Regulating peroxisome–ER contacts via the ACBD5-VAPB tether by FFAT motif phosphorylation and GSK3β

Peroxisomes and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism. They form membrane contacts through interaction of the peroxisomal membrane protein ACBD5 (acyl-coenzyme A–binding domain protein 5) and the ER-resident protein VAPB (vesicle-associated membrane protein–associated protein B). ACBD5 binds to the major sperm protein domain of VAPB via its FFAT-like (two phenylalanines [FF] in an acidic tract) motif. However, molecular mechanisms, which regulate formation of these membrane contact sites, are unknown. Here, we reveal that peroxisome–ER associations via the ACBD5-VAPB tether are regulated by phosphorylation. We show that ACBD5-VAPB binding is phosphatase-sensitive and identify phosphorylation sites in the flanking regions and core of the FFAT-like motif, which alter interaction with VAPB—and thus peroxisome–ER contact sites—differently. Moreover, we demonstrate that GSK3β (glycogen synthase kinase-3 β) regulates this interaction. Our findings reveal for the first time a molecular mechanism for the regulation of peroxisome–ER contacts in mammalian cells and expand the current model of FFAT motifs and VAP interaction.     

Chirality matters: stereo-defined phosphorothioate linkages at the termini of small interfering RNAs improve pharmacology in vivo

A critical challenge for the successful development of RNA interference-based therapeutics therapeutics has been the enhancement of their in vivo metabolic stability. In therapeutically relevant, fully chemically modified small interfering RNAs (siRNAs), modification of the two terminal phosphodiester linkages in each strand of the siRNA duplex with phosphorothioate (PS) is generally sufficient to protect against exonuclease degradation in vivo. Since PS linkages are chiral, we systematically studied the properties of siRNAs containing single chiral PS linkages at each strand terminus. We report an efficient and simple method to introduce chiral PS linkages and demonstrate that Rp diastereomers at the 5′ end and Sp diastereomers at the 3′ end of the antisense siRNA strand improved pharmacokinetic and pharmacodynamic properties in a mouse model. In silico modeling studies provide mechanistic insights into how the Rp isomer at the 5′ end and Sp isomer at the 3′ end of the antisense siRNA enhance Argonaute 2 (Ago2) loading and metabolic stability of siRNAs in a concerted manner.     

Evidence for a small anion-selective channel in the cell wall of Mycobacterium bovis BCG besides a wide cation-selective pore.

Two channels were observed in extracts of whole Mycobacterium bovis BCG cells using organic solvents and detergents. The channels derived from organic solvent treatment had a single-channel conductance of about 4.0 nS in 1 M KCl in lipid bilayer membranes with properties similar to those of the channels discovered previously in Mycobacterium smegmatis and Mycobacterium chelonae. The channel was in its open configuration only at low transmembrane potentials. At higher voltages it switched to closed states that were almost impermeable for ions. Lipid bilayer experiments in the presence of detergent extracts of whole cells revealed another channel with a single-channel conductance of only 780 pS in 1 M KCl. Our results indicate that the mycolic acid layer of M. bovis BCG contains two channels, one is cation-selective and its permeability properties can be finely controlled by cell wall asymmetry or potentials. The other one is anion-selective, has a rather small single-channel conductance and is voltage-insensitive. The concentration of channel-forming proteins in the cell wall seems to be small, which is in agreement with the low cell wall permeability for hydrophilic solutes.     

Roles of non-catalytic subunits in gbetagamma-induced activation of class I phosphoinositide 3-kinase isoforms beta and gamma.

By using purified preparations we show that nanomolar concentrations of Gbetagamma significantly stimulated lipid kinase activity of phosphatidylinositol 3-kinase (PI3K) beta and PI3Kgamma in the presence as well as in the absence of non-catalytic subunits such as p85alpha or p101. Concomitantly, Gbetagamma stimulated autophosphorylation of the catalytic subunit of PI3Kgamma (EC(50), 30 nM; stoichiometry >/=0.6 mol of P(i)/mol of p110gamma), which also occurred in the absence of p101. Surprisingly, we found that p101 affected the lipid substrate preference of PI3Kgamma in its Gbetagamma-stimulated state. With phosphatidylinositol as substrate, p110gamma but not p101/p110gamma was significantly stimulated by Gbetagamma to form PI-3-phosphate (EC(50), 20 nM). The opposite situation was found when PI-4,5-bisphosphate served as substrate. Gbetagamma efficiently and potently (EC(50), 5 nM) activated the p101/p110gamma heterodimer but negligibly stimulated the p110gamma monomer to form PI-3,4,5-trisphosphate. However, this weak stimulatory effect on p110gamma was overcome by excess concentrations of Gbetagamma (EC(50), 100 nM). This finding is in accordance with the in vivo situation, where activated PI3K catalyzes the formation of PI-3,4,5-trisphosphate but not PI-3-phosphate. We conclude that p101 is responsible for PI-4, 5-bisphosphate substrate selectivity of PI3Kgamma by sensitizing p110gamma toward Gbetagamma in the presence of PI-4,5-P(2).     

Ancient Gene Duplication and Differential Gene Flow in Plastid Lineages: The GroEL/Cpn60 Example

Cryptomonads, small biflagellate algae, contain four different genomes. In addition to the nucleus, mitochondrion, and chloroplast is a fourth DNA-containing organelle the nucleomorph. Nucleomorphs result from the successive reduction of the nucleus of an engulfed phototrophic eukaryotic endosymbiont by a secondary eukaryotic host cell. By sequencing the chloroplast genome and the nucleomorph chromosomes, we identified a groEL homologue in the genome of the chloroplast and a related cpn60 in one of the nucleomorph chromosomes. The nucleomorph-encoded Cpn60 and the chloroplast-encoded GroEL correspond in each case to one of the two divergent GroEL homologues in the cyanobacterium Synechocystis sp. PCC6803. The coexistence of divergent groEL/cpn60 genes in different genomes in one cell offers insights into gene transfer from evolving chloroplasts to cell nuclei and convergent gene evolution in chlorophyll a/b versus chlorophyll a/c/phycobilin eukaryotic lineages. Received: 24 April 1998 / Accepted: 12 June 1998