Mobility of the IsiA chlorophyll-binding protein in cyanobacterial thylakoid membranes

We are using fluorescence recovery after photobleaching (FRAP) to probe the dynamics of thylakoid membranes in vivo in cells of the cyanobacterium Synechococcus sp. PCC7942. We have shown previously that the light-harvesting phycobilisomes diffuse quite rapidly on the thylakoid membrane surface. However, the photosystem II core complexes appear completely immobile. This raises the possibility that all of the membrane integral protein complexes in the thylakoid membrane are locked into a rather rigid array. Alternatively, it is possible that photosystem II is specifically anchored in the membrane, with other membrane proteins able to diffuse around it. We have now resolved this question by studying the diffusion of a second integral membrane protein, the IsiA chlorophyll-binding protein. IsiA is induced under iron starvation and some other stress conditions. In iron-stressed cyanobacterial cells, a high proportion of chlorophyll fluorescence comes from IsiA. This makes it straightforward to examine the diffusion of IsiA by FRAP. We find that the complex is mobile with a mean diffusion coefficient of approximately 3 x 10(-11) cm(2) s(-1). Thus it is clear that some thylakoid membrane proteins are mobile and that there must be a specific anchor that prevents photosystem II diffusion. We discuss the implications for the structure and function of the cyanobacterial thylakoid membrane.     

Shapes of bilayer vesicles with membrane embedded molecules

The interdependence of the lateral distribution of molecules which are embedded in a membrane (such as integral membrane proteins) and the shape of a cell with no internal structure (such as phospholipid vesicles or mammalian erythrocytes) has been studied. The coupling of the lateral distribution of the molecules and the cell shape is introduced by considering that the energy of the membrane embedded molecule at a given site of the membrane depends on the curvature of the membrane at that site. Direct interactions between embedded molecules are not considered. A simple expression for the interaction of the membrane embedded molecule with the local membrane curvature is proposed. Starting from this interaction, the consistently related expressions for the free energy and for the distribution function of the embedded molecules are derived. The equilibrium cell shape and the corresponding lateral distribution of the membrane embedded molecules are determined by minimization of the membrane free energy which includes the free energy of the membrane embedded molecules and the membrane elastic energy. The resulting inhomogeneous distribution of the membrane embedded molecules affects the cell shape in a nontrivial manner. In particular, it is shown that the shape corresponding to the absolute energy minimum at given cell volume and membrane area may be elliptically non-axisymmetric, in contrast to the case of a laterally homogeneous membrane where it is axisymmetric.     

Mechanical and functional aspects of membrane skeletons

Membrane skeletons can be characterized as cytoskeletal structures lying parallel to the bilayer part of cellular and organelle membranes. Typical examples are spectrin network and actin-myosin cortex. We approach the problem of elucidating the function of membrane skeletons by theoretically analyzing mechanical models of the cellular behavior. Membranes of different physical and chemical properties are considered. In erythrocytes and some organelles membrane bilayers are smooth and simply underlaid or overlaid by membrane skeletons. It is argued that there the role of a membrane skeleton is, either, to keep the membrane composition laterally homogeneous as it is in the case of the erythrocyte, or, that it is involved in the processes of the lateral separation of integral membrane proteins as it is happening in the case of some intermediate steps of the vesicular membrane trafficking. In the second type of membranes the bilayer part is ruffled and folded, and there the membrane skeletons play a role in the determination of the cortical tension. Here we explore in more detail the mechanical behavior of a cell with such properties of its boundary. The shape transformations are described which occur under the influence (i) of different external forces, i.e., when an originally spherical cell is aspirated into the micropipette or when such a cell is adsorbed on a flat surface, and (ii) of different internal forces on the cell boundary exerted by the cytoskeletal elements.     

CHANGES IN THICKNESS OF THE RED BLOOD CORPUSCLE MEMBRANE

Measurements of the static capacity per cm.² of membrane for the red corpuscle as changed when the cells are made spherical by the addition of lecithin or rose bengal, show a slight increase of capacity, indicating a thinning of the membrane, although the change is not large enough to make it certain that it is real. Furthermore, the membrane capacity shows a slight decrease when spherical cells are swollen in hypotonic saline, indicating a thickening of the membrane, although the change is hardly outside the experimental error. The fact that there is no increase in capacity lends support to the theory that as the cell swells the membrane does not stretch but new material comes from the interior of the cell to make a new portion of the membrane.     

Fishing new proteins in the twilight zone of genomes: the test case of outer membrane proteins in Escherichia coli K12, Escherichia coli O157:H7, and other Gram-negative bacteria

We address the problem of clustering the whole protein content of genomes into three different categories-globular, all-alpha, and all-beta membrane proteins-with the aim of fishing new membrane proteins in the pool of nonannotated proteins (twilight zone). The focus is then mainly on outer membrane proteins. This is performed by using an integrated suite of programs (Hunter) specifically developed for predicting the occurrence of signal peptides in proteins of Gram-negative bacteria and the topography of all-alpha and all-beta membrane proteins. Hunter is tested on the well and partially annotated proteins (2160 and 760, respectively) of Escherichia coli K 12 scoring as high as 95.6% in the correct assignment of each chain to the category. Of the remaining 1253 nonannotated sequences, 1099 are predicted globular, 136 are all-alpha, and 18 are all-beta membrane proteins. In Escherichia coli 0157:H7 we filtered 1901 nonannotated proteins. Our analysis classifies 1564 globular chains, 327 inner membrane proteins, and 10 outer membrane proteins. With Hunter, new membrane proteins are added to the list of putative membrane proteins of Gram-negative bacteria. The content of outer membrane proteins per genome (nine are analyzed) ranges from 1.5% to 2.4%, and it is one order of magnitude lower than that of inner membrane proteins. The finding is particularly relevant when it is considered that this is the first large-scale analysis based on validated tools that can predict the content of outer membrane proteins in a genome and can allow cross-comparison of the same protein type between different species.     

A Multilabel Model Based on Chou’s Pseudo–Amino Acid Composition for Identifying Membrane Proteins with Both Single and Multiple Functional Types

Predicting membrane protein type is a meaningful task because this kind of information is very useful to explain the function of membrane proteins. Due to the explosion of new protein sequences discovered, it is highly desired to develop efficient computation tools for quickly and accurately predicting the membrane type for a given protein sequence. Even though several membrane predictors have been developed, they can only deal with the membrane proteins which belong to the single membrane type. The fact is that there are membrane proteins belonging to two or more than two types. To solve this problem, a system for predicting membrane protein sequences with single or multiple types is proposed. Pseudo–amino acid composition, which has proven to be a very efficient tool in representing protein sequences, and a multilabel KNN algorithm are used to compose this prediction engine. The results of this initial study are encouraging.     

Predicting membrane protein type by functional domain composition and pseudo-amino acid composition

Given the sequence of a protein, how can we predict whether it is a membrane protein or non-membrane protein? If it is, what membrane protein type it belongs to? Since these questions are closely relevant to the function of an uncharacterized protein, their importance is self-evident. Particularly, with the explosion of protein sequences entering into databanks and the relatively much slower progress in using biochemical experiments to determine their functions, it is highly desired to develop an automated method that can be used to give a fast answers to these questions. By hybridizing the functional domain (FunD) and pseudo-amino acid composition (PseAA), a new strategy called FunD-PseAA predictor was introduced. To test the power of the predictor, a highly non-homologous data set was constructed where none of proteins has 25% sequence identity to any other. The overall success rates obtained with the FunD-PseAA predictor on such a data set by the jackknife cross-validation test was 85% for the case in identifying membrane protein and non-membrane protein, and 91% in identifying the membrane protein type among the following 5 categories: (1) type-1 membrane protein, (2) type-2 membrane protein, (3) multipass transmembrane protein, (4) lipid chain-anchored membrane protein, and (5) GPI-anchored membrane protein. These rates are much higher than those obtained by the other methods on the same stringent data set, indicating that the FunD-PseAA predictor may become a useful high throughput tool in bioinformatics and proteomics.     

MEMBRANES OF MESOPHYLL CELLS OF NICOTIANA RUSTICA AND PHASEOLUS VULGARIS WITH PARTICULAR REFERENCE TO THE CHLOROPLAST

Weier , T. E., and W. W. Thomson . (U. California, Davis.) Membranes of mesophyll cells of Nicotiana rustica and Phaseolus vulgaris with particular reference to the chloroplast. Amer. Jour. Bot. 49(8): 807–820. Illus. 1962.—The endoplasmic reticulum in mesophyll cells is represented by short lengths of irregularly disposed, paired membranes. It is occasionally associated with a typically double nuclear envelope. Groups of irregularly parallel, paired membranes suggesting disorganized dictyosomes occur infrequently. Mitochondria are unevenly distributed in mesophyll; they are large and have sparse tubular cristae around their periphery. In the great majority of instances the bounding membrane is diffusely stained with KMnO₄. When it is sharp and distinct, it may be double as usually pictured, or it may have well-delineated stretches of a single membrane bounding 25–50% of its circumference. The tonoplast and ectoplast are very fragile, the former appearing as a single dark line. In young leaves the ectoplast is visualized as a continuous single membrane adjacent to the cell wall, but in our micrographs of mature leaves it is always discontinuous. The plastid membrane sometimes is distinctly double, having 2 dark components bounding a light component. In the great majority of cases, however, this membrane is either a solid dark line, or the clear component of the double membrane is crossed by delicate dark lines giving the membrane a braided, or scalariform appearance. The various appearances of the membrane may intergrade with each other. The width of the plastid membrane is variable, ranging from 200 to 400 A. The inner component may invaginate into the stroma, and bodies may form in the clear space between the 2 outer membrane components. Micrographs suggest that these bodies, and others formed by small masses of stroma, may be expelled into the hyaloplasm, where they exist as spherical single-membraned particulates. The reality of the variable structure of the plastid membrane is discussed in light of concepts of membrane activity, molecular structure, and the relation of these factors to possible artifacts.     

Interactions of lipids and proteins: Some general principles

Methods to describe the binding of phospholipids to membrane proteins are described. It is shown that it is difficult to obtain estimates of the number of phospholipids bound to the surface of a membrane protein from ESR experiments in which plots of free to bound spin label (y) vs. molar ratio of lipid to protein are extrapolated toy=0. The relative advantages and disadvantages of ESR and fluorescence methods for measuring relative binding constants of phospholipids to membrane proteins are discussed. The particular problems associated with comparing binding constants of molecules of very different sizes (e.g., fatty acids and cardiolipin) are described and equations are presented to account for these problems. The possible effects of membrane viscosity and thickness on activity of membrane proteins are discussed, but it is concluded that effects of phospholipid structure on activity can only be understood in terms of a reasonably complete kinetic model for the protein.     

Looking for probes of gated channels: studies of the inhibition of glucose and choline transport in erythrocytes

Two seemingly contradictory sets of observations have been made in studies of biological transport, which are essential for our understanding of the transport mechanism: carriers are integral membrane proteins, which span the membrane and are not free to rotate across the membrane; carriers appear to function like a ferryboat, with a substrate binding site moving back and forth from one side of the membrane to the other. To reconcile these facts, it is necessary to postulated gated channels connecting the substrate site with the two membrane surfaces: the channels are arranged so that as one opens the other closes, with the result that the substrate site is alternately accessible from opposite sides of the membrane. Based on these properties, the following distinguishing features of molecules specifically bound in the channels may be predicted: if sufficiently bulky, they inhibit transport; they bind outside the substrate site (though adjacent to it), they bind asymmetrically either to the outward-facing carrier and on the outer surface of the membrane, or to the inward-facing carrier and on the inner surface of the membrane. The asymmetrical inhibition of the glucose and choline transport systems of erythrocytes by various inhibitors is examined, and the behavior in every case is found to conform with these criteria. From the results it may be concluded that the glucose carrier binds cytochalasin B in the inner gated channel and phloretin and tetrathionate in the outer gated channel.(ABSTRACT TRUNCATED AT 250 WORDS)     

SNAP25, but not syntaxin 1A, recycles via an ARF6-regulated pathway in neuroendocrine cells

Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins mediate cellular membrane fusion events and provide a level of specificity to donor-acceptor membrane interactions. However, the trafficking pathways by which individual SNARE proteins are targeted to specific membrane compartments are not well understood. In neuroendocrine cells, synaptosome-associated protein of 25 kDa (SNAP25) is localized to the plasma membrane where it functions in regulated secretory vesicle exocytosis, but it is also found on intracellular membranes. We identified a dynamic recycling pathway for SNAP25 in PC12 cells through which plasma membrane SNAP25 recycles in approximately 3 h. Approximately 20% of the SNAP25 resides in a perinuclear recycling endosome-trans-Golgi network (TGN) compartment from which it recycles back to the plasma membrane. SNAP25 internalization occurs by constitutive, dynamin-independent endocytosis that is distinct from the dynamin-dependent endocytosis that retrieves secretory vesicle constituents after exocytosis. Endocytosis of SNAP25 is regulated by ADP-ribosylation factor (ARF)6 (through phosphatidylinositol bisphosphate synthesis) and is dependent upon F-actin. SNAP25 endosomes, which exclude the plasma membrane SNARE syntaxin 1A, merge with those derived from clathrin-dependent endocytosis containing endosomal syntaxin 13. Our results characterize a robust ARF6-dependent internalization mechanism that maintains an intracellular pool of SNAP25, which is compatible with possible intracellular roles for SNAP25 in neuroendocrine cells.     

Interaction of amphipathic peptides mediated by elastic membrane deformations

Amphipathic alpha-helical peptides are perspective antimicrobial drugs. These peptides are partially embedded into the membrane to a shallow depth so that the longitudinal axis of the helix is parallel to the plane of the membrane or deviates from it by a small angle. In the framework of theory of elasticity of liquid crystals, adapted to lipid membranes, we calculated the energy of deformations occurring near the peptides partially embedded into the membrane. The energy of deformations is minimal when two peptides are parallel to each other and stay at a distance of about 5 nm. This configuration is stable with respect to small parallel displacements of the peptides and with respect to small variation of the angle between their axes both in the plane of the membrane and in the perpendicular direction. As a result of deformation the average thickness of the membrane decreases. The distribution of the elastic energy density has a maximum in the middle between the peptides. This region is the most likely place for formation of the through pores in the membrane. Since the equilibrium distance between the peptides is relatively large, it is assumed that the originally appearing pore should be purely lipidic.     

Milk lipid and protein traffic in mammary epithelial cells: joint and independent pathways

In mammary epithelial cells, milk lipids and proteins are synthesised in the same compartment, the endoplasmic reticulum. Lipids, carried through the cytoplasm, associate with the apical membrane which then pinches off and releases the lipid globule. Proteins, carried through membrane compartments are released in the lumen after fusion of secretory vesicles with the apical membrane. These processes assure a relatively constant composition of milk but it is not known whether lipid and protein secretion are linked. The protein composition of the milk fat globule membrane and the stimulatory effects of prolactin and oxytocin on lipid and protein secretion suggest that these processes are coupled and co-regulated. However, it is possible to observe a dissociation between the formation and the secretion of the two constituents, during differentiation and in various experimental conditions, and this suggests that coupling is not strictly required.     

The Role of the Membrane in Transporter Folding and Activity

The synthesis, folding, and function of membrane transport proteins are critical factors for defining cellular physiology. Since the stability of these proteins evolved amidst the lipid bilayer, it is no surprise that we are finding that many of these membrane proteins demonstrate coupling of their structure or activity in some way to the membrane. More and more transporter structures are being determined with some information about the surrounding membrane, and computational modeling is providing further molecular details about these solvation structures. Thus, the field is moving towards identifying which molecular mechanisms - lipid interactions, membrane perturbations, differential solvation, and bulk membrane effects - are involved in linking membrane energetics to transporter stability and function. In this review, we present an overview of these mechanisms and the growing evidence that the lipid bilayer is a major determinant of the fold, form, and function of membrane transport proteins in membranes.     

Cell surface localization and processing of the ComG proteins, required for DNA binding during transformation of Bacillus subtilis

The comG operon of Bacillus subtilis encodes seven proteins essential for the binding of transforming DNA to the competent cell surface. We have explored the processing of the ComG proteins and the cellular localization of six of them. All of the proteins were found to be membrane associated. The four proteins with N-terminal sequence motifs typical of type 4 prepilins (ComGC, GD, GE and GG) are processed by a pathway that requires the product of comC , also an essential competence gene. The unprocessed forms of ComGC and GD behave like integral membrane proteins. Pre-ComGG differs from pre-ComGC and pre-ComGD, in that it is accessible to proteolysis only from the cytoplasmic face of the membrane and at least a portion of it behaves like a peripheral membrane protein. The mature forms of these proteins are translocated to the outer face of the membrane and are liberated when peptidoglycan is hydrolysed by lysozyme or mutanolysin. ComGG exists in part as a disulphide-cross-linked homodimer in vivo . ComGC was found to possess an intramolecular disulphide bond. The previously identified homodimer form of this protein is not stabilized by disulphide bond formation. ComGF behaves as an integral membrane protein, while ComGA, a putative ATPase, is located on the inner face of the membrane as a peripheral membrane protein. Possible roles of the ComG proteins in DNA binding to the competent cell surface are discussed in the light of these and other results.     

p103 and A60: novel proteins of the neuronal membrane-associated cytoskeleton.

Associated with the neuronal plasma membrane are cytoskeletal proteins which probably control the specialization of the membrane into axonal and dendritic domains. Specialized isoforms of the proteins spectrin and ankyrin are located in each region and provide molecular mechanisms for locating specific transmembrane proteins at required points. However, spectrin and ankyrin were defined by extensions of the model for the erythrocyte membrane, an analogy unlikely to provide a complete account of the neuronal membrane skeleton. We have defined two new proteins of the neuronal membrane skeleton, designated p103 and A60. p103 is enriched in post-synaptic densities and binds with high affinity to integral membrane proteins--we suggest that it may have a role in linking the cytoskeleton to synaptic glycoproteins. A60 is a 60 kDa axonal protein, which appears to form a lining to the axolemma. It is almost exclusively axonal, although some neurons (such as Purkinje cells) appear to contain it in the cell body and initial dendrite segment. A60 binds both ankyrin and neurofilaments, and may have a role in transmitting information critical to axonal morphology to the membrane.     

Geometry preserving projections algorithm for predicting membrane protein types

Given a new uncharacterized protein sequence, a biologist may want to know whether it is a membrane protein or not? If it is, which membrane protein type it belongs to? Knowing the type of an uncharacterized membrane protein often provides useful clues for finding the biological function of the query protein, developing the computational methods to address these questions can be really helpful. In this study, a sequence encoding scheme based on combing pseudo position-specific score matrix (PsePSSM) and dipeptide composition (DC) is introduced to represent protein samples. However, this sequence encoding scheme would correspond to a very high dimensional feature vector. A dimensionality reduction algorithm, the so-called geometry preserving projections (GPP) is introduced to extract the key features from the high-dimensional space and reduce the original high-dimensional vector to a lower-dimensional one. Finally, the K-nearest neighbor (K-NN) and support vector machine (SVM) classifiers are employed to identify the types of membrane proteins based on their reduced low-dimensional features. Our jackknife and independent dataset test results thus obtained are quite encouraging, which indicate that the above methods are used effectively to deal with this complicated problem of predicting the membrane protein type.     

Hormonally regulated phosphoprotein of turkey erythrocytes: localization to plasma membrane

The catecholamine-stimulated cotransport of sodium and potassium ions across the plasma membrane of the turkey erythrocyte was previously found to be associated with increased 32P incorporation into a high molecular weight protein. To determine the subcellular localization of this phosphorylated protein, which we have termed goblin, a new method has been developed for isolation of pure plasma membranes from turkey erythrocytes. With this method, it has been demonstrated that goblin is located in the plasma membrane. Goblin is not extracted by solutions of low or high ionic strength but is partially extracted by nonionic detergents, indicating that it is not a component of turkey erythrocyte spectrin and suggesting that it may be an intrinsic protein of the plasma membrane. The data are compatible with a possible role for goblin in the hormonal control of ion movements across the plasma membrane.     

An ensemble of support vector machines for predicting the membrane protein type directly from the amino acid sequence

Given a particular membrane protein, it is very important to know which membrane type it belongs to because this kind of information can provide clues for better understanding its function. In this work, we propose a system for predicting the membrane protein type directly from the amino acid sequence. The feature extraction step is based on an encoding technique that combines the physicochemical amino acid properties with the residue couple model. The residue couple model is a method inspired by Chou’s quasi-sequence-order model that extracts the features by utilizing the sequence order effect indirectly. A set of support vector machines, each trained using a different physicochemical amino acid property combined with the residue couple model, are combined by vote rule. The success rate obtained by our system on a difficult dataset, where the sequences in a given membrane type have a low sequence identity to any other proteins of the same membrane type, are quite high, indicating that the proposed method, where the features are extracted directly from the amino acid sequence, is a feasible system for predicting the membrane protein type.