Deconstructing membrane traffic

Intracellular movement of proteins and lipids between organelles is usually described in terms of cargo, carriers, traffic and docking, familiar terms that imply parallels to human activities. Over the past century, scientists have been criticized for constructing hypotheses that reflect too much of their current political and cultural values. In this article, concepts of membrane traffic are re-examined to see whether they reflect the cell’s view of the world or our own.     

Deconstructing membrane traffic

Intracellular movement of proteins and lipids between organelles is usually described in terms of cargo, carriers, traffic and docking, familiar terms that imply parallels to human activities. Over the past century, scientists have been criticized for constructing hypotheses that reflect too much of their current political and cultural values. In this article, concepts of membrane traffic are re-examined to see whether they reflect the cell’s view of the world or our own.     

Clathrin and AP1B: Key roles in basolateral trafficking through trans-endosomal routes

Research following introduction of the MDCK model system to study epithelial polarity (1978) led to an initial paradigm that posited independent roles of the trans Golgi network (TGN) and recycling endosomes (RE) in the generation of, respectively, biosynthetic and recycling routes of plasma membrane (PM) proteins to apical and basolateral PM domains. This model dominated the field for 20 years. However, studies over the past decade and the discovery of the involvement of clathrin and clathrin adaptors in protein trafficking to the basolateral PM has led to a new paradigm. TGN and RE are now believed to cooperate closely in both biosynthetic and recycling trafficking routes. Here, we critically review these recent advances and the questions that remain unanswered.     

Observations on the origins of plasma membrane in rat deciduoma

Summary An NADH-ferricyanide reductase activity resistant to inactivation by cytochemical procedures was examined during decidualization of rat endometrium. Resistant activity was restricted to plasma membranes, distal elements of the Golgi apparatus, and discoid cisternae and cytoplasmic vesicles of decidual cells of endometrium of the pseudopregnant rat on days 3, 4, 5, 7, and 9, after mating. The procedure reduced or eliminated any evidence of NADH-ferricyanide reductase activity from other cellular components such as endoplasmic reticulum, nuclei, and mitochondria. The observations of the glutaraldehyde-resistant reductase in both plasma membranes and discoid cisternae may indicate a role for the latter in the biosynthesis of plasma membranes during decidualization when massive cell proliferation and membrane biosynthesis occur. The origin of the discoid cisternae is tentatively ascribed to the mature faces of the Golgi apparatus.Work supported in part by a grant from the NIH CA1880101 to D.J.M.     

rCNT2 extracellular cysteines,Cys615 and Cys649, are important for maturation and sorting to the plasma membrane

rCNT2 is a purine-preferring concentrative nucleoside transporter implicated in the regulation of extracellular adenosine levels and purinergic signaling. This study addressed the analysis of the CNT2 C-terminus tail as a domain likely to be implicated in transporter sorting. The topological mapping of this segment revealed that Cys⁶¹⁵ and Cys⁶⁴⁹ are important residues for the proper trafficking of CNT2 to the plasma membrane. The inhibition of protein disulfide isomerase (PDI) and ER glycosidase I and II impaired rCNT2 trafficking to the cell surface, similarly to Cys⁶¹⁵ and Cys⁶⁴⁹ mutants. The present work suggests these two cysteine residues are relevant for the proper sorting of the transporter and its functional performance.     

Alterations of wheat root plasma membrane lipid composition induced by copper stress result in changed physicochemical properties of plasma membrane lipid vesicles

A response when wheat is grown in excess copper is an altered lipid composition of the root plasma membrane (PM). With detailed characterisation of the root PM lipid composition of the copper-treated plants as a basis, in the present study, model systems were used to gain a wider understanding about membrane behaviour, and the impact of a changed lipid composition.PMs from root cells of plants grown in excess copper (50 μM Cu²⁺) and control (0.3 μM Cu²⁺) were isolated using the two-phase partitioning method. Membrane vesicles were prepared of total lipids extracts from the isolated PMs, and also reference vesicles of phosphatidylcholine (PC). In a series of tests, the vesicle permeability for glucose and for protons was analysed. The vesicles show that copper stress reduced the permeability for glucose of the lipid bilayer barrier. When vesicles from stressed plants were modified by addition of lipids to resemble vesicles from control plants, the permeability for glucose was very similar to that of vesicles from control plants. The permeability for protons did not change upon stress.Electron paramagnetic resonance (EPR) of the lipid vesicles spin probed with n-doxylstearic acid (nDSA) was used to explore the lipid rotational freedom at different depth of the bilayer. The EPR measurements supported the permeability data, indicating that the copper stress resulted in more tightly packed bilayers of the PMs with reduced acyl chain motion.     

Plasma membrane regeneration in the myxomyceteBadhamia utricularis

Summary Plasmodial strands of the myxomyceteBadhamia utricularis were injured and, after fixation and sectioning, examined using light and transmission electron microscopy. Injury results in the formation of a cytoplasmic droplet that rapidly regenerates a plasma membrane by the apparent fusion of vesicles at its surface. During the same time several structurally distinct layers form within the droplet. Some of these internal layers, which persist for several minutes after injury, may also arise from vesicle fusion. These results have differences from those reported for the relatedPhysarum polycephalum.     

Identification of the Plasma Membrane Proteolipid Protein as a Constituent of Brain Coated Vesicles and Synaptic Plasma Membrane

We have analyzed brain coated vesicles and synaptic plasma membrane for the presence of the plasma membrane proteolipid protein. Coated vesicles were isolated from calf brain gray matter with a final purification on Sephacryl S-1000 and reisolated twice by chromatography to ensure homogeneity. Fractions were analyzed by gel electrophoresis, immunoblotting for clathrin heavy chain, and by electron microscopy. Using an immunoblotting assay we were able to demonstrate the presence of the plasma membrane proteolipid protein in these coated vesicles at a significant level (i.e., approximately 1% of the bilayer protein of these vesicles). Reisolation of coated vesicles did not diminish the concentration of the protein in this fraction. Removal of the clathrin coat proteins or exposure of the coated vesicles to 0.1 M Na2CO3 showed that the plasma membrane proteolipid protein is not removed during uncoating and lysis but is intrinsic to the membrane bilayer of these vesicles. These studies demonstrate that plasma membrane proteolipid protein represents a significant amount of the bilayer protein of coated vesicles, suggesting that these vesicles may be a transport vehicle for the intracellular movement of the plasma membrane proteolipid protein. Isolation of synaptic plasma membranes proteolipid adult rat brain and estimation of the plasma membrane proteolipid protein content using the immunoblotting method confirmed earlier studies that show this protein is present in this membrane fraction at high levels as well (approximately 1-2%). The level of this protein in the synaptic plasma membrane suggests that the synaptic plasma membrane is one major site to which these vesicles may be targeted or from which the protein is being retrieved.     

Phospholipase A2 antagonists inhibit constitutive retrograde membrane traffic to the endoplasmic reticulum

Eukaryotic cells contain a variety of cytoplasmic Ca²⁺-dependent and Ca²⁺-independent phospholipase A₂s (PLA₂s; EC 2.3.1.2.3). However, the physiological roles for many of these ubiquitously-expressed enzymes is unclear or not known. Recently, pharmacological studies have suggested a role for Ca²⁺-independent PLA₂ (iPLA₂) enzymes in governing intracellular membrane trafficking events in general and regulating brefeldin A (BFA)-stimulated membrane tubulation and Golgi-to-endoplasmic reticulum (ER) retrograde membrane trafficking, in particular. Here, we extend these studies to show that membrane-permeant iPLA₂ antagonists potently inhibit the normal, constitutive retrograde membrane trafficking from the trans -Golgi network (TGN), Golgi complex, and the ERGIC-53-positive ER-Golgi-intermediate compartment (ERGIC), which occurs in the absence of BFA. Taken together, these results suggest that iPLA₂ enzymes play a general role in regulating, or directly mediating, multiple mammalian membrane trafficking events.     

Expression of a receptor kinase in Arabidopsis roots is stimulated by the basidiomycete Piriformospora indica and the protein accumulates in Triton X-100 insoluble plasma membrane microdomains

Piriformospora indica, an endophytic fungus of the Sebacinaceae family, colonises the roots of a wide variety of plant species and promotes their growth, in a manner similar to mycorrhizal fungi. We demonstrate that the fungus also interacts with the non-mycorrhizal host Arabidopsis thaliana. Promotion of root growth was detectable even before noticeable root colonization, and was accompanied by a massive transfer of phosphate from the media to the aerial parts of the seedlings. During the recognition period of both organisms, the message for a receptor kinase with leucine-rich repeats is transiently upregulated. The kinase is located in Triton X-100-insoluble plasma membrane microdomains. Thus, this is one of the earliest events of a plant root in response to a fungus reported to date.     

Extracting sequence motifs and the phylogenetic features of SNARE-dependent membrane traffic

The SNARE proteins are required for membrane fusion during intracellular vesicular transport and for its specificity. Only the unique combination of SNARE proteins (cognates) can be bound and can lead to membrane fusion, although the characteristics of the possible specificity of the binding combinations encoded in the SNARE sequences have not yet been determined. We discovered by whole genome sequence analysis that sequence motifs (conserved sequences) in the SNARE motif domains for each protein group correspond to localization sites or transport pathways. We claim that these motifs reflect the specificity of the binding combinations of SNARE motif domains. Using these motifs, we could classify SNARE proteins from 48 organisms into their localization sites or transport pathways. The classification result shows that more than 10 SNARE subgroups are kingdom specific and that the SNARE paralogs involved in the plasma membrane-related transport pathways have developed greater variations in higher animals and higher plants than those involved in the endoplasmic reticulum-related transport pathways throughout eukaryotic evolution.     

SNARE proteins in membrane trafficking

SNAREs are the core machinery mediating membrane fusion. In this review, we provide an update on the recent progress on SNAREs regulating membrane fusion events, especially the more detailed fusion processes dissected by well‐developed biophysical methods and in vitro single molecule analysis approaches. We also briefly summarize the relevant research from Chinese laboratories and highlight the significant contributions on our understanding of SNARE‐mediated membrane trafficking from scientists in China.      

Membrane traffic during axon development

Brain formation requires the establishment of complex neural circuits between a diverse array of neuronal subtypes in an intricate and ever changing microenvironment and yet with a large degree of specificity and reproducibility. In the last three decades, mounting evidence has established that neuronal development relies on the coordinated regulation of gene expression, cytoskeletal dynamics, and membrane trafficking. Membrane trafficking has been considered important in that it brings new membrane and proteins to the plasma membrane of developing neurons and because it also generates and maintains the polarized distribution of proteins into neuronal subdomains. More recently, accumulating evidence suggests that membrane trafficking may have an even more active role during development by regulating the distribution and degree of activation of a wide variety of proteins located in plasma membrane subdomains and endosomes. In this article the evidence supporting the different roles of membrane trafficking during axonal development, particularly focusing on the role of SNAREs and Rabs was reviewed. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1185–1200, 2016     

Phosphoinositides: regulators of membrane traffic and protein function

Phosphoinositides serve as important spatio-temporal regulators of intracellular trafficking and cell signalling events. In addition to their recognition by specific phosphoinositide binding domains present within cytoplasmic adaptor proteins or membrane integral channels and transporters phosphoinositides may affect membrane transport by eliciting conformational changes within proteins or by regulating enzymatic activities. During adaptor-mediated membrane traffic phosphoinositides form part of coincidence detection systems that aid in targeting pools of specific phosphoinositides to select intracellular transport pathways. In this review, we discuss potential mechanisms for conferring selectivity onto the phosphoinositide code as well as possible avenues for future research.     

Securin and separase modulate membrane traffic by affecting endosomal acidification

Securin and separase play a key role in sister chromatid separation during anaphase. However, a growing body of evidence suggests that in addition to regulating chromosome segregation, securin and separase display functions implicated in membrane traffic in Caenorhabditis elegans and Drosophila. Here we show that in mammalian cells both securin and separase associate with membranes and that depletion of either protein causes robust swelling of the trans-Golgi network (TGN) along with the appearance of large endocytic vesicles in the perinuclear region. These changes are accompanied by diminished constitutive protein secretion as well as impaired receptor recycling and degradation. Unexpectedly, cells depleted of securin or separase display defective acidification of early endosomes and increased membrane recruitment of vacuolar (V-) ATPase complexes, mimicking the effect of the specific V-ATPase inhibitor Bafilomycin A1. Taken together, our findings identify a new functional role of securin and separase in the modulation of membrane traffic and protein secretion that implicates regulation of V-ATPase assembly and function.     

A rice lipid transfer protein binds to plasma membrane proteinaceous sites

Nonspecific lipid transfer protein (nsLTP) is usually basic and secreted low-molecular-mass protein in plants. The 3-D structure of nsLTP1 resembles that of elicitin produced by the plant pathogen Phytophthora cryptogea, which can bind to the plant plasma membrane putative receptor and activate the downstream responses. It is inferred that nsLTP1 may have similar binding sites on the plasma membranes. In this work, rice recombinant protein TRX-nsLTP110 labeled with ¹²⁵I was shown to bind to rice plasma membrane preparations in a saturable curve, with an apparent K_d of 13.6 nM and B_max of 150 fmol/mg proteins. Competition experiments revealed that the binding of TRX-nsLTP110 was specific, in contrast to the nonspecific binding of the fusion tag thioredoxin. Protease treatment assay showed that the binding sites were proteinaceous. Our results suggest that the binding sites of nsLTPs on plasma membranes may be ubiquitous in the plant kingdom. They may be competed out from the binding sites under pathogen attack, supporting a role for nsLTP1 in host defense response to pathogens.     

Adenosine, a cytoprotective autocoid: effects of adenosine on neutrophil plasma membrane viscosity and chemoattractant receptor display

At inflammatory sites neutrophils are stimulated to produce a variety of toxic agents, yet rarely harm the endothelium across which they migrate. We have recently found that endothelium releases adenosine which, acting via receptors on the surface of human neutrophils, inhibits generation of toxic metabolites by stimulated neutrophils but, paradoxically, promotes chemotaxis. Agents which diminish plasma membrane viscosity affect neutrophil function similarly, possibly by modulating chemoattractant receptor number or affinity. We therefore determined whether adenosine receptor agonists modulate neutrophil function by decreasing membrane viscosity and/or chaning the affinity of chemoattractant (N-fMet-Leu-Phe, FMLP) receptors. Surprisingly, 5′-(N-ethylcar☐amido)adenosine (NECA, 10 μM), the most potent agonist at neutrophil adenosine receptors, increased plasma membrane viscosity, as measured by fluorescence anisotropy of the plasma membrane specific probe 1-(4-trimethylaminophenyl)-6-diphenyl-1,3,5-hexatriene (TMA-DPH), in unstimulated neutrophils from a mean microviscosity of 1.67 ± 0.02 (S.E.) to 1.80 ± 0.02 (p < 0.001) while inosine (10 μM), a poor adenosine receptor agonist, had no effect (1.73 ± 0.04, p =n.s. vs. control, p < 0.01 vs. NECA). Adenosine receptor agonists increased plasma membrane viscosity in neutrophils with the same order of potency previously seen for inhibition of superoxide anion generation and enhancement of chemotaxis (NECA > adenosine = N⁶-phenylisopropyladenosine). The adenosine receptor antagonist 8-(p-sulfophenyl)theophylline reversed the effect of NECA on plasma membrane viscosity. Unlike other agents which modulate plasma membrane viscosity, NECA (10 μM) did not significantly change the number or affinity of [³H]FMLP binding sites on neutrophils. In contrast to the hypothesis of Yuli et al. these results indicate that occupancy of adenosine receptors on neutrophils increases plasma membrane viscosity without affecting chemoattractant receptor display.     

Inhibition of SNARE-mediated membrane traffic impairs cell migration

Cell migration occurs as a highly-regulated cycle of cell polarization, membrane extension at the leading edge, adhesion, contraction of the cell body, and release from the extracellular matrix at the trailing edge. In this study, we investigated the involvement of SNARE-mediated membrane trafficking in cell migration. Using a dominant-negative form of the enzyme N-ethylmaleimide-sensitive factor as a general inhibitor of SNARE-mediated membrane traffic and tetanus toxin as a specific inhibitor of VAMP3/cellubrevin, we conducted transwell migration assays and determined that serum-induced migration of CHO-K1 cells is dependant upon SNARE function. Both VAMP3-mediated and VAMP3-independent traffic were involved in regulating this cell migration. Inhibition of SNARE-mediated membrane traffic led to a decrease in the protrusion of lamellipodia at the leading edge of migrating cells. Additionally, the reduction in cell migration resulting from the inhibition of SNARE function was accompanied by perturbation of a Rab11-containing alpha(5)beta(1) integrin compartment and a decrease in cell surface alpha(5)beta(1) without alteration to total cellular integrin levels. Together, these observations suggest that inhibition of SNARE-mediated traffic interferes with the intracellular distribution of integrins and with the membrane remodeling that contributes to lamellipodial extension during cell migration.