Accumulation of an Antidepressant in Vesiculogenic Membranes of Yeast Cells Triggers Autophagy

Many antidepressants are cationic amphipaths, which spontaneously accumulate in natural or reconstituted membranes in the absence of their specific protein targets. However, the clinical relevance of cellular membrane accumulation by antidepressants in the human brain is unknown and hotly debated. Here we take a novel, evolutionarily informed approach to studying the effects of the selective-serotonin reuptake inhibitor sertraline/Zoloft® on cell physiology in the model eukaryote Saccharomyces cerevisiae (budding yeast), which lacks a serotonin transporter entirely. We biochemically and pharmacologically characterized cellular uptake and subcellular distribution of radiolabeled sertraline, and in parallel performed a quantitative ultrastructural analysis of organellar membrane homeostasis in untreated vs. sertraline-treated cells. These experiments have revealed that sertraline enters yeast cells and then reshapes vesiculogenic membranes by a complex process. Internalization of the neutral species proceeds by simple diffusion, is accelerated by proton motive forces generated by the vacuolar H+-ATPase, but is counteracted by energy-dependent xenobiotic efflux pumps. At equilibrium, a small fraction (10–15%) of reprotonated sertraline is soluble while the bulk (90–85%) partitions into organellar membranes by adsorption to interfacial anionic sites or by intercalation into the hydrophobic phase of the bilayer. Asymmetric accumulation of sertraline in vesiculogenic membranes leads to local membrane curvature stresses that trigger an adaptive autophagic response. In mutants with altered clathrin function, this adaptive response is associated with increased lipid droplet formation. Our data not only support the notion of a serotonin transporter-independent component of antidepressant function, but also enable a conceptual framework for characterizing the physiological states associated with chronic but not acute antidepressant administration in a model eukaryote

A common mechanism of action for three mood-stabilizing drugs

Lithium, carbamazepine and valproic acid are effective mood-stabilizing treatments for bipolar affective disorder. The molecular mechanisms underlying the actions of these drugs and the illness itself are unknown. Berridge and colleagues suggested that inositol depletion may be the way that lithium works in bipolar affective disorder, but others have suggested that glycogen synthase kinase (GSK3) may be the relevant target. The action of valproic acid has been linked to both inositol depletion and to inhibition of histone deacetylase (HDAC). We show here that all three drugs inhibit the collapse of sensory neuron growth cones and increase growth cone area. These effects do not depend on GSK3 or HDAC inhibition. Inositol, however, reverses the effects of the drugs on growth cones, thus implicating inositol depletion in their action. Moreover, the development of Dictyostelium is sensitive to lithium and to valproic acid, but resistance to both is conferred by deletion of the gene that codes for prolyl oligopeptidase, which also regulates inositol metabolism. Inhibitors of prolyl oligopeptidase reverse the effects of all three drugs on sensory neuron growth cone area and collapse. These results suggest a molecular basis for both bipolar affective disorder and its treatment

Determining the Lipid-Binding Specificity of SMP Domains: An ERMES Subunit as a Case Study.

Membrane contact sites between the endoplasmic reticulum (ER) and mitochondria function as a central hub for the exchange of phospholipids and calcium. The yeast Endoplasmic Reticulum-Mitochondrion Encounter Structure (ERMES) complex is composed of five subunits that tether the ER and mitochondria. Three ERMES subunits (i.e., Mdm12, Mmm1, and Mdm34) contain the synaptotagmin-like mitochondrial lipid-binding protein (SMP) domain. The SMP domain belongs to the tubular lipid-binding protein (TULIP) superfamily, which consists of ubiquitous lipid scavenging and transfer proteins. Herein, we describe the methods for expression and purification of recombinant Mdm12, a bona fide SMP-containing protein, together with the subsequent identification of its bound phospholipids by high-performance thin-layer chromatography (HPTLC) and the characterization of its lipid exchange and transfer functions using lipid displacement and liposome flotation in vitro assays with liposomes as model biological membranes. These methods can be applied to the study and characterization of novel lipid-binding and lipid-transfer proteins