Proteins of the Bcl-2 protein family, including pro-apoptotic Bax and anti-apoptotic Bcl-xL, are critical for mitochondrial-mediated apoptosis regulation. Since yeast lacks obvious orthologs of Bcl-2 family members, heterologous expression of these proteins has been used to investigate their molecular and functional aspects. Active Bax is involved in the formation of mitochondrial outer membrane pores, through which cytochrome c (cyt c) is released, triggering a cascade of downstream apoptotic events. However, when in its inactive form, Bax is largely cytosolic or weakly bound to mitochondria. Given the central role of Bax in apoptosis, studies aiming to understand its regulation are of paramount importance towards its exploitation as a therapeutic target. So far, studies taking advantage of heterologous expression of human Bax in yeast to unveil regulation of Bax activation have relied on the use of artificial mutated or mitochondrial tagged Bax for its activation, rather than the wild type Bax (Bax α). Here, we found that cell death could be triggered in yeast cells heterologoulsy expressing Bax α with concentrations of acetic acid that are not lethal to wild type cells. This was associated with Bax mitochondrial translocation and cyt c release, closely resembling the natural Bax function in the cellular context. This regulated cell death process was reverted by co-expression with Bcl-xL, but not with Bcl-xLΔC, and in the absence of Rim11p, the yeast ortholog of mammalian GSK3β. This novel system mimics human Bax α regulation by GSK3β and can therefore be used as a platform to uncover novel Bax regulators and explore its therapeutic modulation.
Membrane voltage arises from the transport of ions through ion-translocating ATPases, ion-coupled transport of solutes, and ion channels, and is an integral part of the bioenergetic “currency” of the membrane. The dynamics of membrane voltage—so-called action, systemic, and variation potentials—have also led to a recognition of their contributions to signal transduction, both within cells and across tissues. Here, we review the origins of our understanding of membrane voltage and its place as a central element in regulating transport and signal transmission. We stress the importance of understanding voltage as a common intermediate that acts both as a driving force for transport—an electrical “substrate”—and as a product of charge flux across the membrane, thereby interconnecting all charge-carrying transport across the membrane. The voltage interconnection is vital to signaling via second messengers that rely on ion flux, including cytosolic free Ca2+, H+, and the synthesis of reactive oxygen species generated by integral membrane, respiratory burst oxidases. These characteristics inform on the ways in which long-distance voltage signals and voltage oscillations give rise to unique gene expression patterns and influence physiological, developmental, and adaptive responses such as systemic acquired resistance to pathogens and to insect herbivory.Membrane voltage serves as a platform coordinating ion flux to transmit and transduce biological signals. Advances
The biophysics of transport that determine membrane voltage are well-described with quantitative flux equations.
In the models of the guard cell and the giant algae Chara and Nitella these charge-transporting processes accurately describe and predict physiological behavior, including the coupling of membrane voltage oscillations with ion flux, [Ca2+]i, pH, their consequences for cellular osmotic adjustments, and their spatial propagation.
Unlike neuronal and other animal tissues, action potentials in plants are mediated by a temporal sequence of ion flux through Ca2+ and Cl- channels with voltage recovery driven by ion flux through K+ channels. The interplay of channel-mediated ion flux and changes in H+-ATPase activity are likely responsible for the slower propagation of variation and systemic potentials.
In terrestrial plants, membrane voltage transients may propagate along vascular traces, both through the parenchymal cells lining the xylem and through the phloem. Propagation of such voltage transients is associated with glutamate receptor-like channels that may contribute to plasma membrane Ca2+ flux and [Ca2+]i elevations.
Changes in [Ca2+]i, pH, and reactive oxygen species are key mediators that translate voltage signals into physiological, developmental, and adaptive responses in plant tissues.
Processing of large numbers smaples of plant tissue samples for molecular mapping and gene tagging requires methods that are
quick, simple, and cheap, and that eventually can be automated. Organic solvents used for DNA extraction can represent a significant
proportion of the overall cost. In this study we examined dichloromethane as a replacement for chloroform to be used in combination
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