The stoichiometry of P2X2/6 receptor heteromers depends on relative subunit expression levels

Fast synaptic transmission involves the operation of ionotropic receptors, which are often composed of at least two types of subunit. We have developed a method, based on atomic force microscopy imaging to determine the stoichiometry and subunit arrangement within ionotropic receptors. We showed recently that the P2X(2) receptor for ATP is expressed as a trimer but that the P2X(6) subunit is unable to oligomerize. In this study we addressed the subunit stoichiometry of heteromers containing both P2X(2) and P2X(6) subunits. We transfected tsA 201 cells with both P2X(2) and P2X(6) subunits, bearing different epitope tags. We manipulated the transfection conditions so that either P2X(2) or P2X(6) was the predominant subunit expressed. By atomic force microscopy imaging of isolated receptors decorated with antiepitope antibodies, we demonstrate that when expression of the P2X(2) subunit predominates, the receptors contain primarily 2 x P2X(2) subunits and 1 x P2X(6) subunit. In contrast, when the P2X(6) subunit predominates, the subunit stoichiometry of the receptors is reversed. Our results show that the composition of P2X receptor heteromers is plastic and dependent on the relative subunit expression levels. We suggest that this property of receptor assembly might introduce an additional layer of subtlety into P2X receptor signaling

Seipin oligomers can interact directly with AGPAT2 and lipin 1, physically scaffolding critical regulators of adipogenesis

This work was supported by a Merit Scholarship from the Islamic Development Bank (to M.M.U.T.), The Agency for Science, Technology and Research, Singapore (A*STAR) (M.F.M.S), the Medical Research Council (MRC) [NIRG GO800203 and Research Grant MR/L002620/1 (to J.J.R.), Program GrantG09000554 (to S.O.R)], The Wellcome Trust [078986/Z/06/Z (to S.O.R.)], the MRC Centre for Obesity and Related Metabolic Disorders (MRC-CORD) [GO600717] and the NIHR Comprehensive Biomedical Research Centre [CG50826].Peer reviewedPublisher PD

Analysis of naturally occurring mutations in the human lipodystrophy protein seipin reveals multiple potential pathogenic mechanisms

Peer reviewedPublisher PD

AFM imaging reveals the assembly of a P2X receptor complex containing P2X2, P2X4 and P2X6 subunits

Seven P2X purinergic receptor subunits have been identified: P2X1-P2X7. All except P2X6 assemble as homotrimers, and six heteromeric receptors (P2X1/2, P2X1/4, P2X1/5, P2X2/3, P2X2/6 and P2X4/6) have been described. In addition, P2X4 homomers associate with P2X2 or P2X7 homomers as dimers of trimers. The various P2X receptors show individual functional properties, suggesting distinct physiological roles. The overlapping expression of P2X2, P2X4 and P2X6 subunits has been shown in different cell types, and functional analysis of P2X receptors in Leydig cells suggests that the three subunits interact

Sar1 GTPase Activity Is Regulated by Membrane Curvature.

The majority of biosynthetic secretory proteins initiate their journey through the endomembrane system from specific subdomains of the endoplasmic reticulum. At these locations, coated transport carriers are generated, with the Sar1 GTPase playing a critical role in membrane bending, recruitment of coat components, and nascent vesicle formation. How these events are appropriately coordinated remains poorly understood. Here, we demonstrate that Sar1 acts as the curvature-sensing component of the COPII coat complex and highlight the ability of Sar1 to bind more avidly to membranes of high curvature. Additionally, using an atomic force microscopy-based approach, we further show that the intrinsic GTPase activity of Sar1 is necessary for remodeling lipid bilayers. Consistent with this idea, Sar1-mediated membrane remodeling is dramatically accelerated in the presence of its guanine nucleotide-activating protein (GAP), Sec23-Sec24, and blocked upon addition of guanosine-5'-[(β,γ)-imido]triphosphate, a poorly hydrolysable analog of GTP. Our results also indicate that Sar1 GTPase activity is stimulated by membranes that exhibit elevated curvature, potentially enabling Sar1 membrane scission activity to be spatially restricted to highly bent membranes that are characteristic of a bud neck. Taken together, our data support a stepwise model in which the amino-terminal amphipathic helix of GTP-bound Sar1 stably penetrates the endoplasmic reticulum membrane, promoting local membrane deformation. As membrane bending increases, Sar1 membrane binding is elevated, ultimately culminating in GTP hydrolysis, which may destabilize the bilayer sufficiently to facilitate membrane fission.This work was supported by grants from the NIH (GM110567 and GM088151 to AA). IM, RMH and JME were supported by a grant from the Biotechnology and Biological Sciences Research Council (BB/J018236/1). ERC is an Investigator of the Howard Hughes Medical Institute. We thank Elizabeth Miller for providing purified yeast COPII components, Subhanjan Mondal and Said Goueli at Promega Corporation for providing us access to the GTPase-Glo system ahead of release, and members of the Audhya lab for critically reading this manuscript.This is the final version of the article. It first appeared from the American Society for Biochemistry and Molecular Biology via http://dx.doi.org/10.1074/jbc.M115.67228

Syncollin is an antibacterial polypeptide

Funder: AstraZeneca; Id: http://dx.doi.org/10.13039/100004325Funder: David James Studentship, Department of Pharmacology, University of CambridgeAbstract: Syncollin is a 16‐kDa protein found predominantly in the zymogen granules of pancreatic acinar cells, with expression at lower levels in intestinal epithelial cells and neutrophils. Here, we used Strep‐tagged syncollin isolated from the supernatant of transiently transfected mammalian cells to test the hypothesis that syncollin has antibacterial properties, which might enable it to play a role in host defence in the gut and possibly elsewhere. We show that syncollin is an exceptionally thermostable protein with a circular dichroism spectrum consistent with a predominantly beta‐sheet structure. Syncollin binds to bacterial peptidoglycan and restricts the growth of representative Gram‐positive (Lactococcus lactis) and Gram‐negative (Escherichia coli) bacteria. Syncollin induces propidium iodide uptake into E. coli (but not L. lactis), indicating permeabilisation of the bacterial membrane. It also causes surface structural damage in both L. lactis and E. coli, as visualised by scanning electron microscopy. We propose that syncollin is a previously unidentified member of a large group of antimicrobial polypeptides that control the gut microbiome. Take Aways: Syncollin is a 16‐kDa protein found in pancreatic zymogen granules. Syncollin is highly thermostable and has a predominantly beta‐sheet structure. Syncollin binds peptidoglycan and restricts the growth of L. lactis and E. coli. Syncollin causes propidium iodide uptake into E. coli (but not L. lactis). Syncollin causes surface structural damage in both L. lactis and E. coli

Exocytotic fusion pores are composed of both lipids and proteins.

During exocytosis, fusion pores form the first aqueous connection that allows escape of neurotransmitters and hormones from secretory vesicles. Although it is well established that SNARE proteins catalyze fusion, the structure and composition of fusion pores remain unknown. Here, we exploited the rigid framework and defined size of nanodiscs to interrogate the properties of reconstituted fusion pores, using the neurotransmitter glutamate as a content-mixing marker. Efficient Ca(2+)-stimulated bilayer fusion, and glutamate release, occurred with approximately two molecules of mouse synaptobrevin 2 reconstituted into ∼6-nm nanodiscs. The transmembrane domains of SNARE proteins assumed distinct roles in lipid mixing versus content release and were exposed to polar solvent during fusion. Additionally, tryptophan substitutions at specific positions in these transmembrane domains decreased glutamate flux. Together, these findings indicate that the fusion pore is a hybrid structure composed of both lipids and proteins.We thank Gerhard Wagner for providing the MSP∆1D1H4-H6 plasmid. This study was supported by a grant from the US National Institutes of Health (MH061876). H.B. is supported by a postdoctoral fellowship from Human Frontier Science Program. B.C. and M.P.G are supported by funding from the US National Institutes of Health (R01 GM084140). P.J. is supported by Kidney Research UK. J.M.E. is supported by the Biotechnology and Biological Sciences Research Council (BB/J018236/1) and Kidney Research UK. E.R.C. is supported as an Investigator of the Howard Hughes Medical Institute.This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/nsmb.314

The Vesicle Priming Factor CAPS Functions as a Homodimer via C2 Domain Interactions to Promote Regulated Vesicle Exocytosis.

Neurotransmitters and peptide hormones are secreted by regulated vesicle exocytosis. CAPS (also known as CADPS) is a 145-kDa cytosolic and peripheral membrane protein required for vesicle docking and priming steps that precede Ca2+-triggered vesicle exocytosis. CAPS binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and SNARE proteins and is proposed to promote SNARE protein complex assembly for vesicle docking and priming. We characterized purified soluble CAPS as mainly monomer in equilibrium with small amounts of dimer. However, the active form of CAPS bound to PC12 cell membranes or to liposomes containing PI(4,5)P2 and Q-SNARE proteins was mainly dimer. CAPS dimer formation required its C2 domain based on mutation or deletion studies. Moreover, C2 domain mutations or deletions resulted in a loss of CAPS function in regulated vesicle exocytosis, indicating that dimerization is essential for CAPS function. Comparison of the CAPS C2 domain to a structurally defined Munc13-1 C2A domain dimer revealed conserved residues involved in CAPS dimerization. We conclude that CAPS functions as a C2 domain-mediated dimer in regulated vesicle exocytosis. The unique tandem C2-PH domain of CAPS may serve as a PI(4,5)P2-triggered switch for dimerization. CAPS dimerization may be coupled to oligomeric SNARE complex assembly for vesicle docking and priming