Biology subjects

Allen, W. J.

Publications and source records attributed to Allen, W. J..

11 recordsLinked to original sources

bioRxiv2026

Partner fidelity with root symbionts impacts plant performance in the face of changing above- and belowground community context

Global environmental changes generate novel communities. Some species adapt to novel community contexts by demonstrating interaction fidelity, or consistently interacting with the same species, while others demonstrate interaction flexibility, or interacting with whichever partners are available (i.e., rewiring). However, the drivers and benefits of fidelity are unclear. Here, we use data from a large-scale mesocosm experiment to determine whether plant characteristics (e.g., provenance, functional group) and community contexts impact plant interaction fidelity to belowground mutualist and antagonist partners (i.e., root fungi and bacteria), and how this fidelity relates to plant performance. We found that the plant-antagonist relationships exhibited higher fidelity than plant-mutualist relationships with the strength of fidelity depending on a combination of plant characteristics and community context. Conversely, plant-mutualist fidelity impacted plant performance with competing effects depending on plant provenance and aboveground herbivore presence. Our study advances understanding of how species interactions influence plant performance in a changing world. Statement of authorshipAMM, JDT, JMT and DBS designed the concept. All authors contributed to refining the ideas. AM coded the analysis to produce the results with contributions from JDT, DBS, and BBM. HRL and DBS helped AMM with the statistical analysis. LPW, WJA, IAD, and JMT designed the experiment while LPW and WJA conducted the experiment. AMM wrote the first draft of the manuscript with assistance from all authors. IAD, JMT, and JDT obtained funding. All authors contributed to further editing of the manuscript.

ecology↗

bioRxiv2024

A whole cell luminescence-based screen for inhibitors of the bacterial Sec machinery

There is a pressing need for new antibiotics to combat rising resistance to those already in use. The bacterial general secretion (Sec) system has long been considered a good target for novel antimicrobials thanks to its irreplacable role in maintaining cell envelope integrity; yet the lack of a robust, high-throughput method to screen for Sec inhibition has so far hampered efforts to realise this potential. Here, we have adapted our recently-developed in vitro assay for Sec activity - based on the split NanoLuc luciferase - to work at scale and in living cells. A simple counterscreen allows compounds that specifically target Sec to be distinguished from those with other effects on cellular function. As proof of principle we have applied this assay to a library of 5000 compounds and identified a handful of moderately effective in vivo inhibitors of Sec. We therefore anticipate that the methods presented here will be scalable to larger compound libraries, in the ultimate quest for Sec inhibitors with clinically relevant properties.

biochemistry↗

bioRxiv2021

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

Nearly all mitochondrial proteins need to be targeted for import from the cytosol. For the majority, the first port of call is the translocase of the outer membrane (TOM complex), followed by a procession of alternative molecular machines, conducting transport to their final destination. The pre-sequence translocase of the inner-membrane (TIM23-complex) imports proteins with cleavable pre-sequences, and comes in two flavours: the TIM23SORT complex mediates inner mitochondrial membrane (IMM) protein insertion and the TIM23MOTOR complex delivers proteins to the matrix. Progress in understanding these transport mechanisms has 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 {Delta}{psi} and ATP hydrolysis, the two main driving forces for import 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 PCP import time: passage of 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 not total amount of PCP imported - emphasising the importance of collecting rapid kinetic data, achieved here, to elucidate mechanistic detail. The apparent distinction between transport through the two membranes (passage through TOM is substantially complete before PCP-TIM engagement) is in contrast with the current view that import occurs through TOM and TIM in a single continuous step. 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 fitness.

biochemistry↗

bioRxiv2020

Refined measurement of SecA-driven protein transport reveals indirect coupling to ATP turnover

The universally conserved Sec system is the primary method cells utilise to transport proteins across membranes. Until recently, measuring the activity - a prerequisite for understanding how biological systems works - has been limited to discontinuous protein transport assays with poor time resolution, or used as reporters large, non-natural tags that interfere with the process. The development of an assay based on a split super-bright luciferase (NanoLuc) changed this. Here, we exploit this technology to unpick the steps that constitute post-translational transport in bacteria. Under the conditions deployed, transport of the model pre-protein substrate proSpy occurs at 200 amino acids per minute with the data best fit by a series of large, [~]30 amino acid, steps each coupled to many (100s) ATP hydrolysis events. Prior to that, there is no evidence for a distinct, rate-limiting initiation event. Kinetic modelling suggests that SecA-driven transport activity is facilitated by the substrate (polypeptide) concentration gradient - in keeping with classical membrane transporters. Furthermore, the features we describe are consistent with a non-deterministic motor mechanism, such as a Brownian ratchet.

biochemistry↗

bioRxiv2019

HDX-MS reveals nucleotide-regulated, anti-correlated opening and closure of SecA and SecY channels of the bacterial translocon

The bacterial Sec translocon is a multi-component protein complex responsible for translocating diverse proteins across the plasma membrane. For post-translational protein translocation, the Sec-channel - SecYEG - associates with the motor protein SecA to mediate the ATP-dependent transport of unfolded pre-proteins across the membrane. Based on the structure of the machinery, combined with ensemble and single molecule analysis, a diffusional based Brownian ratchet mechanism for protein secretion has been proposed [Allen et al. eLife 2016;5:e15598]. However, the conformational dynamics required to facilitate this mechanism have not yet been fully resolved. Here, we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal striking nucleotide-dependent conformational changes in the Sec protein-channel. In addition to the ATP-dependent opening of SecY, reported previously, we observe a counteracting, also ATP-dependent, constriction of SecA around the mature regions of the pre-protein. Thus, ATP binding causes SecY to open and SecA to close, while ATP hydrolysis has the opposite effect. This alternating behaviour could help impose the directionality of the Brownian ratchet for protein transport through the Sec machinery, and possibly in translocation systems elsewhere. The results highlight the power of HDX-MS for interrogating the dynamic mechanisms of diverse membrane proteins; including their interactions with small molecules such as nucleotides (ATPases and GTPases) and inhibitors (e.g. antibiotics).

biophysics↗

bioRxiv2019

A ‘proton ratchet’ for coupling the membrane potential to protein transport

Transport of proteins across and into membranes is a fundamental biological process with the vast majority being conducted by the ubiquitous Sec machinery. In bacteria, this is usually achieved when the SecY-complex engages the cytosolic ATPase SecA (secretion) or translating ribosomes (insertion). Great strides have been made towards understanding the mechanism of protein translocation. Yet, important questions remain - notably, the nature of the individual steps that constitute transport, and how the proton-motive force (PMF) across the plasma membrane contributes. Here, we apply a recently developed high-resolution protein transport assay to explore these questions. We find that pre-protein transport is limited primarily by the diffusion of arginine residues across the membrane, particularly in the context of bulky hydrophobic sequences. This specific effect of arginine, caused by its positive charge, is mitigated for lysine which can be deprotonated and transported across the membrane in its neutral form. These observations have interesting implications for the mechanism of protein secretion, suggesting a simple mechanism by which PMF can aid transport, and enabling a proton ratchet, wherein re-protonation of exiting lysine residues prevents channel re-entry, biasing transport in the outward direction.

biochemistry↗

bioRxiv2018

A High-Resolution Luminescent Assay For Rapid And Continuous Monitoring Of Protein Translocation Across Biological Membranes

Protein translocation is a fundamental process in biology. Major gaps in our understanding of this process arises due the poor sensitivity, low time-resolution and irreproducibility of translocation assays. To address this, we applied NanoLuc split-luciferase to produce a new strategy for measuring protein transport. The system reduces the timescale of data collection from days to minutes, and allows continuous acquisition with a time-resolution in the order of seconds - yielding kinetics parameters suitable for mechanistic elucidation and mathematical fitting. To demonstrate its versatility, we implemented and validated the assay in vitro and in vivo for the bacterial Sec system, and the mitochondrial protein import apparatus. Overall, this technology represents a major step forward, providing a powerful new tool for fundamental mechanistic enquiry of protein translocation and for inhibitor (drug) screening, with an intensity and rigour unattainable through classical methods.

biochemistry↗

bioRxiv2018

Dynamic action of the Sec machinery during initiation, protein translocation and termination revealed by single molecule fluorescence

Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. This is mediated, for the majority of proteins, by the highly conserved Sec machinery. The bacterial translocon - SecYMKEG - resides in the plasma membrane, where translocation is driven through rounds of ATP hydrolysis by the cytoplasmic SecA ATPase, and the proton motive force (PMF). We have used single molecule Forster resonance energy transfer (FRET) alongside a combination of confocal and total internal reflection microscopy to gain access to SecY pore dynamics and translocation kinetics on timescales spanning milliseconds to minutes. This allows us to dissect and characterise the translocation process in unprecedented detail. We show that SecA, signal sequence, pre-protein and ATP hydrolysis each have important and specific roles in unlocking and opening the Sec channel, priming it for transport. After channel opening, translocation proceeds in two phases: an initiation phase independent of substrate length, and a length-dependent transport phase with an intrinsic translocation rate of ~ 40 amino acids per second for the model pre-protein substrate proOmpA. The initiation and translocation phases are both coupled to ATP hydrolysis while termination is ATP-independent. Distributions of translocation rates reflect the stochastic nature of the translocation process and are consistent with the recently proposed Brownian ratchet model [Allen et al. doi: 10.7554/eLife.15598]. The results allow us unparalleled access to the kinetics of the complex reaction and provide a framework for understanding the molecular mechanism of protein secretion.

biophysics↗

bioRxiv2017

ATP-induced asymmetric pre-protein folding: a potential driver of protein translocation?

The transport of proteins across membranes is a fundamental and essential process, achieved in every cell by the Sec translocon. In prokaryotes, SecYEG associates with the motor protein SecA to carry out ATP-driven pre-protein secretion - a vital step in the biogenesis of most periplasmic, outer membrane and secreted proteins. Structural data of the SecA-SecYEG complex has provided considerable insight into underlying mechanism of this process. Previously, we have proposed a Brownian ratchet model for protein translocation, whereby the free energy of ATP binding and hydrolysis favours the progression of pre-protein across the membrane from the cytosol toward the outside [Allen, Corey et al. eLife 2016]. Here, we use atomistic molecular dynamics simulation of a SecA-SecYEG complex engaged with preprotein to further address the mechanism underlying this process. The data describe pre-protein secondary structure formation within the channel, which exhibits a nucleotide-dependent asymmetry between the cytoplasmic and exterior cavities. The results suggest ATP-dependent pre-protein transport is partly driven by pre-protein secondary structure formation. The model previously described, and refined here, could easily be adapted for the transport of proteins across various other membranes, such as the endoplasmic reticular and mitochondrial inner membranes.

biophysics↗

bioRxiv2017

Identification and functional analyses of cardiolipin binding sites on the bacterial Sec translocase

The transport of proteins across or into membranes is a vital biological process, achieved in every cell by the conserved Sec machinery. In bacteria, SecYEG combines with the SecA motor protein for secretion of pre-proteins across the plasma membrane, powered by ATP hydrolysis and the trans-membrane proton-motive-force (PMF). The activities of SecYEG and SecA are modulated by membrane lipids, particularly by cardiolipin, a specialised phospholipid known to associate with a range of energy-transducing machines. Here, we identify two specific cardiolipin binding sites on the Thermotoga maritima SecA-SecYEG complex, through application of coarse-grained molecular dynamics simulations. We validate the computational data and demonstrate the conserved nature of the binding sites using in vitro mutagenesis, native mass spectrometry and biochemical analysis of Escherichia coli SecYEG. The results show that the two sites account for the preponderance of functional cardiolipin binding to SecYEG, and mediate its roles in ATPase and protein transport activity. In addition, we demonstrate an important role for cardiolipin in the conferral of PMF-stimulation of protein transport. The apparent transient nature of the CL interaction might facilitate proton exchange with the Sec machinery and thereby stimulate protein transport, by an as yet unknown mechanism. This study demonstrates the power of coupling the high predictive ability of coarse-grained simulation with experimental analyses, towards investigation of both the nature and functional implications of protein-lipid interactions.\n\nSignificance StatementMany proteins are located in lipid membranes surrounding cells and cellular organelles. The membrane can impart important structural and functional effects on the protein, making understanding of this interaction critical. Here, we apply computational simulation to the identification of conserved lipid binding sites on an important highly conserved bacterial membrane protein, the Sec translocase (SecA-SecYEG), which uses ATP and the proton motive force (PMF) to secrete proteins across the bacterial plasma membrane. We experimentally validate and reveal the conserved nature of these binding sites, and use functional analyses to investigate the biological significance of this interaction. We demonstrate that these interactions are specific, transient, and critical for both ATP- and PMF- driven protein secretion.

biophysics↗

bioRxiv2017

Intraspecific variation in indirect plant-soil feedbacks as a driver of a wetland plant invasion

Plant-soil feedbacks (PSFs) can influence plant competition via direct interactions with pathogens and mutualists or indirectly via apparent competition/mutualisms (i.e., spillover to cooccurring plants) and soil legacy effects. Presently, it is unknown how intraspecific variation in PSFs interacts with the environment (e.g., nutrient availability) to influence competition between native and invasive plants. We conducted a fully crossed multi-factor greenhouse experiment to determine the effects of soil biota, interspecific competition, and nutrient availability on biomass of replicate populations from one native and two invasive lineages of common reed (Phragmites australis) and a single lineage of native smooth cordgrass (Spartina alterniflora). Harmful soil biota consistently dominated PSFs involving all three P. australis lineages, reducing biomass by 10%, regardless of nutrient availability or S. alterniflora presence as a competitor. Spillover of soil biota derived from the rhizosphere of the two invasive P. australis lineages reduced S. alterniflora biomass by 7%, whereas soil biota from the native P. australis lineage increased S. alterniflora biomass by 6%. Interestingly, regardless of lineage, P. australis soil biota negatively affected S. alterniflora biomass when grown alone (i.e., a soil legacy), but had a positive impact when grown with P. australis, suggesting that P. australis is preferred by harmful generalist soil biota or facilitates S. alterniflora via spillover (i.e., apparent mutualism). Soil biota also reduced the negative impacts of interspecific competition on S. alterniflora by 13%, although it remained competitively inferior to P. australis across all treatments. Moreover, competitive interactions and the response to nutrients did not differ among P. australis lineages, indicating that interspecific competition and nutrient deposition may not be key drivers of P. australis invasion in North America. Taken together, although soil biota, interspecific competition, and nutrient availability appear to have no direct impact on the success of invasive P. australis lineages in North America, indirect spillover and soil legacies from P. australis occur and may have important implications for co-occurring native species and restoration of invaded habitats. Our study integrates multiple factors linked to plant invasions, highlighting that indirect interactions are likely commonplace in driving successful invasions and their impacts on the local community.

ecology↗