Assembly of signaling machinery at the postsynaptic membrane.

The postsynaptic membrane and the subsynaptic cell compartment are specialized for inter- and intracellular signaling. Recent work has focused on the role of synaptic activity in regulating the surface distribution of neurotransmitter receptors. In addition, several components of secondary signaling pathways involved in the long-term regulation of synaptic efficacy have been identified.     

Localization of polyphosphates at the outside of the yeast cell plasma membrane

Under appropriate experimental conditions toluidine blue is bound to the yeast cell surface, without penetrating into the cells. Based on experimental observations it is highly probable that the dye is bound to polyphosphates, localized outside the plasma membrane. The probable localization of polyphosphates outside the plasma membrane is important in the context of the proposed involvement of polyphosphates in glucose transport in yeast.     

Functional patchworking at the plasma membrane

Lipids and proteins are not evenly distributed within the plasma membrane (PM), but instead segregate laterally into many specialized microdomains whose functional relevance is not clear. In this issue, Busto et al ( 2018 ) demonstrate that substrate flux through a nutrient transporter drives the lateral relocation of the transporter between specific microdomains at the yeast PM, suggesting that regulating the lateral plasma membrane compartmentalization for individual proteins could be a general process for cellular response to environmental conditions.     

Live cell micropatterning reveals the dynamics of signaling complexes at the plasma membrane

Interactions of proteins in the plasma membrane are notoriously challenging to study under physiological conditions. We report in this paper a generic approach for spatial organization of plasma membrane proteins into micropatterns as a tool for visualizing and quantifying interactions with extracellular, intracellular, and transmembrane proteins in live cells. Based on a protein-repellent poly(ethylene glycol) polymer brush, micropatterned surface functionalization with the HaloTag ligand for capturing HaloTag fusion proteins and RGD peptides promoting cell adhesion was devised. Efficient micropatterning of the type I interferon (IFN) receptor subunit IFNAR2 fused to the HaloTag was achieved, and highly specific IFN binding to the receptor was detected. The dynamics of this interaction could be quantified on the single molecule level, and IFN-induced receptor dimerization in micropatterns could be monitored. Assembly of active signaling complexes was confirmed by immunostaining of phosphorylated Janus family kinases, and the interaction dynamics of cytosolic effector proteins recruited to the receptor complex were unambiguously quantified by fluorescence recovery after photobleaching.     

Secretion of Plasmodium falciparum rhoptry protein into the plasma membrane of host erythrocytes

The rhoptry is an organelle of the malarial merozoite which has been suggested to play a role in parasite invasion of its host cell, the erythrocyte. A monoclonal antibody selected for reactivity with this organelle identifies a parasite synthesized protein of 110 kD. From biosynthetic labeling experiments it was demonstrated that the protein is synthesized midway through the erythrocytic cycle (the trophozoite stage) but immunofluorescence indicates the protein is not localized in the organelle until the final stage (segmenter stage) of intraerythrocytic development. Immunoelectron microscopy shows that the protein is localized in the matrix of the rhoptry organelle and on membranous whorls secreted from the merozoite. mAb recognition of the protein is dithiothreitol (DTT) labile, indicating that the conformation of the epitope is dependent on a disulfide linkage. During erythrocyte reinvasion by the extracellular merozoite, immunofluorescence shows the rhoptry protein discharging from the merozoite and spreading around the surface of the erythrocyte. The protein is located in the plasma membrane of the newly invaded erythrocyte. These studies suggest that the 110-kD rhoptry protein is inserted into the membrane of the host erythrocyte during merozoite invasion.     

The ESCRT machinery: From the plasma membrane to endosomes and back again

Abstract

The manipulation and reorganization of lipid bilayers are required for diverse cellular processes, ranging from organelle biogenesis to cytokinetic abscission, and often involves transient membrane disruption. A set of membrane-associated proteins collectively known as the endosomal sorting complex required for transport (ESCRT) machinery has been implicated in membrane scission steps, which transform a single, continuous bilayer into two distinct bilayers, while simultaneously segregating cargo throughout the process. Components of the ESCRT pathway, which include 5 distinct protein complexes and an array of accessory factors, each serve discrete functions. This review focuses on the molecular mechanisms by which the ESCRT proteins facilitate cargo sequestration and membrane remodeling and highlights their unique roles in cellular homeostasis.     

Electrostatic interactions at the plasma membrane.

Hydrogen-ion titration has been used to detect the presence of charged groups on the human red-cell plasma membrane. The findings are discussed in terms of the effect of the local environment on electrostatic interactions between the charged groups.     

Protein quality control at the plasma membrane

Cellular proteostasis (or protein homeostasis) depends on the timely folding and disposal of conformationally damaged polypeptides during their life span at all subcellular locations. This process is particularly important for membrane proteins confined to the cell surface with crucial regulatory role in cellular homoeostasis and intercellular communication. Accumulating evidences indicate that membrane proteins exported from the endoplasmic reticulum (ER) are subjected to peripheral quality control (QC) along the late secretory and endocytic pathways, as well as at the plasma membrane (PM). Recently identified components of the PM QC recognition and effector mechanisms responsible for ubiquitination and lysosomal degradation of conformationally damaged PM proteins uncovered striking similarities to and differences from that of the ER QC machinery. Possible implications of the peripheral protein QC activity in phenotypic modulation of conformational diseases are also outlined.     

Iron at the cell surface controls both DNA synthesis and plasma membrane redox system

Summary Addition of the impermeable iron II chelator bathophenanthroline disulfonate (BPS) to cultured Chinese hamster lung fibroblast (CCL 39 cells) inhibits DNA synthesis but not protein synthesis or cytoplasmic alkalinization, when cell growth is initiated with growth factors such as EGF plus insulin, thrombin, or ceruloplasmin. The BPS inhibition is reversed by addition of stoichiometric ferrous iron at stoichiometric concentration. BPS does not inhibit cell growth stimulated by fetal calf serum. The effect of the BPS differs from the inhibition of growth by hydroxyurea which acts on the ribonucleotide reductase. The BPS treatment leads to release of iron from the cells as determined by BPS iron II complex formation over 90 min. Cells treated with BPS just during starvation period cannot re-initiate DNA synthesis after mitogen stimulation even if BPS is removed from the medium and cells are previously washed. BPS treatment also inhibits transplasma membrane electron which is restored by incubation of cells with 10 M ferric ammonium citrate. Growth factor stimulation of DNA synthesis is restored by addition of 1 M ferrous ammonium sulfate or ferric ammonium citrate, or 0.1 M diferric transferrin. Copper, cobalt, nickel, zinc, gallium, aluminum, or apotransferrin cannot restore the activity. The BPS effect is consistent with removal of iron from a site on the cell surface which controls electron transport and DNA synthesis.Abbreviations BCS bathocuproine disulfonate - BPS bathophenan-throline disulfonate - CUP ceruloplasmin - FCS fetal calf serum - Fe₂Tf diferric transferrin - EGF epidermal growth factor - HU hydroxyurea - THR -thrombin     

Secretion of VEGF-165 has unique characteristics,including shedding from the plasma membrane

Vascular endothelial growth factor (VEGF) is a critical regulator of endothelial cell differentiation and vasculogenesis during both development and tumor vascularization. VEGF-165 is a major form that is secreted from the cells via a poorly characterized pathway. Here we use green fluorescent protein– and epitope-tagged VEGF-165 and find that its early trafficking between the endoplasmic reticulum and the Golgi requires the small GTP-binding proteins Sar1 and Arf1 and that its glycosylation in the Golgi compartment is necessary for efficient post-Golgi transport and secretion from the cells. The relative temperature insensitivity of VEGF secretion and its Sar1 and Arf1 inhibitory profiles distinguish it from other cargoes using the “constitutive” secretory pathway. Prominent features of VEGF secretion are the retention of the protein on the outer surface of the plasma membrane and the stimulation of its secretion by Ca²⁺ and protein kinase C. Of importance, shedding of VEGF-165 from the cell surface together with other membrane components appears to be a unique feature by which some VEGF is delivered to the surroundings to exert its known biological actions. Understanding VEGF trafficking can reveal additional means by which tumor vascularization can be inhibited by pharmacological interventions.     

High affinity RGD-binding sites at the plasma membrane of Arabidopsis thaliana links the cell wall

The heptapeptide Tyr-Gly- Arg-Gly-Asp- Ser-Pro containing the sequence Arg-Gly-Asp (RGD – the essential structure recognised by animal cells in substrate adhesion molecules) was tested on epidermal cells of onion and cultured cells of Arabidopsis upon plasmolysis. Dramatic changes were observed on both types of cells following treatment: on onion cells, Hechtian strands linking the cell wall to the membrane were lost, while Arabidopsis cells changed from concave to convex plasmolysis. A control heptapeptide Tyr-Gly-Asp-Gly-Arg-Ser-Pro had no effect on the shape of plasmolysed cells. Protoplasts isolated from Arabidopsis cells agglutinate in the presence of ProNectinF, a genetically engineered protein of 72 kDa containing 13 RGD sequences: several protoplasts may adhere to a single molecule of ProNectinF. The addition of the RGD-heptapeptide disrupted the adhesion between the protoplasts. Purified plasma membrane from Arabidopsis cells exhibits specific binding sites for the iodinated RGD-heptapeptide. The binding is saturable, reversible, and two types of high affinity sites (K_d1 1 nM, and K_d2 40 nM) can be discerned. Competitive inhibition by several structurally related peptides and proteins noted the specific requirement for the RGD sequence. Thus, the RGD-binding activity of Arabidopsis fulfils the adhesion features of integrins, i.e. peptide specificity, subcellular location, and involvement in plasma membrane-cell wall attachments.     

Identification of a new fatty acid synthesis-transport machinery at the peroxisomal membrane

The neurodegenerative disease X-linked adrenoleukodystrophy (X-ALD) is characterized by the abnormal accumulation of very long chain fatty acids. Mutations in the gene encoding the peroxisomal ATP-binding cassette half-transporter, adrenoleukodystrophy protein (ALDP), are the primary cause of X-ALD. To gain a better understanding of ALDP dysfunction, we searched for interaction partners of ALDP and identified binary interactions to proteins with functions in fatty acid synthesis (ACLY, FASN, and ACC) and activation (FATP4), constituting a thus far unknown fatty acid synthesis-transport machinery at the cytoplasmic side of the peroxisomal membrane. This machinery adds to the knowledge of the complex mechanisms of peroxisomal fatty acid metabolism at a molecular level and elucidates potential epigenetic mechanisms as regulatory processes in the pathogenesis and thus the clinical course of X-ALD.     

Secretion and membrane assembly

Cytoplasmic proteins undergo rapid and stable folding which buries their apolar segments. In contrast, precursors of secreted and membrane proteins have apolar segments which are recognized by chaperones and membrane receptors to distinguish them from soluble proteins.     

Does ATP cross the cell plasma membrane.

Although there is an abundance of evidence which indicates that ATP is released as well as taken up by cells, the concept that ATP cannot cross the cell membrane has tended to prevail. This article reviews the evidence for the release as well as uptake of ATP by cells. The evidence presented by various investigators clearly indicates that ATP can cross the cell membrane and suggests that the release and uptake of ATP are physiological processes.     

Dynamin-like protein 1 at the Golgi complex: A novel component of the sorting/targeting machinery en route to the plasma membrane

The final step in the liberation of secretory vesicles from the trans-Golgi network (TGN) involves the mechanical action of the large GTPase dynamin as well as conserved dynamin-independent fission mechanisms, e.g. mediated by Brefeldin A-dependent ADP-ribosylated substrate (BARS). Another member of the dynamin family is the mammalian dynamin-like protein 1 (DLP1/Drp1) that is known to constrict and tubulate membranes, and to divide mitochondria and peroxisomes. Here, we examined a potential role for DLP1 at the Golgi complex. DLP1 localized to the Golgi complex in some but not all cell lines tested, thus explaining controversial reports on its cellular distribution. After silencing of DLP1, an accumulation of the apical reporter protein YFP-GL-GPI, but not the basolateral reporter VSVG-SP-GFP at the Golgi complex was observed. A reduction in the transport of YFP-GL-GPI to the plasma membrane was confirmed by surface immunoprecipitation and TGN-exit assays. In contrast, YFP-GL-GPI trafficking was not disturbed in cells silenced for BARS, which is involved in basolateral sorting and trafficking of VSVG-SP-GFP in COS-7 cells. Our data indicate a new role for DLP1 at the Golgi complex and thus a role for DLP1 as a novel component of the apical sorting machinery at the TGN is discussed.