Membrane-deformation ability of ANKHD1 is involved in the early endosome enlargement

Ankyrin-repeat domains (ARDs) are conserved in large numbers of proteins. ARDs are composed of various numbers of ankyrin repeats (ANKs). ARDs often adopt curved structures reminiscent of the Bin-Amphiphysin-Rvs (BAR) domain, which is the dimeric scaffold for membrane tubulation. BAR domains sometimes have amphipathic helices for membrane tubulation and vesiculation. However, it is unclear whether ARD-containing proteins exhibit similar membrane deformation properties. We found that the ARD of ankyrin repeat and KH domain-containing protein 1 (ANKHD1) dimerizes and deforms membranes into tubules and vesicles. Among 25 ANKs of ANKHD1, the first 15 ANKs can form a dimer, and the latter 10 ANKs enable membrane tubulation and vesiculation through an adjacent amphipathic helix and a predicted curved structure with a positively charged surface, analogous to BAR domains. Knockdown and localization of ANKHD1 suggested its involvement in the negative regulation of early endosome enlargement owing to its membrane vesiculation

Measurement of caveolin-1 densities in the cell membrane for quantification of caveolar deformation after exposure to hypotonic membrane tension

Caveolae are abundant flask-shaped invaginations of plasma membranes that buffer membrane tension through their deformation. Few quantitative studies on the deformation of caveolae have been reported. Each caveola contains approximately 150 caveolin-1 proteins. In this study, we estimated the extent of caveolar deformation by measuring the density of caveolin-1 projected onto a two-dimensional (2D) plane. The caveolin-1 in a flattened caveola is assumed to have approximately one-quarter of the density of the caveolin-1 in a flask-shaped caveola. The proportion of one-quarter-density caveolin-1 increased after increasing the tension of the plasma membrane through hypo-osmotic treatment. The one-quarter-density caveolin-1 was soluble in detergent and formed a continuous population with the caveolin-1 in the caveolae of cells under isotonic culture. The distinct, dispersed lower-density caveolin-1 was soluble in detergent and increased after the application of tension, suggesting that the hypo-osmotic tension induced the dispersion of caveolin-1 from the caveolae, possibly through flattened caveolar intermediates.Peer reviewe

Importance of the conserved nucleotides around the tRNA-like structure of Escherichia coli transfer-messenger RNA for protein tagging

A bacterial RNA functioning as both tRNA and mRNA, transfer-messenger RNA (tmRNA) rescues stalled ribosomes and clears the cell of incomplete polypeptides. For function, Escherichia coli tmRNA requires an elaborate interplay between a tRNA-like structure and an internal mRNA domain that are connected by a 295 nt long compact secondary structure. The tRNA-like structure is surrounded by 16 unpaired nt, including 10 residues that are >95% conserved among the known 140 tmRNA sequences. All these residues were mutated to define their putative role(s) in trans-translation. Both the extent of aminoacylation and the alanine incorporation into the tag sequence, reflecting the two functions of tmRNA, were measured in vitro for all variants. As anticipated from the low sequence conservation, mutating positions 8–12 and position 15 affects neither aminoacylation nor protein tagging. Mutating a set of two conserved positions 13 and 14 abolishes both functions. Probing the solution conformation indicates that this defective mutant adopts an alternate conformation of its acceptor stem that is no more aminoacylatable, and thus inactive in protein tagging. Selected point mutations at the conserved nucleotide stretches 16–20 and 333–335 seriously impair protein tagging with only minor changes in their solution conformations and aminoacylation. Point mutations at conserved positions 19 and 334 abolish trans-translation and 70S ribosome binding, although retaining nearly normal aminoacylation capacities. Two proteins that are known to interact with tmRNA were purified, and their interactions with the defective RNA variants were examined in vitro. Based on phylogenetic and functional data, an additional structural motif consisting of a quartet composed of non-Watson–Crick base pairs 5′-YGAC-3′:5′-GGAC-3′ involving some of the conserved nucleotides next to the tRNA-like portion is proposed. Overall, the highly conserved nucleotides around the tRNA-like portion are maintained for both structural and functional requirements during evolution

Regulation of caveolae through cholesterol-depletion dependent tubulation by PACSIN2/Syndapin II

The membrane shaping ability of PACSIN2 via its FCH-BAR (F-BAR) domain has been shown to be essential for caveolar morphogenesis, presumably through the shaping of the caveolar neck. Caveolar membrane contains abundant levels of cholesterol. However, the role of cholesterol in PACSIN2-mediated membrane deformation remains unclear. We show that the binding of PACSIN2 to the membrane could be negatively regulated by the amount of cholesterol in the membrane. We prepared a reconstituted membrane based on the lipid composition of caveolae. The reconstituted membrane with cholesterol had a weaker affinity to the F-BAR domain of PACSIN2 than the membrane without cholesterol, presumably due to a decrease in electrostatic charge density. Consistently, the acute depletion of cholesterol from the plasma membrane resulted in the appearance of PACSIN2-localized tubules with caveolin-1 at their tips, suggesting that the presence of cholesterol inhibited the prominent membrane tubulation by PACSIN2. The tubules induced by PACSIN2 were suggested to be an intermediate of caveolae endocytosis. Consistently, the removal of caveolae from the plasma membrane upon cholesterol depletion was diminished in the cells deficient in PACSIN2. These data suggested that PACSIN2 mediated the caveolae internalization dependently on the amount of cholesterol at the plasma membrane, providing a possible mechanism for the cholesterol-dependent regulation of caveolae

TRPV4 channel activity is modulated by direct interaction of the ankyrin domain to PI(4,5)P2

[プレスリリース]バイオサイエンス研究科分子医学細胞生物学研究室の末次志郎教授らの研究グループが生理的に重要なイオンを運ぶ通り道TRPV4の新たな制御機構を解明（2014/09/29）Mutations in the ankyrin repeat domain (ARD) of ​TRPV4 are responsible for several channelopathies, including Charcot–Marie–Tooth disease type 2C and congenital distal and scapuloperoneal spinal muscular atrophy. However, the molecular pathogenesis mediated by these mutations remains elusive, mainly due to limited understanding of the ​TRPV4 ARD function. Here we show that phosphoinositide binding to the ​TRPV4 ARD leads to suppression of the channel activity. Among the phosphoinositides, phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) most potently binds to the ​TRPV4 ARD. The crystal structure of the ​TRPV4 ARD in complex with ​inositol-1,4,5-trisphosphate, the head-group of PI(4,5)P2, and the molecular-dynamics simulations revealed the PI(4,5)P2-binding amino-acid residues. The ​TRPV4 channel activities were increased by titration or hydrolysis of membrane PI(4,5)P2. Notably, disease-associated ​TRPV4 mutations that cause a gain-of-function phenotype abolished PI(4,5)P2 binding and PI(4,5)P2 sensitivity. These findings identify ​TRPV4 ARD as a lipid-binding domain in which interactions with PI(4,5)P2 normalize the channel activity in ​TRPV4