Biology subjects

Allen, W. J.

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

4 recordsLinked to original sources

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↗