Regulation of the V-ATPase along the Endocytic Pathway Occurs through Reversible Subunit Association and Membrane Localization

The lumen of endosomal organelles becomes increasingly acidic when going from the cell surface to lysosomes. Luminal pH thereby regulates important processes such as the release of internalized ligands from their receptor or the activation of lysosomal enzymes. The main player in endosomal acidification is the vacuolar ATPase (V-ATPase), a multi-subunit transmembrane complex that pumps protons from the cytoplasm to the lumen of organelles, or to the outside of the cell. The active V-ATPase is composed of two multi-subunit domains, the transmembrane V0 and the cytoplasmic V1. Here we found that the ratio of membrane associated V1/Vo varies along the endocytic pathway, the relative abundance of V1 being higher on late endosomes than on early endosomes, providing an explanation for the higher acidity of late endosomes. We also found that all membrane-bound V-ATPase subunits were associated with detergent resistant membranes (DRM) isolated from late endosomes, raising the possibility that association with lipid-raft like domains also plays a role in regulating the activity of the proton pump. In support of this, we found that treatment of cells with U18666A, a drug that leads to the accumulation of cholesterol in late endosomes, affected acidification of late endosome. Altogether our findings indicate that the activity of the vATPase in the endocytic pathway is regulated both by reversible association/dissociation and the interaction with specific lipid environments

Proteomic Analysis of S-Acylated Proteins in Human B Cells Reveals Palmitoylation of the Immune Regulators CD20 and CD23

S-palmitoylation is a reversible post-translational modification important for controlling the membrane targeting and function of numerous membrane proteins with diverse roles in signalling, scaffolding, and trafficking. We sought to identify novel palmitoylated proteins in B lymphocytes using acyl-biotin exchange chemistry, coupled with differential analysis by liquid-chromatography tandem mass spectrometry. In total, we identified 57 novel palmitoylated protein candidates from human EBV-transformed lymphoid cells. Two of them, namely CD20 and CD23 (low affinity immunoglobulin epsilon Fc receptor), are immune regulators that are effective/potential therapeutic targets for haematological malignancies, autoimmune diseases and allergic disorders. Palmitoylation of CD20 and CD23 was confirmed by heterologous expression of alanine mutants coupled with bioorthogonal metabolic labeling. This study demonstrates a new subset of palmitoylated proteins in B cells, illustrating the ubiquitous role of protein palmitoylation in immune regulation

Transport of Anthocyanins and other Flavonoids by the Arabidopsis ATP-Binding Cassette Transporter AtABCC2

Flavonoids have important developmental, physiological, and ecological roles in plants and are primarily stored in the large central vacuole. Here we show that both an ATP-binding cassette (ABC) transporter(s) and an H+-antiporter(s) are involved in the uptake of cyanidin 3-O-glucoside (C3G) by Arabidopsis vacuolar membrane-enriched vesicles. We also demonstrate that vesicles isolated from yeast expressing the ABC protein AtABCC2 are capable of MgATP-dependent uptake of C3G and other anthocyanins. The uptake of C3G by AtABCC2 depended on the co-transport of glutathione (GSH). C3G was not altered during transport and a GSH conjugate was not formed. Vesicles from yeast expressing AtABCC2 also transported flavone and flavonol glucosides. We performed ligand docking studies to a homology model of AtABCC2 and probed the putative binding sites of C3G and GSH through site-directed mutagenesis and functional studies. These studies identified residues important for substrate recognition and transport activity in AtABCC2, and suggest that C3G and GSH bind closely, mutually enhancing each other’s binding. In conclusion, we suggest that AtABCC2 along with possibly other ABCC proteins are involved in the vacuolar transport of anthocyanins and other flavonoids in the vegetative tissue of Arabidopsis

Functionalization of corroles: Formylcorroles

Porphyrins and metalloporphyrins can be readily functionalized at the meso-(5,10,15,20)-positions by using a variety of reagents, the Vilsmeier formylation reaction being universally regarded as the prototypical example. On the other hand, the literature shows that corroles and metallocorroles are unusually resistant to most functionalization procedures which are common to porphyrins. The first example of peripheral functionalization of the corrole ring is now reported. Octamethyl-corrole 1 readily reacts with the Vilsmeier reagent (POCl3/DMF), but the product is the 10-dimethylaminomethene derivative 2, and not the expected meso-formyl derivatives 3 or 4, as confirmed by a single crystal X-ray crystallographic study. Corrole 5, where the 10-meso-position is substituted, affords the similar product 6. When the reaction is extended to corrole 7 two different products, 8 and 9, are obtained wherein the substitution is performed at both 5- and 10-meso-positions. Unlike the case of porphyrins, no reaction occurs at the β-unsubstituted pyrrolic positions. Coordination of cobalt to the dimethylaminomethenylcorroles 2 and 5 causes both metalation and tautomerization/hydrolysis to afford the originally targeted meso-formylcorrolato complexes in almost quantitative yields. Attempts to formylate cobalt complex 14 using TFA/trimethylorthoformate affords the corresponding β-formyl complexes 15 and 16, the ratio of which depends on the reaction time. meso-Formylcorroles are also obtained by way of a new direct synthesis, described herein, using a prefunctionalized a,c-biladiene 22

New chemistry of oxophlorins (oxyporphyrins) and their pi-radicals

Syntheses of novel 15-substituted-oxophlorins via the MacDonald condensation of diformyl-dipyrroketones and 5-substituted-dipyrromethanes are described. The electronic and steric features of the 15-substituent enable facile control over the oxidation potential of the oxophlorins. Introduction of an electron-withdrawing group efficiently minimizes the formation of oxophlorin pi-radicals. Stabilization of neutral pi radicals is promoted by hyperconjugation with a 15-tert-butyl group. A sterically induced stabilization of a novel non-aromatic tautomer of oxophlorin, the so-called "iso-oxophlorin" is demonstrated. These species exist also as 15-iso-oxophlorins upon complexation to divalent metals. Radical formation, enhanced by mild oxidants such as K3FeCN6, yielded pure oligomers and stereospecific supramolecular arrays by radical dimerizations taking place at the 10- and 10'-positions, (C) 1999 Elsevier Science Ltd. All rights reserved