloroplasts. IM30 is most likely involved in thylakoid membrane biogenesis and/or maintenance, and has recently been shown to function as a membrane fusion protein in presence of Mg2+ However, the precise role of Mg2+ in this process and its impact on the structure and function of IM30 remains unknown. Here, we show that Mg2+ binds directly to IM30 with a binding affinity of ∼1 mm Mg2+ binding compacts the IM30 structure coupled with an increase in the thermodynamic stability of the proteins' secondary, tertiary, and quaternary structures. Furthermore, the structural alterations trigger IM30 double ring formation in vitro because of increased exposure of hydrophobic surface regions. However, in vivo Mg2+-triggered exposure of hydrophobic surface regions most likely modulates membrane binding and induces membrane fusion.

What is Vipp1 good for?

While Vipp1 (also known as IM30) clearly is essential for proper biogenesis of thylakoid membranes in chloroplasts and cyanobacteria, the exact function of Vipp1/IM30 still remains unclear. The recent in vivo study of Gutu et al. now demonstrates that Vipp1/IM30 forms localized puncta specifically at highly curved membrane regions at the cell periphery. These Vipp1/IM30 puncta were found being highly dynamic under normal growth conditions, while it has recently been shown that they stably associate with membranes under high-light conditions. These observations, together with the observation that other Vipp1/IM30 homologous proteins also form puncta under stress conditions, indicate a protective function of these proteins at stressed membrane regions. However, Gutu et al. additionally show that Vipp1/IM30 is of special importance when growing cells are shifted from dark to light growth conditions, which could be explained by light stress, by thylakoid membrane dynamics and/or by photosystem biogenesis, which is being discussed in this article.

Mg2+ homeostasis and transport in cyanobacteria - at the crossroads of bacterial and chloroplast Mg2+ import.

Magnesium cation (Mg2+) is the most abundant divalent cation in living cells, where it is required for various intracellular functions. In chloroplasts and cyanobacteria, established photosynthetic model systems, Mg2+ is the central ion in chlorophylls, and Mg2+ flux across the thylakoid membrane is required for counterbalancing the light-induced generation of a ΔpH across the thylakoid membrane. Yet, not much is known about Mg2+ homoeostasis, transport and distribution within cyanobacteria. However, Mg2+ transport across membranes has been studied in non-photosynthetic bacteria, and first observations and findings are reported for chloroplasts. Cyanobacterial cytoplasmic membranes appear to contain the well-characterized Mg2+ channels CorA and/or MgtE, which both facilitate transmembrane Mg2+ flux down the electrochemical gradient. Both Mg2+ channels are typical for non-photosynthetic bacteria. Furthermore, Mg2+ transporters of the MgtA/B family are also present in the cytoplasmic membrane to mediate active Mg2+ import into the bacterial cell. While the cytoplasmic membrane of cyanobacteria resembles a 'classical' bacterial membrane, essentially nothing is known about Mg2+ channels and/or transporters in thylakoid membranes of cyanobacteria or chloroplasts. As discussed here, at least one Mg2+ channelling protein must be localized within thylakoid membranes. Thus, either one of the 'typical' bacterial Mg2+ channels has a dual localization in the cytoplasmic plus the thylakoid membrane, or another, yet unidentified channel is present in cyanobacterial thylakoid membranes.

Against Expectations: Unassisted RNA Adsorption onto Negatively Charged Lipid Bilayers.

The composition and physicochemical properties of biological membranes can be altered by diverse membrane integral and peripheral proteins as well as by small molecules, natural and synthetic. Diverse oligonucleotides have been shown to electrostatically interact with cationic and bivalent ion loaded zwitterionic liposomes, leading to the formation of oligonucleotide-liposome aggregates. However, interaction of RNAs with other membrane surfaces remains ill understood. We used the nonnatural RNA10 to investigate RNA binding to anionic and net-uncharged membrane surfaces. RNA10 had initially been selected in a screen for nonnatural RNA motives that bind to phosphatidylcholine liposomes in the presence of Mg2+. Here we show that interaction of defined RNA molecules with membrane surfaces crucially depends on electrostatic surface properties. Furthermore, RNA10 electrostatically binds to anionic lipid bilayers in the absence of Mg2+ or other bivalent cations, and this interaction leads to measurably changed physicochemical properties of the bilayer and the oligonucleotide. Thus, the structure of polyanionic RNA can be modulated via contact with negatively charged membrane surfaces and vice versa.

Covalently Linking Oligomerization-Impaired GlpF Protomers Does Not Completely Re-establish Wild-Type Channel Activity.

Integral membrane proteins of the aquaporin family facilitate rapid water flux across cellular membranes in all domains of life. Although the water-conducting pore is clearly defined in an aquaporin monomer, all aquaporins assemble into stable tetramers. In order to investigate the role of protomer⁻protomer interactions, we analyzed the activity of heterotetramers containing increasing fractions of mutated monomers, which have an impaired oligomerization propensity and activity. In order to enforce interaction between the protomers, we designed and analyzed a genetically fused homotetramer of GlpF, the aquaglyceroporin of the bacterium Escherichia coli (E. coli). However, increasing fractions of the oligomerization-impaired mutant GlpF E43A affected the activity of the GlpF heterotetramer in a nearly linear manner, indicating that the reduced protein activity, caused by the introduced mutations, cannot be fully compensated by simply covalently linking the monomers. Taken together, the results underline the importance of exactly positioned monomer⁻monomer contacts in an assembled GlpF tetramer.

The GlpF residue Trp219 is part of an amino-acid cluster crucial for aquaglyceroporin oligomerization and function.

The vestibule loop regions of aquaglyceroporins are involved in accumulation of glycerol inside the channel pore. Even though most loop regions do not show high sequence similarity among aquaglyceroporins, loop E is highly conserved in aquaglyceroporins, but not in members of the homologous aquaporins. Specifically, a tryptophan residue is extremely conserved within this loop. We have investigated the role of this residue (Trp219) that deeply protrudes into the protein and potentially interacts with adjacent loops, using the E. coli aqualgyeroporin GlpF as a model. Replacement of Trp219 affects the activity of GlpF and impairs the stability of the tetrameric protein. Furthermore, we have identified an amino acid cluster involving Trp219 that stabilizes the GlpF tetramer. Based on our results we propose that Trp219 is key for formation of a defined vestibule structure, which is crucial for glycerol accumulation as well as for the stability of the active GlpF tetramer.

The IM30/Vipp1 C-terminus associates with the lipid bilayer and modulates membrane fusion.

IM30/Vipp1 proteins are crucial for thylakoid membrane biogenesis in chloroplasts and cyanobacteria. A characteristic C-terminal extension distinguishes these proteins from the homologous bacterial PspA proteins, and this extension has been discussed to be key for the IM30/Vipp1 activity. Here we report that the extension of the Synechocystis IM30 protein is indispensable, and argue that both, the N-terminal PspA-domain as well as the C-terminal extension are needed in order for the IM30 protein to conduct its in vivo function. In vitro, we show that the PspA-domain of IM30 is vital for stability/folding and oligomer formation of IM30 as well as for IM30-triggered membrane fusion. In contrast, the IM30 C-terminal domain is involved in and necessary to stabilize defined contacts to negatively charged membrane surfaces, and to modulate the IM30-induced membrane fusion activity. Although the two IM30 protein domains have distinct functional roles, only together they enable IM30 to work properly.

Organization into Higher Ordered Ring Structures Counteracts Membrane Binding of IM30, a Protein Associated with Inner Membranes in Chloroplasts and Cyanobacteria.

The IM30 (inner membrane-associated protein of 30 kDa), also known as the Vipp1 (vesicle-inducing protein in plastids 1), has a crucial role in thylakoid membrane biogenesis and maintenance. Recent results suggest that the protein binds peripherally to membranes containing negatively charged lipids. However, although IM30 monomers interact and assemble into large oligomeric ring complexes with different numbers of monomers, it is still an open question whether ring formation is crucial for membrane interaction. Here we show that binding of IM30 rings to negatively charged phosphatidylglycerol membrane surfaces results in a higher ordered membrane state, both in the head group and in the inner core region of the lipid bilayer. Furthermore, by using gold nanorods covered with phosphatidylglycerol layers and single particle spectroscopy, we show that not only IM30 rings but also lower oligomeric IM30 structures interact with membranes, although with higher affinity. Thus, ring formation is not crucial for, and even counteracts, membrane interaction of IM30.