Sorting the trash:Micronucleophagy gets selective

During micronucleophagy, the nucleolus is targeted by autophagic degradation, but although nucleolar proteins are recycled, ribosomal DNA is spared. Mostofa et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201706164) reveal that the separation of these two nucleolar components is mediated by the CLIP and cohibin complexes and is vital for cell survival during starvation

Membrane Potentials Regulating GPCRs:Insights From Experiments and Molecular Dynamics Simulation

G-protein coupled receptors (GPCRs) form the largest class of membrane proteins in humans and the targets of most present drugs. Membrane potential is one of the defining characteristics of living cells. Recent work has shown that the membrane voltage, and changes thereof, modulates signal transduction and ligand binding in GPCRs. As it may allow differential signalling patterns depending on tissue, cell type, and the excitation status of excitable cells, GPCR voltage sensitivity could have important implications for their pharmacology. This review summarises recent experimental insights on GPCR voltage regulation and the role of molecular dynamics simulations in identifying the structural basis of GPCR voltage-sensing. We discuss the potential significance for drug design on GPCR targets from excitable and non-excitable cells

Lipids modulate the conformational dynamics of a secondary multidrug transporter

Direct interactions with lipids have emerged as key determinants of the folding, structure and function of membrane proteins, but an understanding of how lipids modulate protein dynamics is still lacking. Here, we systematically explored the effects of lipids on the conformational dynamics of the proton-powered multidrug transporter LmrP from Lactococcus lactis, using the pattern of distances between spin-label pairs previously shown to report on alternating access of the protein. We uncovered, at the molecular level, how the lipid headgroups shape the conformational-energy landscape of the transporter. The model emerging from our data suggests a direct interaction between lipid headgroups and a conserved motif of charged residues that control the conformational equilibrium through an interplay of electrostatic interactions within the protein. Together, our data lay the foundation for a comprehensive model of secondary multidrug transport in lipid bilayers

A comparison of SMA (styrene maleic acid) and DIBMA (di-isobutylene maleic acid) for membrane protein purification

The use of styrene maleic acid co-polymer (SMA) for membrane protein extraction and purification has grown in recent years. SMA inserts in the membrane and assembles into small discs of bilayer encircled by polymer, termed SMA lipid particles (SMALPs). This allows purification of membrane proteins whilst maintaining their lipid bilayer environment. SMALPs offer several improvements over conventional detergent approaches, however there are limitations, most notably a sensitivity to low pH and divalent cations. Recently it was shown that the aliphatic diisobutylene-maleic acid (DIBMA) copolymer, was also able to directly solubilise membranes forming DIBMALPs (DIBMA lipid particles), and that this polymer overcame some of the limitations of SMA. In this study the ability of DIBMA to solubilise and purify functional membrane proteins has been compared to SMA. It was found that DIBMA is able to solubilise several different membrane proteins from different expression systems, however for some proteins it gives a lower yield and lower degree of purity than SMA. DIBMA extracted G protein-coupled receptors retain ligand- and G protein-binding. DIBMALPS are larger than SMALPs and display a decreased sensitivity to magnesium. However the stability of DIBMALPs appears to be lower than SMALPs. The lower purity and lower stability are likely linked to the larger size of the DIBMALP particle. However, this also offers a potentially less rigid lipid environment which may be more amenable to protein dynamics. Therefore the optimal choice of polymer will depend on which features of a protein are to be investigated

Examining the stability of membrane proteins within SMALPs

Amphipathic co-polymers such as styrene-maleic acid (SMA) have gained popularity over the last few years due to their ability and ease of use in solubilising and purifying membrane proteins in comparison to conventional methods of extraction such as detergents. SMA2000 is widely used for membrane protein studies and is considered as the optimal polymer for this technique. In this study a side-by-side comparison of SMA2000 with the polymer SZ30010 was carried out as both these polymers have similar styrene:maleic acid ratios and average molecular weights. Ability to solubilise, purify and stabilise membrane proteins was tested using three structurally different membrane proteins. Our results show that both polymers can be used to extract membrane proteins at a comparable efficiency to conventional detergent dodecylmaltoside (DDM). SZ30010 was found to give a similar protein yield and, SMALP disc size as SMA2000, and both polymers offered an increased purity and increased thermostability compared to DDM. Further investigation was conducted to investigate SMALP sensitivity to divalent cations. It was found that the sensitivity is polymer specific and not dependent on the protein encapsulated. Neither is it affected by the concentration of SMALPs. Larger divalent cations such as Co2+ and Zn2+ resulted in an increased sensitivity

Infection-Associated Nuclear Degeneration in the Rice Blast Fungus Magnaporthe oryzae Requires Non-Selective Macro-Autophagy

addresses: School of Biosciences, University of Exeter, Exeter, Devon, United Kingdom.notes: PMCID: PMC3308974Freely-available open access article.The rice blast fungus Magnaporthe oryzae elaborates a specialized infection structure called an appressorium to breach the rice leaf surface and gain access to plant tissue. Appressorium development is controlled by cell cycle progression, and a single round of nuclear division occurs prior to appressorium formation. Mitosis is always followed by programmed cell death of the spore from which the appressorium develops. Nuclear degeneration in the spore is known to be essential for plant infection, but the precise mechanism by which it occurs is not known

Microautophagy of the Nucleus Coincides with a Vacuolar Diffusion Barrier at Nuclear–Vacuolar Junctions

Nuclear-vacuolar (NV) junctions are organelle contact sites in yeast. They exclude nuclear pores from the organelle interface. On the vacuolar side, a lipid-dependent process excludes specific membrane proteins, such as V-ATPase, from the contact site. This suggests that NV junctions establish selective diffusion barriers

Expression and purification of recombinant G protein-coupled receptors: A review

Given their extensive role in cell signalling, GPCRs are significant drug targets; despite this, many of these receptors have limited or no available prophylaxis. Novel drug design and discovery significantly rely on structure determination, of which GPCRs are typically elusive. Progress has been made thus far to produce sufficient quantity and quality of protein for downstream analysis. As such, this review highlights the systems available for recombinant GPCR expression, with consideration of their advantages and disadvantages, as well as examples of receptors successfully expressed in these systems. Additionally, an overview is given on the use of detergents and the styrene maleic acid (SMA) co-polymer for membrane solubilisation, as well as purification techniques

ATP synthase: evolution, energetics, and membrane interactions

The synthesis of ATP, life's 'universal energy currency', is the most prevalent chemical reaction in biological systems, and is responsible for fueling nearly all cellular processes, from nerve impulse propagation to DNA synthesis. ATP synthases, the family of enzymes that carry out this endless task, are nearly as ubiquitous as the energy-laden molecule they are responsible for making. The F-type ATP synthase (F-ATPase) is found in every domain of life, and is believed to predate the divergence of these lineages over 1.5 billion years ago. These enzymes have therefore facilitated the survival of organisms in a wide range of habitats, ranging from the deep-sea thermal vents to the human intestine. In this review, we present an overview of the current knowledge of the structure and function of F-type ATPases, highlighting several adaptations that have been characterized across taxa. We emphasize the importance of studying these features within the context of the enzyme's particular lipid environment: Just as the interactions between an organism and its physical environment shape its evolutionary trajectory, ATPases are impacted by the membranes within which they reside. We argue that a comprehensive understanding of the structure, function, and evolution of membrane proteins -- including ATP synthase -- requires such an integrative approach.Comment: Review article; 29 pages, 6 figures/1 tabl