Cell-free systems to study vesicular transport along the secretory and endocytic pathways

Proteins bound for the cell surface, lysosomes, and secretory storage granules share a common pathway of intracellular transport. After their synthesis and translocation into the endoplasmic reticulum, these proteins traverse the secretory pathway by a series of vesicular transfers. Similarly, nutrient and signaling molecules enter cells by endocytosis, and move through the endocytic pathway by passage from one membrane-bound compartment to another. Little is known about the mechanisms by which proteins are collected into transport vesicles, or how these vesicles form, identify their targets, and subsequently fuse with their target membranes. An important advance toward our understanding these processes has come from the establishment of cell-free systems that reconstitute vesicular transfers in vitro. It is now possible to measure, in vitro, the transport of proteins from the endoplasmic reticulum to the Golgi, between Golgi cisternae, and the formation of transport vesicles en route from the trans Golgi network to the cell surface. Along the endocytic pathway, cell-free systems are available to study clathrin-coated vesicle formation, early endosome fusion, and the fusion of late endosomes with lysosomes. Moreover, the selective movement of receptors between late endosomes and the trans Golgi network has also been reconstituted. The molecular mechanisms of vesicular transport are now amenable to elucidation.     

The structures of BtuCD and MscS and their implications for transporter and channel function

The passage of most molecules across biological membranes is mediated by specialized integral membrane proteins known as channels and transporters. Although these transport families encompass a wide range of functions, molecular architectures and mechanisms, there are common elements that must be incorporated within their structures, namely the translocation pathway, ligand specificity elements and regulatory sensors to control the rate of ligand flow across the membrane. This minireview discusses aspects of the structure and mechanism of two bacterial transport systems, the stretch-activated mechanosensitive channel of small conductance (MscS) and the ATP-dependent vitamin B12 uptake system (BtuCD), emphasizing their general implications for transporter function.     

The use of membrane vesicles in transport studies

Transport-competent plasma membrane vesicles isolated from mammalian cells provide a system to investigate mechanisms and regulation of nutrient and ion transport systems. The characteristics of membrane vesicle systems to study transport in erythrocytes, renal and epithelial membranes, Ehrlich ascites cells, and mouse fibroblasts are discussed. Studies of Na+-stimulated and Na+-independent amino acid and glucose transport in these systems are evaluated, with emphasis on experimental verification of concepts stated in the Na+ gradient hypothesis. Nucleoside, phosphate, and calcium transport systems in plasma membrane vesicles from mouse fibroblast cultures are discussed. Also, current biochemical approaches to investigate mechanisms of regulation of nutrient transport systems by hormones or cellular proliferative state are described.     

Synthesis and biosynthetic trafficking of membrane lipids

Eukaryotic cells can synthesize thousands of different lipid molecules that are incorporated into their membranes. This involves the activity of hundreds of enzymes with the task of creating lipid diversity. In addition, there are several, typically redundant, mechanisms to transport lipids from their site of synthesis to other cellular membranes. Biosynthetic lipid transport helps to ensure that each cellular compartment will have its characteristic lipid composition that supports the functions of the associated proteins. In this article, we provide an overview of the biosynthesis of the major lipid constituents of cell membranes, that is, glycerophospholipids, sphingolipids, and sterols, and discuss the mechanisms by which these newly synthesized lipids are delivered to their target membranes.     

The lipid network

Natural cell membranes are composed of a remarkable variety of lipids, which provide specific biophysical properties to support membrane protein function. An improved understanding of this complexity of membrane composition may also allow the design of membrane active drugs. Crafting a relevant model of a cell membrane with controlled composition is becoming an art, with the ability to reveal the molecular mechanisms of biological processes and lead to better treatment of pathologies. By matching physiological observations from in vivo experiments to high-resolution information, more easily obtained from in vitro studies, complex interactions at the lipid interface are determined. The role of the lipid network in biological membranes is, therefore, the subject of increasing attention.     

The Balance of Fluid and Osmotic Pressures across Active Biological Membranes with Application to the Corneal Endothelium

The movement of fluid and solutes across biological membranes facilitates the transport of nutrients for living organisms and maintains the fluid and osmotic pressures in biological systems. Understanding the pressure balances across membranes is crucial for studying fluid and electrolyte homeostasis in living systems, and is an area of active research. In this study, a set of enhanced Kedem-Katchalsky (KK) equations is proposed to describe fluxes of water and solutes across biological membranes, and is applied to analyze the relationship between fluid and osmotic pressures, accounting for active transport mechanisms that propel substances against their concentration gradients and for fixed charges that alter ionic distributions in separated environments. The equilibrium analysis demonstrates that the proposed theory recovers the Donnan osmotic pressure and can predict the correct fluid pressure difference across membranes, a result which cannot be achieved by existing KK theories due to the neglect of fixed charges. The steady-state analysis on active membranes suggests a new pressure mechanism which balances the fluid pressure together with the osmotic pressure. The source of this pressure arises from active ionic fluxes and from interactions between solvent and solutes in membrane transport. We apply the proposed theory to study the transendothelial fluid pressure in the in vivo cornea, which is a crucial factor maintaining the hydration and transparency of the tissue. The results show the importance of the proposed pressure mechanism in mediating stromal fluid pressure and provide a new interpretation of the pressure modulation mechanism in the in vivo cornea.     

Constant change: dynamic regulation of membrane transport by calcium signalling networks keeps plants in tune with their environment

Despite substantial variation and irregularities in their environment, plants must conform to spatiotemporal demands on the molecular composition of their cytosol. Cell membranes are the major interface between organisms and their environment and the basis for controlling the contents and intracellular organization of the cell. Membrane transport proteins (MTPs) govern the flow of molecules across membranes, and their activities are closely monitored and regulated by cell signalling networks. By continuously adjusting MTP activities, plants can mitigate the effects of environmental perturbations, but effective implementation of this strategy is reliant on precise coordination among transport systems that reside in distinct cell types and membranes. Here, we examine the role of calcium signalling in the coordination of membrane transport, with an emphasis on potassium transport. Potassium is an exceptionally abundant and mobile ion in plants, and plant potassium transport has been intensively studied for decades. Classic and recent studies have underscored the importance of calcium in plant environmental responses and membrane transport regulation. In reviewing recent advances in our understanding of the coding and decoding of calcium signals, we highlight established and emerging roles of calcium signalling in coordinating membrane transport among multiple subcellular locations and distinct transport systems in plants, drawing examples from the CBL‐CIPK signalling network. By synthesizing classical studies and recent findings, we aim to provide timely insights on the role of calcium signalling networks in the modulation of membrane transport and its importance in plant environmental responses.     

Biological processes in organised media

Embedding a simple Michaelis-Menten enzyme in a gel slice may allow the catalysis of not only scalar processes but also vectorial ones, including uphill transport of a substrate between two compartments, and may make it seem as if two enzymes or transporters are present or as if an allosterically controlled enzyme/transporter is operating. The values of kinetic parameters of an enzyme in a partially hydrophobic environment are usually different from those actually measured in a homogeneous aqueous solution. This implies that fitting kinetic data (expressed in reciprocal co-ordinates) from in vivo studies of enzymes or transporters to two straight lines or a sigmoidal curve does not prove the existence of two different membrane mechanisms or allosteric control. In the artificial transport systems described here, a functional asymmetry was sufficient to induce uphill transport, therefore, although the active transport systems characterised so far correspond to proteins asymmetrically anchored in a membrane, the past or present existence of structurally symmetrical systems of transport in vivo cannot be excluded. The fact that oscillations can be induced in studies of the maintenance of the electrical potential of frog skin by addition of lithium allowed evaluation of several parameters fundamental to the functioning of the system in vivo (e.g., relative volumes of internal compartments, characteristic times of ionic exchanges between compartments). Hence, under conditions that approach real biological complexity, increasing the complexity of the behaviour of the system may provide information that cannot be obtained by a conventional, reductionist approach.     

Linking transport and translation of mRNAs with endosomes and mitochondria

In eukaryotic cells, proteins are targeted to their final subcellular locations with precise timing. A key underlying mechanism is the active transport of cognate mRNAs, which in many systems can be linked intimately to membrane trafficking. A prominent example is the long‐distance endosomal transport of mRNAs and their local translation. Here, we describe current highlights of fundamental mechanisms of the underlying transport process as well as of biological functions ranging from endosperm development in plants to fungal pathogenicity and neuronal processes. Translation of endosome‐associated mRNAs often occurs at the cytoplasmic surface of endosomes, a process that is needed for membrane‐assisted formation of heteromeric protein complexes and for accurate subcellular targeting of proteins. Importantly, endosome‐coupled translation of mRNAs encoding mitochondrial proteins, for example, seems to be particularly important for efficient organelle import and for regulating subcellular mitochondrial activity. In essence, these findings reveal a new mechanism of loading newly synthesised proteins onto endocytic membranes enabling intimate crosstalk between organelles. The novel link between endosomes and mitochondria adds an inspiring new level of complexity to trafficking and organelle biology.     

A new approach in the kinetics of biological transport the potential of reversible inhibition studies

Kinetic equations are derived for reversible inhibition of both active and facilitated transport systems for seven common experimental arrangements. It is shown that the unique features of transport kinetics may be exploited to give new kinds of information. It is also shown that the familiar rules of enzyme kinetics, though often applied to transport, can be seriously misleading. The analysis leads to the following general conclusions: (1) A competitive mechanism frequently gives rise to non-competitive kinetics, depending on the experimental design, but a non-competitive mechanism never produces competitive kinetics. (2) Inhibition studies on exchange diffusion at equilibrium in non-active systems or in the final steady state in active systems are the only unambiguous kinetic tests to distinguish competitive from non-competitive mechanisms. (3) Substrate analogs that are bound to the carrier and transported are readily distinguished by inhibition kinetics from those not transported, even though both may rapidly enter the cell by another route. (4) Even in non-active systems competitive inhibitors commonly have far different affinities for the substrate sites on the two membranes faces: where sufficient non-polarity allows their penetration into the cell, inhibition kinetics readily establish such sidedness in their action. (5) Inhibition kinetics of the mixed competitive and non-competitive type result from moderately asymmetrical binding of inhibitor at the substrate site. (6) Asymmetry is a necessary feature of active transport; hence studies of inhibition kinetics should provide important insights into its mechanism.     

Structure of a bacterial type IV secretion core complex at subnanometre resolution

Type IV secretion (T4S) systems are able to transport DNAs and/or proteins through the membranes of bacteria. They form large multiprotein complexes consisting of 12 proteins termed VirB1‐11 and VirD4. VirB7, 9 and 10 assemble into a 1.07 MegaDalton membrane‐spanning core complex (CC), around which all other components assemble. This complex is made of two parts, the O‐layer inserted in the outer membrane and the I‐layer inserted in the inner membrane. While the structure of the O‐layer has been solved by X‐ray crystallography, there is no detailed structural information on the I‐layer. Using high‐resolution cryo‐electron microscopy and molecular modelling combined with biochemical approaches, we determined the I‐layer structure and located its various components in the electron density. Our results provide new structural insights on the CC, from which the essential features of T4S system mechanisms can be derived.     

Complementary biophysical tools to investigate lipid specificity in the interaction between bioactive molecules and the plasma membrane: A review

Plasma membranes are complex entities common to all living cells. The basic principle of their organization appears very simple, but they are actually of high complexity and represent very dynamic structures. The interactions between bioactive molecules and lipids are important for numerous processes, from drug bioavailability to viral fusion. The cell membrane is a carefully balanced environment and any change inflicted upon its structure by a bioactive molecule must be considered in conjunction with the overall effect that this may have on the function and integrity of the membrane. Conceptually, understanding the molecular mechanisms by which bioactive molecules interact with cell membranes is of fundamental importance.     

The network of calcium regulation in muscle

In this review the molecular characteristics and reaction mechanisms of different Ca(2+) transport systems associated with various membranes in muscle cells will be summarized. The following topics will be discussed in detail: a brief history of early observations concerning maintenance and regulation of cellular Ca(2+) homeostasis, characterization of the Ca(2+) pumps residing in plasma membranes and sarco(endo)plasmic reticulum, mitochondrial Ca(2+) transport, Ca(2+)-binding proteins, coordinated expression of Ca(2+) transport systems, a general background of muscle excitation-contraction coupling with emphasis to the calcium release channels of plasma membrane and sarcoplasmic reticulum, the structure and function of dihydropyridine and ryanodine receptors of skeletal and cardiac muscles, and finally their disposition in various types of muscles.     

Nucleoside transporters of parasitic protozoa

Protozoan parasites are incapable of synthesizing purine nucleotides de novo and so must salvage preformed purines from their hosts. This process of purine acquisition is initiated by the translocation of preformed host purines across parasite or host membranes. Here, we report upon the identification and isolation of DNAs encoding parasite nucleoside transporters and the functional characterization of these proteins in various expression systems. These potential approaches provide a powerful approach for a thorough molecular and biochemical dissection of nucleoside transport in protozoan parasites.     

Capturing the nanoscale complexity of cellular membranes in supported lipid bilayers

The lateral mobility of cell membranes plays an important role in cell signaling, governing the rate at which embedded proteins can interact with other biomolecules. The past two decades have seen a dramatic transformation in understanding of this environment, as the mechanisms and potential implications of nanoscale structure of these systems has become accessible to theoretical and experimental investigation. In particular, emerging micro- and nano-scale fabrication techniques have made possible the direct manipulation of model membranes at the scales relevant to these biological processes. This review focuses on recent advances in nanopatterning of supported lipid bilayers, capturing the impact of membrane nanostructure on molecular diffusion and providing a powerful platform for further investigation of the role of this spatial complexity on cell signaling.     

H1 helix of colicin U causes phospholipid membrane permeation

In light of an increasing number of antibiotic-resistant bacterial strains, it is essential to understand an action imposed by various antimicrobial agents on bacteria at the molecular level. One of the leading mechanisms of killing bacteria is related to the alteration of their plasmatic membrane. We study bio-inspired peptides originating from natural antimicrobial proteins colicins, which can disrupt membranes of bacterial cells. Namely, we focus on the α-helix H1 of colicin U, produced by bacterium Shigella boydii, and compare it with analogous peptides derived from two different colicins. To address the behavior of the peptides in biological membranes, we employ a combination of molecular simulations and experiments. We use molecular dynamics simulations to show that all three peptides are stable in model zwitterionic and negatively charged phospholipid membranes. At the molecular level, their embedment leads to the formation of membrane defects, membrane permeation for water, and, for negatively charged lipids, membrane poration. These effects are caused by the presence of polar moieties in the considered peptides. Importantly, simulations demonstrate that even monomeric H1 peptides can form toroidal pores. At the macroscopic level, we employ experimental co-sedimentation and fluorescence leakage assays. We show that the H1 peptide of colicin U incorporates into phospholipid vesicles and disrupts their membranes, causing leakage, in agreement with the molecular simulations. These insights obtained for model systems seem important for understanding the mechanisms of antimicrobial action of natural bacteriocins and for future exploration of small bio-inspired peptides able to disrupt bacterial membranes.     

Mitochondria and Ca(2+) signaling

Mitochondria play a central role in cell homeostasis. Amongst others, one of the important functions of mitochondria is to integrate its metabolic response with one of the major signaling pathways - the Ca²⁺ signaling. Mitochondria are capable to sense the levels of cytosolic Ca²⁺ and generate mitochondrial Ca²⁺ responses. Specific mechanisms for both Ca²⁺ uptake and Ca²⁺ release exist in the mitochondrial membranes. In turn, the mitochondrial Ca²⁺ signals are able to produce changes in the mitochondrial function and metabolism, which provide the required level of functional integration. This essay reviews briefly the current available information regarding the mitochondrial Ca²⁺ transport systems and some of the functional consequences of mitochondrial Ca²⁺ uptake