Initial Steps in the Assembly of the Vacuole-Type H+-ATPase1

The plant vacuole is acidified by a complex multimeric enzyme, the vacuole-type H+-ATPase (V-ATPase). The initial association of ATPase subunits on membranes was studied using an in vitro assembly assay. The V-ATPase assembled onto microsomes when V-ATPase subunits were supplied. However, when the A or B subunit or the proteolipid were supplied individually, only the proteolipid associated with membranes. By using poly(A+) RNA depleted in the B subunit and proteolipid subunit mRNA, we demonstrated A subunit association with membranes at substoichiometric amounts of the B subunit or the 16-kD proteolipid. These data suggest that poly(A+) RNA-encoded proteins are required to catalyze the A subunit membrane assembly. Initial events were further studied by in vivo protein labeling. Consistent with a temporal ordering of V-ATPase assembly, membranes contained only the A subunit at early times; at later times both the A and B subunits were found on the membranes. A large-mass ATPase complex was not efficiently formed in the absence of membranes. Together, these data support a model whereby the A subunit is first assembled onto the membrane, followed by the B subunit.

Stepwise assembly of the lipid-linked oligosaccharide in the endoplasmic reticulum of Saccharomyces cerevisiae: identification of the ALG9 gene encoding a putative mannosyl transferase.

The core oligosaccharide Glc3Man9GlcNAc2 is assembled at the membrane of the endoplasmic reticulum on the lipid carrier dolichyl pyrophosphate and transferred to selected asparagine residues of nascent polypeptide chains. This transfer is catalyzed by the oligosaccharyl transferase complex. Based on the synthetic phenotype of the oligosaccharyl transferase mutation wbp1 in combination with a deficiency in the assembly pathway of the oligosaccharide in Saccharomyces cerevisiae, we have identified the novel ALG9 gene. We conclude that this locus encodes a putative mannosyl transferase because deletion of the gene led to accumulation of lipid-linked Man6GlcNAc2 in vivo and to hypoglycosylation of secreted proteins. Using an approach combining genetic and biochemical techniques, we show that the assembly of the lipid-linked core oligosaccharide in the lumen of the endoplasmic reticulum occurs in a stepwise fashion.

Characterization of a second secreted IgE isoform and identification of an asymmetric pathway of IgE assembly.

A number of alternatively spliced epsilon transcripts have been detected in IgE-producing B cells, in addition to the mRNAs encoding the classical membrane and secreted IgE heavy (H) chains. In a recent study, we examined the protein products of three of these alternatively spliced isoforms and found that they are intracellularly retained and degraded because of their inability to assemble into complete IgE molecules. We have now similarly examined a more recently described epsilon mRNA species that is generated by splicing between a donor splice site immediately upstream of the stop codon in the H-chain constant region exon 4 (CH4) and an acceptor site located in the 3' part of the second membrane exon. We show that this isoform is efficiently secreted by both plasma cells and B lymphocytes and therefore represents a second secreted IgE isoform (epsilon S2). The epsilon S2 H chain is only six amino acids longer than the classical secreted Ig H chain (epsilon S1) and contains a C-terminal cysteine, which is a characteristic sequence feature of mu and alpha H chains. However, unlike IgM and IgA, the epsilon S2 C-terminal cysteine (Cys-554) does not induce polymerization of H2L2 molecules (where L is light chain), but rather creates a disulfide bond between the two H chains that increases the rate of association into covalently bound H2L2 monomers. This C-terminal cysteine also does not function as an intracellular retention element because the epsilon S2 isoform was secreted in amounts equal to that of the epsilon S1, both in B lymphocytes and in plasma cells. The epsilon S2 H chains secreted by B lymphocytes differed from the epsilon S1 H chains in the extent of glycosylation. Interestingly, a difference in glycosylation between B-lymphocytes and plasma cells was also noted for both isoforms. The presence of the Cys-554 also allowed the identification of a distinctive asymmetric pathway of IgE assembly, common to both types of epsilon H chains.

Inhibition of function in Xenopus oocytes of the inwardly rectifying G-protein-activated atrial K channel (GIRK1) by overexpression of a membrane-attached form of the C-terminal tail.

Coexpression in Xenopus oocytes of the inwardly rectifying guanine nucleotide binding (G)-protein-gated K channel GIRK1 with a myristoylated modification of the (putative) cytosolic C-terminal tail [GIRK1 aa 183-501 fused in-frame to aa 1-15 of p60src and denoted src+ (183-501)] leads to a high degree of inhibition of the inward G-protein-gated K+ current. The nonmyristoylated segment, src- (183-501), is not active. Although some interference with assembly is not precluded, the evidence indicates that the main mechanism of inhibition is interference with functional activation of the channel by G proteins. In part, the tail functions as a blocking particle similar to a "Shaker ball"; it may also function by competing for the available supply of free G beta gamma liberated by hormone activation of a seven-helix receptor. The non-G-protein-gated weak inward rectifier ROMK1 is less effectively inhibited, and a Shaker K channel was not inhibited. Immunological assays show the presence of a high concentration of src+ (183-501) in the plasma membrane and the absence of any membrane forms for the nonmyristoylated segment.

Regulated assembly of tight junctions by protein kinase C.

We have previously shown that protein phosphorylation plays an important role in the sorting and assembly of tight junctions. We have now examined in detail the role of protein kinases in intercellular junction biogenesis by using a combination of highly specific and broad-spectrum inhibitors that act by independent mechanisms. Our data indicate that protein kinase C (PKC) is required for the proper assembly of tight junctions. Low concentrations of the specific inhibitor of PKC, calphostin C, markedly inhibited development of transepithelial electrical resistance, a functional measure of tight-junction biogenesis. The effect of PKC inhibitors on the development of tight junctions, as measured by resistance, was paralleled by a delay in the sorting of the tight-junction protein, zona occludens 1 (ZO-1), to the tight junction. The assembly of desmosomes and the adherens junction were not detectably affected, as determined by immunocytochemical analysis. In addition, ZO-1 was phosphorylated subsequent to the initiation of cell-cell contact, and treatment with calphostin C prevented approximately 85% of the phosphorylation increase. Furthermore, in vitro measurements indicate that ZO-1 may be a direct target of PKC. Moreover, membrane-associated PKC activity more than doubled during junction assembly, and immunocytochemical analysis revealed a pool of PKC zeta that appeared to colocalize with ZO-1 at the tight junction. A preformed complex containing ZO-1, ZO-2, p130, as well as 330- and 65-kDa phosphoproteins was detected by coimmunoprecipitation in both the presence and absence of cell-cell contact. Identity of the 330- and 65-kDa phosphoproteins remains to be determined, but the 65-kDa protein may be occludin. The mass of this complex and the incorporation of ZO-1 into the Triton X-100-insoluble cytoskeleton were not PKC dependent.

Performance Assessment of a Polymer Electrolyte Membrane Electrochemical Reactor under Alkaline Conditions – A Case Study with the Electrooxidation of Alcohols

A novel polymer electrolyte membrane electrochemical reactor (PEMER) configuration has been employed for the direct electrooxidation of propargyl alcohol (PGA), a model primary alcohol, towards its carboxylic acid derivatives in alkaline medium. The PEMER configuration comprised of an anode and cathode based on nanoparticulate Ni and Pt electrocatalysts, respectively, supported on carbonaceous substrates. The electrooxidation of PGA was performed in 1.0 M NaOH, where a cathode based on a gas diffusion electrode was manufactured for the reduction of oxygen in alkaline conditions. The performance of a novel alkaline anion-exchange membrane based on Chitosan (CS) and Poly(vinyl) alcohol (PVA) in a 50:50 composition ratio doped with a 5 wt.% of poly (4-vinylpyridine) organic ionomer cross-linked, methyl chloride quaternary salt resin (4VP) was assessed as solid polymer electrolyte. The influence of 4VP anionic ionomer loading of 7, 12 and 20 wt.% incorporated into the electrocatalytic layers was examined by SEM and cyclic voltammetry (CV) upon the optimisation of the electroactive area, the mechanical stability and cohesion of the catalytic ink onto the carbonaceous substrate for both electrodes. The performance of the 4VP/CS:PVA membrane was compared with the commercial alkaline anion-exchange membrane FAA −a membrane generally used in direct alcohol alkaline fuel cells- in terms of polarisation plots in alkaline conditions. Furthermore, preparative electrolyses of the electrooxidation of PGA was performed under alkaline conditions of 1 M NaOH at constant current density of 20 mA cm−2 using a PEMER configuration to provide proof of the principle of the feasibility of the electrooxidation of other alcohols in alkaline media. PGA conversion to Z isomers of 3-(2-propynoxy)-2-propenoic acid (Z-PPA) was circa 0.77, with average current efficiency of 0.32. Alkaline stability of the membranes within the PEMER configuration was finally evaluated after the electrooxidation of PGA.

H-ras, K-ras, and inner plasma membrane raft proteins operate in nanoclusters with differential dependence on the actin cytoskeleton

Plasma membrane compartmentalization imposes lateral segregation on membrane proteins that is important for regulating signal transduction. We use computational modeling of immunogold spatial point patterns on intact plasma membrane sheets to test different models of inner plasma membrane organization. We find compartmentalization at the nanoscale level but show that a classical raft model of preexisting stable domains into which lipid raft proteins partition is incompatible with the spatial point patterns generated by the immunogold labeling of a palmitoylated raft marker protein. Rather, approximate to 30% of the raft protein exists in cholesterol-dependent nanoclusters, with approximate to 70% distributed as monomers. The cluster/monomer ratio (number of proteins in clusters/number of proteins outside clusters) is independent of expression level. H-rasG12V and K-rasG12V proteins also operate in nanoclusters with fixed cluster/monomer ratios that are independent of expression level. Detailed calibration of the immunogold imaging protocol suggests that radii of raft and RasG12V protein nanoclusters may be as small as 11 and 6 nm, respectively, and shows that the nanoclusters contain small numbers (6.0-7.7) of proteins. Raft nanoclusters do not form if the actin cytoskeleton is disassembled. The formation of K-rasG12V but not H-rasG12V nanoclusters also is actin-dependent. K-rasG12V but not H-rasG12V signaling is abrogated by actin cytoskeleton disassembly, which shows that nanoclustering is critical for Ras function. These findings argue against stable preexisting domains on the inner plasma membrane in favor of dynamic actively regulated nanoclusters similar to those proposed for the outer plasma membrane. RasG12V nanoclusters may facilitate the assembly of essential signal transduction complexes.

Ras plasma membrane signalling platforms

The plasma membrane is a complex, dynamic structure that provides platforms for the assembly of many signal transduction pathways. These platforms have the capacity to impose an additional level of regulation on cell signalling networks. In this review, we will consider specifically how Ras proteins interact with the plasma membrane. The focus will be on recent studies that provide novel spatial and dynamic insights into the micro-environments that different Ras proteins utilize for signal transduction. We will correlate these recent studies suggesting Ras proteins might operate within a heterogeneous plasma membrane with earlier biochemical work on Ras signal transduction.

Molecular dissection of the Munc18c/syntaxin4 interaction: Implications for regulation of membrane trafficking

Sec1p/Munc18 (SM) proteins are believed to play an integral role in vesicle transport through their interaction with SNAREs. Different SM proteins have been shown to interact with SNAREs via different mechanisms, leading to the conclusion that their function has diverged. To further explore this notion, in this study, we have examined the molecular interactions between Munc18c and its cognate SNAREs as these molecules are ubiquitously expressed in mammals and likely regulate a universal plasma membrane trafficking step. Thus, Munc18c binds to monomeric syntaxin4 and the N-terminal 29 amino acids of syntaxin4 are necessary for this interaction. We identified key residues in Munc18c and syntaxin4 that determine the N-terminal interaction and that are consistent with the N-terminal binding mode of yeast proteins Sly1p and Sed5p. In addition, Munc18c binds to the syntaxin4/SNAP23/VAMP2 SNARE complex. Pre-assembly of the syntaxin4/Munc18c dimer accelerates the formation of SNARE complex compared to assembly with syntaxin4 alone. These data suggest that Munc18c interacts with its cognate SNAREs in a manner that resembles the yeast proteins Sly1p and Sed5p rather than the mammalian neuronal proteins Munc18a and syntaxin1a. The Munc18c-SNARE interactions described here imply that Munc18c could play a positive regulatory role in SNARE assembly.

Analytical electron microscopy of proton exchange membrane fuel cells

Microtome sections of proton exchange membrane cells produce a wide range of information ranging from macroscopic distribution of components through specimens in which the detailed distribution of catalyst particles can be observed. Using modern data management practices it is possible to combine information at different scales and correlate processing and performance data. Analytical electron microscopy reveals the compositional variations across used cells at the electrolyte/electrode interface. In particular analytical techniques indicate that sulphur concentrations are likely to diminish at the interface Nafion/anode interface. © 2006 Elsevier B.V. All rights reserved.

A proteo-liposome system for the analysis of the intracellular interactome of membrane proteins using amyloid precursor protein as a model

Transmembrane proteins play crucial roles in many important physiological processes. The intracellular domain of membrane proteins is key for their function by interacting with a wide variety of cytosolic proteins. It is therefore important to examine this interaction. A recently developed method to study these interactions, based on the use of liposomes as a model membrane, involves the covalent coupling of the cytoplasmic domains of membrane proteins to the liposome membrane. This allows for the analysis of interaction partners requiring both protein and membrane lipid binding. This thesis further establishes the liposome recruitment system and utilises it to examine the intracellular interactome of the amyloid precursor protein (APP), most well-known for its proteolytic cleavage that results in the production and accumulation of amyloid beta fragments, the main constituent of amyloid plaques in Alzheimer’s disease pathology. Despite this, the physiological function of APP remains largely unclear. Through the use of the proteo-liposome recruitment system two novel interactions of APP’s intracellular domain (AICD) are examined with a view to gaining a greater insight into APP’s physiological function. One of these novel interactions is between AICD and the mTOR complex, a serine/threonine protein kinase that integrates signals from nutrients and growth factors. The kinase domain of mTOR directly binds to AICD and the N-terminal amino acids of AICD are crucial for this interaction. The second novel interaction is between AICD and the endosomal PIKfyve complex, a lipid kinase involved in the production of phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2) from phosphatidylinositol-3-phosphate, which has a role in controlling ensdosome dynamics. The scaffold protein Vac14 of the PIKfyve complex binds directly to AICD and the C-terminus of AICD is important for its interaction with the PIKfyve complex. Using a recently developed intracellular PI(3,5)P2 probe it is shown that APP controls the formation of PI(3,5)P2 positive vesicular structures and that the PIKfyve complex is involved in the trafficking and degradation of APP. Both of these novel APP interactors have important implications of both APP function and Alzheimer’s disease. The proteo-liposome recruitment method is further validated through its use to examine the recruitment and assembly of the AP-2/clathrin coat from purified components to two membrane proteins containing different sorting motifs. Taken together this thesis highlights the proteo-liposome recruitment system as a valuable tool for the study of membrane proteins intracellular interactome. It allows for the mimicking of the protein in its native configuration therefore identifying weaker interactions that are not detected by more conventional methods and also detecting interactions that are mediated by membrane phospholipids.

Actin dynamics provides membrane tension to merge fusing vesicles into the plasma membrane

Vesicle fusion is executed via formation of an Ω-shaped structure (Ω-profile), followed by closure (kiss-and-run) or merging of the Ω-profile into the plasma membrane (full fusion). Although Ω-profile closure limits release but recycles vesicles economically, Ω-profile merging facilitates release but couples to classical endocytosis for recycling. Despite its crucial role in determining exocytosis/endocytosis modes, how Ω-profile merging is mediated is poorly understood in endocrine cells and neurons containing small ∼30-300 nm vesicles. Here, using confocal and super-resolution STED imaging, force measurements, pharmacology and gene knockout, we show that dynamic assembly of filamentous actin, involving ATP hydrolysis, N-WASP and formin, mediates Ω-profile merging by providing sufficient plasma membrane tension to shrink the Ω-profile in neuroendocrine chromaffin cells containing ∼300 nm vesicles. Actin-directed compounds also induce Ω-profile accumulation at lamprey synaptic active zones, suggesting that actin may mediate Ω-profile merging at synapses. These results uncover molecular and biophysical mechanisms underlying Ω-profile merging.

How curvature-generating proteins build scaffolds on membrane nanotubes

Bin/Amphiphysin/Rvs (BAR) domain proteins control the curvature of lipid membranes in endocytosis, trafficking, cell motility, the formation of complex sub-cellular structures, and many other cellular phenomena. They form three-dimensional assemblies, which act as molecular scaffolds to reshape the membrane and alter its mechanical properties. It is unknown, however, how a protein scaffold forms and how BAR domains interact in these assemblies at protein densities relevant for a cell. In this work, we employ various experimental, theoretical and simulation approaches to explore how BAR proteins organize to form a scaffold on a membrane nanotube. By combining quantitative microscopy with analytical modeling, we demonstrate that a highly curving BAR protein endophilin nucleates its scaffolds at the ends of a membrane tube, contrary to a weaker curving protein centaurin, which binds evenly along the tube’s length. Our work implies that the nature of local protein-membrane interactions can affect the specific localization of proteins on membrane-remodeling sites. Furthermore, we show that amphipathic helices are dispensable in forming protein scaffolds. Finally, we explore a possible molecular structure of a BAR-domain scaffold using coarse-grained molecular dynamics simulations. Together with fluorescence microscopy, the simulations show that proteins need only to cover 30–40% of a tube’s surface to form a rigid assembly. Our work provides mechanical and structural insights into the way BAR proteins may sculpt the membrane as a high-order cooperative assembly in important biological processes. 

Epitaxially Grown Strained Pentacene Thin Film on Graphene Membrane

Organic-graphene system has emerged as a new platform for various applications such as flexible organic photovoltaics and organic light emitting diodes. Due to its important implication in charge transport, the study and reliable control of molecular packing structures at the graphene-molecule interface are of great importance for successful incorporation of graphene in related organic devices. Here, an ideal membrane of suspended graphene as a molecular assembly template is utilized to investigate thin-film epitaxial behaviors. Using transmission electron microscopy, two distinct molecular packing structures of pentacene on graphene are found. One observed packing structure is similar to the well-known bulk-phase, which adapts a face-on molecular orientation on graphene substrate. On the other hand, a rare polymorph of pentacene crystal, which shows significant strain along the c-axis, is identified. In particular, the strained film exhibits a specific molecular orientation and a strong azimuthal correlation with underlying graphene. Through ab initio electronic structure calculations, including van der Waals interactions, the unusual polymorph is attributed to the strong graphene-pentacene interaction. The observed strained organic film growth on graphene demonstrates the possibility to tune molecular packing via graphene-molecule interactions.