A bacterial secretosome for regulated envelope biogenesis and quality control

As the first line of defence against antibiotics, the Gram-negative bacterial envelope and its biogenesis are of considerable interest to the microbiological and biomedical communities. All bacterial proteins are synthesised in the cytosol, so inner- and outer-membrane proteins, and periplasmic residents have to be transported to their final destinations via specialised protein machinery. The Sec translocon, a ubiquitous integral inner-membrane (IM) complex, is key to this process as the major gateway for protein transit from the cytosol to the cell envelope; this can be achieved during their translation, or afterwards. Proteins need to be directed to the inner-membrane (usually co-translational), otherwise SecA utilises ATP and the proton-motive-force (PMF) to drive proteins across the membrane post-translationally. These proteins are then picked up by chaperones for folding in the periplasm or delivered to the β-barrel assembly machinery (BAM) for incorporation into the outer-membrane. The core heterotrimeric SecYEG-complex forms the hub for an extensive network of interactions that regulate protein delivery and quality control. Here, we conduct a biochemical exploration of this secretosome: a very large, versatile and inter-changeable assembly with the Sec-translocon at its core; featuring interactions that facilitate secretion (SecDF), inner- and outer-membrane protein insertion (respectively, YidC and BAM), protein folding and quality control (e.g. PpiD, YfgM and FtsH). We propose the dynamic interplay amongst these and other factors act to ensure efficient whole envelope biogenesis, regulated to accommodate the requirements of cell elongation and division. This organisation would be essential for cell wall biogenesis and remodelling and thus its perturbation would be a good strategy for the development of anti-microbials.

Changes in the Proteome in the Development of Chronic Human Papillomavirus Infection—A Prospective Study in HIV Positive and HIV Negative Rwandan Women

Background: Effects on the proteome when a high risk (HR)-HPV infection occurs, when it is cleared and when it becomes chronic were investigated. Moreover, biomarker panels that could identify cervical risk lesions were assessed. Methods: Cytology, HPV screening and proteomics were performed on cervical samples from Rwandan HIV+ and HIV- women at baseline, at 9 months, at 18 months and at 24 months. Biological pathways were identified using the String database. Results: The most significantly affected pathway when an incident HR-HPV infection occurred was neutrophil degranulation, and vesicle-mediated transport was the most significantly affected pathway when an HR-HPV infection was cleared; protein insertion into membrane in chronic HR-HPV lesions and in lesions where HR-HPVs were cleared were compared; and cellular catabolic process in high-grade lesions was compared to that in negative lesions. A four-biomarker panel (EIF1; BLOC1S5; LIMCH1; SGTA) was identified, which was able to distinguish chronic HR-HPV lesions from cleared HR-HPV/negative lesions (sensitivity 100% and specificity 91%). Another four-biomarker panel (ERH; IGKV2-30; TMEM97; DNAJA4) was identified, which was able to distinguish high-grade lesions from low-grade/negative lesions (sensitivity 100% and specificity 81%). Conclusions: We have identified the biological pathways triggered in HR-HPV infection, when HR-HPV becomes chronic and when cervical risk lesions develop. Moreover, we have identified potential biomarkers that may help to identify women with cervical risk lesions.

Synthetic Cell as a Platform for Understanding Membrane-Membrane Interactions

In the pursuit of understanding life, model membranes made of phospholipids were envisaged decades ago as a platform for the bottom-up study of biological processes. Micron-sized lipid vesicles have gained great acceptance as their bilayer membrane resembles the natural cell membrane. Important biological events involving membranes, such as membrane protein insertion, membrane fusion, and intercellular communication, will be highlighted in this review with recent research updates. We will first review different lipid bilayer platforms used for incorporation of integral membrane proteins and challenges associated with their functional reconstitution. We next discuss different methods for reconstitution of membrane fusion and compare their fusion efficiency. Lastly, we will highlight the importance and challenges of intercellular communication between synthetic cells and synthetic cells-to-natural cells. We will summarize the review by highlighting the challenges and opportunities associated with studying membrane–membrane interactions and possible future research directions.

Large-scale conformational changes of FhaC provide insights into the two-partner secretion mechanism

The Two-Partner secretion pathway mediates protein transport across the outer membrane of Gram-negative bacteria. TpsB transporters belong to the Omp85 superfamily, whose members catalyze protein insertion into, or translocation across membranes without external energy sources. They are composed of a transmembrane beta barrel preceded by two periplasmic POTRA domains that bind the incoming protein substrate. Here we used an integrative approach combining in vivo assays, mass spectrometry, nuclear magnetic resonance and electron paramagnetic resonance techniques suitable to detect minor states in heterogeneous populations, to explore transient conformers of the TspB transporter FhaC. This revealed substantial, spontaneous conformational changes with a portion of the POTRA2 domain coming close to the lipid bilayer and surface loops. Specifically, the amphipathic beta hairpin immediately preceding the first barrel strand can insert into the beta barrel. We propose that these motions enlarge the channel and may hoist the substrate into it for secretion. An anchor region at the interface of the beta barrel and the POTRA2 domain stabilizes the transporter in the course of secretion. Our data propose a solution to the conundrum how these proteins mediate protein secretion without the need for cofactors, by utilizing intrinsic protein dynamics.

Subunit cooperation in the Get1/2 receptor promotes tail-anchored membrane protein insertion

The guided entry of tail-anchored protein (GET) pathway, in which the Get3 ATPase delivers an essential class of tail-anchored membrane proteins (TAs) to the Get1/2 receptor at the endoplasmic reticulum, provides a conserved mechanism for TA biogenesis in eukaryotic cells. The membrane-associated events of this pathway remain poorly understood. Here we show that complex assembly between the cytosolic domains (CDs) of Get1 and Get2 strongly enhances the affinity of the individual subunits for Get3•TA, thus enabling efficient capture of the targeting complex. In addition to the known role of Get1CD in remodeling Get3 conformation, two molecular recognition features (MoRFs) in Get2CD induce Get3 opening, and both subunits are required for optimal TA release from Get3. Mutation of the MoRFs attenuates TA insertion into the ER in vivo. Our results demonstrate extensive cooperation between the Get1/2 receptor subunits in the capture and remodeling of the targeting complex, and emphasize the role of MoRFs in receptor function during membrane protein biogenesis.

Towards a molecular mechanism underlying mitochondrial protein import through the TOM-TIM23 complex

Mitochondria contain over a thousand different proteins, which, aside from a few encoded on the mitochondrial genome, are translated in the cytosol and targeted for import. For the majority, the first port of call is the translocase of the outer membrane (TOM-complex); their onward journey is via a procession of alternative molecular machines, conducting transport to their final sub-compartment destination: the outer-mitochondrial membrane (OMM), inner-mitochondrial membrane (IMM), inter-membrane space (IMS) or matrix. The pre-sequence translocase of the inner-membrane (TIM23-complex) is responsible for importing proteins with cleavable pre-sequences, and comes in two distinct forms: the TIM23SORT complex mediates IMM protein insertion and the TIM23MOTOR complex is responsible for matrix import. Progress in understanding these transport mechanisms has, until recently, been hampered by the poor sensitivity and time-resolution of import assays. However, with the development of an assay based on split NanoLuc luciferase, we can now explore this process in greater detail. Here, we apply this new methodology to understand how ∆ψ and ATP hydrolysis, the two main driving forces for transport through the TIM23MOTOR complex, contribute to the import of pre-sequence-containing precursors (PCPs) with varying properties. Notably, we found that two major rate limiting steps define the PCP import time: passage of the PCP across the OMM and initiation of IMM transport by the pre-sequence. The rates of these steps are influenced by PCP properties such as size and net charge, but correlate poorly with the total amount of PCP imported - emphasising the importance of collecting rapid kinetic data to elucidating mechanistic detail. Our results also indicate that PCPs spend very little time in the TIM23 channel - presumably rapid success or failure of import is critical for maintaining mitochondrial health.

The ER transmembrane complex (EMC) can functionally replace the Oxa1 insertase in mitochondria

Two multisubunit protein complexes for membrane protein insertion were recently identified in the endoplasmic reticulum (ER): The guided entry of tail anchor proteins (GET) complex and ER membrane complex (EMC). The structures of both of their hydrophobic core subunits, that are required for the insertion reaction, revealed an overall similarity to the YidC/Oxa1/Alb3 family members found in bacteria, mitochondria and chloroplasts. This suggests that these membrane insertion machineries all share a common ancestry. To test whether these ER proteins can functionally replace Oxa1 in yeast mitochondria, we generated strains that express mitochondria-targeted Get2-Get1 and Emc6-Emc3 fusion proteins in Oxa1 deletion mutants. Interestingly, the Emc6-Emc3 fusion was able to complement an Δoxa1 mutant and restored its respiratory competence. The Emc6-Emc3 fusion promoted the insertion of the mitochondrially encoded protein Cox2 as well as of nuclear encoded inner membrane proteins though was not able to facilitate the assembly of the Atp9 ring. Our observations indicate that protein insertion into the ER is functionally conserved to the insertion mechanism in bacteria and mitochondria and adheres to similar topological principles.

Intra-helical salt bridge contribution to membrane protein insertion

ABSTRACTSalt bridges between negatively (D, E) and positively charged (K, R, H) amino acids play an important role in protein stabilization. This has a more prevalent effect in membrane proteins where polar amino acids are exposed to a very hydrophobic environment. In transmembrane (TM) helices the presence of charged residues can hinder the insertion of the helices into the membrane. This can sometimes be avoided by TM region rearrangements after insertion, but it is also possible that the formation of salt bridges could decrease the cost of membrane integration. However, the presence of intra-helical salt bridges in TM domains and their effect on insertion has not been properly studied yet. In this work, we use an analytical pipeline to study the prevalence of charged pairs of amino acid residues in TM α-helices, which shows that potentially salt-bridge forming pairs are statistically over-represented. We then selected some candidates to experimentally determine the contribution of these electrostatic interactions to the translocon-assisted membrane insertion process. Using both in vitro and in vivo systems, we confirm the presence of intra-helical salt bridges in TM segments during biogenesis and determined that they contribute between 0.5-0.7 kcal/mol to the apparent free energy of membrane insertion (ΔGapp). Our observations suggest that salt bridge interactions can be stabilized during translocon-mediated insertion and thus could be relevant to consider for the future development of membrane protein prediction software.