Selection of Arabidopsis genes encoding secreted and plasma membrane proteins

Secreted and plasma membrane proteins play crucial roles in a variety of physiological and developmental processes of multicellular organisms. Systematic cloning of the genes encoding these proteins is therefore of general interest. An effective method of trapping signal sequences was first described by Tashiro et al. (1993), and a similar yet more efficient method was reported by Klein et al. (1996) and Jacobs et al. (1997). In this study, we carried out the latter yeast-based signal sequence trap to clone genes from Arabidopsis thaliana encoding secreted and plasma membrane proteins. Of 144 sequenced cDNA clones, 18% are identical to previously cloned Arabidopsis thaliana genes, 12% are homologous to genes identified from various organisms, and 46% are novel. All of the isolated genes identical or homologous to previously reported genes are either secreted or plasma membrane proteins, and the remaining novel genes appear to contain functional signal sequences based on computer-aided sequence analysis. The full-length cDNA clones of one homologous gene and another novel gene were isolated and sequenced. The deduced amino acid sequences suggest that the former encodes a secreted protein, and the latter encodes a type 1 membrane protein. These results indicate that the signal sequence trap method is effective and useful for the isolation of plant genes encoding secreted and plasma membrane proteins.     

Mixing and matching during synaptic vesicle endocytosis

The question of how synapses maintain an active recycling pool of synaptic vesicles to support high-frequency synaptic transmission has been a perplexing and often controversial problem. In this issue of Neuron, Fernandez-Alfonso et al. present data indicating that at least two synaptic vesicle proteins, synaptotagmin 1 and VAMP-2, are present in a large pool on the synaptic and axonal plasma membrane and can interchange with recently exocytosed proteins. These findings suggest that a plasma membrane pool of synaptic vesicle proteins provides a reservoir that can facilitate rapid endocytosis.     

Active flows cluster cell surface proteins

The plasma membrane of cells is a dynamic mixture of different lipids, proteins, and sugars. In a recent issue of Cell, Gowrishankar et?al. (2012) propose a model for how the actin cortex may generate and regulate lateral heterogeneity in the plasma membrane by actively clustering cell surface molecules.     

Linkage between cell membrane proteins and actin-based cytoskeleton: the cytoskeletal-driven cellular functions

Asymmetric organization of the plasma membrane and cytosolic organelles is fundamental for a variety of cells, including bacteria, yeast and eukaryotic cells (Nelson, 1992). The degree into which cells polarize is characterized by their ability to create and maintain morphologically and biochemically distinct plasma membrane domains. The generation and maintenance of polarized distribution of membrane components (proteins and lipids) is thus critical to the ability of cells to perform complex activities such as cell-to-cell interactions, vectorial transport and secretion, cellular immunity, development and morphogenesis. Modification of cellular polarity may potentially lead to abnormal cellular activities and various pathological disorders (Molitoris, 1991; Carone et al., 1994; Chen et al., 1995). Our review shows the complex interplay between membrane proteins and the cytoskeletal network in determining the "polarized phenotype" in the cell. We provide evidence that membrane/cytoskeleton interaction is the key to regulation of the vast majority of cellular functions.     

The distinguishing characteristics of the plasma membrane are its exposed proteins.

The exposed proteins of the plasma membrane of normal human lymphocytes and platelets were labelled by using the lactoperoxidase macromolecular probe system. The labelled components were separated into molecular-weight classes by sodium dodecyl sulphate--polyacrylamide-gel electrophoresis. In contrast with the report by Tanner et al. (1974), a comparison of the two cell types showed that the major labelled components in both cell types were glycoproteins and were not identical. It is concluded that the exposed proteins are probably the most distinguishing characteristic of the plasma membrane of differentiated cell types.     

Lipid raft proteome of the human neutrophil azurophil granule

Detergent-resistant membrane domains (DRMs) are present in the membranes of azurophil granules in human neutrophils (Feuk-Lagerstedt et al., J. Leukoc. Biol. 2002, 72, 970). Using a proteomic approach, we have now identified 106 proteins in a DRM preparation from these granule membranes. Among these proteins were the lipid raft structural proteins flotillin-1 and -2, cytoskeletal proteins such as actin, vimentin and tubulin, and membrane fusion promoting proteins like annexins and dysferlin. Our results suggest that the azurophil granule membrane, in similarity to the plasma membrane, is an elaborate structure that takes part in intracellular signaling and functions other than the mere delivery of bactericidal effector molecules to the phagosome.     

Oiling the key hole

Many bacteria have been found to interact with specialized domains, rich in cholesterol and sphingolipids, of the host plasma membrane, termed lipid rafts. The mechanisms that underlie this interaction are starting to be unravelled. In this issue, Hayward et al. show that early effector proteins secreted by type III secretion harbouring Gram-negative bacteria are in fact cholesterol-binding proteins. Combined with other recent findings, this work shows that multiple steps leading to infection by these bacteria depend on raft components: activation of secretion, binding, perforation of the host cell membrane and signalling to trigger bacterial engulfment.     

Ubiquitin Binding in Endocytosis – How Tight Should It Be and Where Does It Happen?

Ubiquitin is an important tag in membrane transport. From studies in yeast, monoubiquitin has been considered sufficient to elicit uptake of cell surface transporters and receptors into endosomes. Two articles in the current issue of Traffic (Hawryluk et al. and Barriere et al.) indicate that stronger binding is required to retain and concentrate cargo in endocytic microdomains of the plasma membrane. High avidity interactions can be obtained by tandemly arrayed ubiquitin interaction motifs (UIM), in proteins such as the endocytic adaptors epsin and Eps15, interacting with polyubiquitin or by UIM-containing proteins binding several ubiquitins brought together through oligomerization of receptors. A controversial issue has been where such interactions take place. One view is that the association of epsin with ubiquitinated cargo is negatively regulated by its interaction with clathrin (Chen H and De Camilli P. Proc Natl Acad Sci USA 2005;102:2766-2771). This contention is now challenged by the articles of Hawryluk et al. and Barriere et al. Hawryluk et al. demonstrate that epsin and Eps15 consistently co-localize with clathrin but never with caveolin.     

Heterotrimeric G protein signaling: Getting inside the cell

Heterotrimeric G proteins have traditionally been thought to transduce signals at the plasma membrane. In this issue, Slessareva et al. (2006) now show that a G protein alpha subunit acts at the endosome to stimulate a phosphatidylinositol 3-kinase to help yeast respond to mating pheromones.     

How do yeast cells sense glucose?

A glucose-sensing mechanism has been described in Saccharomyces cerevisiae that regulates expression of glucose transporter genes. The sensor proteins Snf3 and Rgt2 are homologous to the transporters they regulate. Snf3 and Rgt2 are integral plasma membrane proteins with unique carboxy-terminal domains that are predicted to be localized in the cytoplasm. In a recent paper Ozcan and colleagues [Ozcan S, et al. EMBO J 1998; 17:2556-2773 (Ref. 1)] present evidence that the cytoplasmic domains of Snf3 and Rgt2 are required to transmit a glucose signal. They provide additional evidence to support their earlier assertion [Ozcan S, et al. Proc Natl Acad Sci USA 1996;93:12428-12432 (Ref. 2)] that glucose transport via Snf3 and Rgt2 is not involved in glucose sensing but, rather, that these proteins behave like glucose receptors. Other examples of transporter homologs with regulatory functions have recently been described in fungi as well [Madi L, et al. Genetics 1997; 146:499-508 (Ref. 3). and Didion T, et al. Mol Microbiol 1998;27:643-650 (Ref. 4)]. The identification of this class of nutrient sensors is an important step in elucidating the complex of regulatory mechanisms that leads to adaptation of fungi to different environments.     

New ways to make old walls: bacterial surprises

Two papers in this issue of Cell (Paradis-Bleau et?al., 2010 and Typas et?al., 2010) report that the lipoproteins LpoA and LpoB are required for the synthesis of cell walls in Escherichia coli. Attached to the bacterial outer membrane, these new cell wall components regulate penicillin-binding proteins located at the inner membrane.     

Functional and Evolutionary Analysis of the CASPARIAN STRIP MEMBRANE DOMAIN PROTEIN Family

CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASPs) are four-membrane-span proteins that mediate the deposition of Casparian strips in the endodermis by recruiting the lignin polymerization machinery. CASPs show high stability in their membrane domain, which presents all the hallmarks of a membrane scaffold. Here, we characterized the large family of CASP-like (CASPL) proteins. CASPLs were found in all major divisions of land plants as well as in green algae; homologs outside of the plant kingdom were identified as members of the MARVEL protein family. When ectopically expressed in the endodermis, most CASPLs were able to integrate the CASP membrane domain, which suggests that CASPLs share with CASPs the propensity to form transmembrane scaffolds. Extracellular loops are not necessary for generating the scaffold, since CASP1 was still able to localize correctly when either one of the extracellular loops was deleted. The CASP first extracellular loop was found conserved in euphyllophytes but absent in plants lacking Casparian strips, an observation that may contribute to the study of Casparian strip and root evolution. In Arabidopsis (Arabidopsis thaliana), CASPL showed specific expression in a variety of cell types, such as trichomes, abscission zone cells, peripheral root cap cells, and xylem pole pericycle cells.Biological membranes are conceptually simple structures that may be generated in vitro according to simple physicochemical principles. In vivo, however, membranes are highly complex and host a plethora of proteins that mediate the transfer of molecules and communication across the membrane. Proteins may be trapped in membrane by their transmembrane domains, anchored by lipid tails, or attach to membrane-integral proteins. A further level of complexity is seen when membrane proteins are not equally distributed but occupy only a limited fraction of the available surface (i.e. when they are polarly localized or when they form small membrane subdomains in the micrometer range). The question of how membrane proteins are retained locally and prevented from diffusing freely is of high importance to cell biology. Polarly localized proteins may be retained in their respective domains by membrane fences; in such a situation, polarly localized proteins are mobile in their domains but cannot diffuse through tightly packed scaffold proteins forming a molecular fence within the membrane. Membrane fences delimiting polar domains have been described in different organisms. For example, diffusion between membrane compartments is prevented in budding yeast (Saccharomyces cerevisiae) at the level of the bud neck (Barral et al., 2000; Takizawa et al., 2000); in ciliated vertebrate cells, between ciliary and periciliary membranes (Hu et al., 2010); in epithelial cells, between apical and basolateral membranes (van Meer and Simons, 1986); in neurons, between axon and soma (Kobayashi et al., 1992; Winckler et al., 1999; Nakada et al., 2003); and in spermatozoa, at the level of the annulus (Myles et al., 1984; Nehme et al., 1993). The existence of membrane scaffolds that prevent free protein diffusion has also been described in bacteria (Baldi and Barral, 2012; Schlimpert et al., 2012). In plants, we have shown the existence of a strict membrane fence in the root endodermis, where a median domain splits the cell in two lateral halves occupied by different sets of proteins (Alassimone et al., 2010). The situation in the plant endodermis is analogous to the separation of animal epithelia into apical and basolateral domains; indeed, a parallel between epithelia and endodermal cells has been drawn, despite the different origin of multicellularity in plants and animals (Grebe, 2011).The protein complexes responsible for the formation of membrane fences have been identified. Septins are a family of proteins able to oligomerize and form filaments (Saarikangas and Barral, 2011); their role in the formation of membrane fences has been demonstrated in several organisms and cellular situations, including the yeast bud neck (Barral et al., 2000; Takizawa et al., 2000), animal cilia (Hu et al., 2010), and mammalian spermatozoa (Ihara et al., 2005; Kissel et al., 2005; Kwitny et al., 2010). At the axonal initial segment of neurons, AnkyrinG is necessary to establish and maintain a membrane scaffold where different membrane proteins are immobilized and stabilized (Hedstrom et al., 2008; Sobotzik et al., 2009). In Caulobacter crescentus, the stalk protein Stp forms a complex that prevents diffusion between the cell body and stalk and between stalk compartments. Claudins and occludin are the main components of epithelial tight junctions (Furuse et al., 1993, 1998). Occludins are four-membrane-span proteins and belong to the MARVEL protein family (Sánchez-Pulido et al., 2002), as do Tricellulin and MARVELD3, which are also tight junction-associated proteins (Furuse et al., 1993; Ikenouchi et al., 2005; Steed et al., 2009).In Arabidopsis (Arabidopsis thaliana), our group identified a family of proteins that form a membrane fence in the endodermis (Roppolo et al., 2011). These CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASP1 to CASP5) are four-transmembrane proteins that form a median domain referred to as the Casparian strip membrane domain (CSD). CASPs are initially targeted to the whole plasma membrane, then they are quickly removed from lateral plasma membranes and remain localized exclusively at the CSD; there, they show an extremely low turnover, although they are eventually removed (Roppolo et al., 2011). The membrane proteins NOD26-LIKE INTRINSIC PROTEIN5;1 and BORON TRANSPORTER1 are restricted from diffusing through the CSD and remain polarly localized in the outer and inner lateral membranes, respectively; a fluorescent lipophilic molecule, when integrated in the outer endodermal membrane, was blocked at the level of the CSD and could not diffuse into the inner membrane (Roppolo et al., 2011). Besides making a plasma membrane diffusion barrier, CASPs have an important role in directing the modification of the cell wall juxtaposing their membrane domain: by interacting with secreted peroxidases, they mediate the deposition of lignin and the building up of the Casparian strips (Roppolo et al., 2011; Naseer et al., 2012; Lee et al., 2013). The two CASP activities, making membrane scaffolds and directing a modification of the cell wall, can be uncoupled: indeed, (1) formation of the CASP domain is independent from the deposition of lignin, and (2) interaction between CASPs and peroxidases can take place outside the CSD when CASPs are ectopically expressed (Lee et al., 2013).As CASPs are currently the only known proteins forming membrane fences in plants and because of their essential role in directing a local cell wall modification, we were interested in characterizing the repertoire of a large number of CASP-like (CASPL) proteins in the plant kingdom. Our aim was to provide the molecular basis for the discovery of additional membrane domains in plants and for the identification of proteins involved in local cell wall modifications. We extended our phylogenetic analysis outside of the plant kingdom and found conservation between CASPLs and the MARVEL protein family. Conserved residues are located in transmembrane domains, and we provide evidence suggesting that these domains are involved in CASP localization. We explored the potential use of the CASPL module in plants by investigating CASPL expression patterns and their ability to form membrane domains in the endodermis. Moreover, we related the appearance of the Casparian strips in the plant kingdom to the emergence of a CASP-specific signature that was not found in the genomes of plants lacking Casparian strips.     

Need tension relief fast? Try caveolae

Caveolae are protein-driven membrane invaginations that regulate both the physical and chemical composition of the plasma membrane. Sinha et?al. (2011) now show that caveolae are membrane reservoirs that are used to rapidly buffer against changes in membrane tension.     

Catch the KIF5B train to the apical surface

As epithelial cells become polarized, they develop new pathways to send proteins to the apical or basolateral domains of their plasma membrane. In this issue of Developmental Cell, Jaulin et al. (2007) show that as polarity develops, there is a shift in the kinesin motor protein used to transport an apical protein to the cell surface.     

Isolation and partial characterization of the plasma membrane of the sea urchin egg

The cell surface complex (Detering et al., 1977, J. Cell Biol. 75, 899-914) of the sea urchin egg consists of two subcellular organelles: the plasma membrane, containing associated peripheral proteins and the vitelline layer, and the cortical vesicles. We have now developed a method of isolating the plasma membrane from this complex and have undertaken its biochemical characterization. Enzymatic assays of the cell surface complex revealed the presence of a plasma membrane marker enzyme, ouabain-sensitive Na+/K+ ATPase, as well as two cortical granule markers, proteoesterase and ovoperoxidase. After separation from the cortical vesicles and purification on a sucrose gradient, the purified plasma membranes are recovered as large sheets devoid of cortical vesicles. The purified plasma membranes are highly enriched in the Na+/K+ ATPase but contain only very low levels of the proteoesterase and ovoperoxidase. Ultrastructurally, the purified plasma membrane is characterized as large sheets containing a "fluffy" proteinaceous layer on the external surface, which probably represent peripheral proteins, including remnants of the vitelline layer. Extraction of these membranes with Kl removes these peripheral proteins and causes the membrane sheets to vesiculate. Polyacrylamide gel electrophoresis of the cell surface complex, plasma membranes, and Kl-extracted membranes indicates that the plasma membrane contains five to six major proteins species, as well as a large number of minor species, that are not extractable with Kl. The vitelline layer and other peripheral membrane components account for a large proportion of the membrane-associated protein and are represented by at least six to seven polypeptide components. The phospholipid composition of the Kl-extracted membranes is unique, being very rich in phosphatidylethanolamine and phosphatidylinositol. Cholesterol was found to be a major component of the plasma membrane. Before Kl extraction, the purified plasma membranes retain the same species-specific sperm binding property that is found in the intact egg. This observation indicates that the sperm receptor mechanisms remain functional in the isolated, cortical vesicle-free membrane preparation.     

Localization of proteins, specifically binding highly-repetitive sequences of DNA in human spermatozoids

Immunoblot revealed in spermatozoa alpha-satellite (sat) DNA-specific centromere protein B (CENP-B) and p70 (Enukashvily et al., 2000), a membrane telomere binding protein (MTBP/TRF2) (Podgornaya et al., 2000), and Alu-binding protein p68 (Lukyanov et al., 2000). The localization of some of these proteins in spermatozoa was defined using indirect immunofluorescence. Spermatozoa were fixed in methanol/acetic acid 3:1, or prior to fixation were treated with 5 mM heparin and 10 mM DTT. The heparin/DTT treatment causes the nuclear membrane destruction and a partial chromatin decondensation. In non-treated spermatozoa fluorescent signals from all ABs are registered near the membrane, with MTBP/TRF2 being localized closer to the acrosome than sat-DNA-specific proteins. In the treated spermatozoa MTBP/TRF2 was partially lost, whereas part of CENP-B and sat-p70 remained in contact with membrane. Another part of sat-binding proteins reveals a dot-like staining pattern, with dots confined to the DAPI-stained chromatin area, inside a nuclei. This is in partial agreement with the pattern of telomere and CEN position revealed by FISH. Commonly MTBP has a near membrane localization, being lost when the nuclear membrane is destroyed. Centromere-binding proteins are arranged in the order from the nuclear membrane towards the nuclear center, with CENP-B being situated more peripherally but not in the middle of the nucleus. This discrepancy may be explained by the fact, that some proteins are not associated with the appropriate sequences in a spermatozoon. Possibly, such a distribution of proteins may reflect their role in unpacking the paternal genetic material in a zygote.     

Desmosome assembly in MDCK epithelial cells does not require the presence of functional microtubules.

Desmosomes, complex multisubunit structures that assemble at sites of cell-cell contact, are important components of the epithelial junctional complex. Desmosome assembly requires the coordinated interaction at the plasma membrane of at least 8 cytoplasmic and integral membrane proteins organized into two structurally and functionally distinct domains, the cytoplasmic plaque and membrane core. Previous studies (Pasdar et al., J. Cell Biol., 113:645-655) provided evidence that cytokeratin filaments and microtubules may regulate transfer and assembly of cytoplasmic plaque and membrane core proteins, respectively. To determine directly the role of microtubules in these processes, Madin-Darby canine kidney (MDCK) cells were treated with nocodazole or colchicine to disrupt the microtubular network. Biochemical analysis of the different components of the cytoplasmic plaque and membrane core domains revealed little or no effect of nocodazole or colchicine on the kinetics of synthesis, post-translational modifications, transfer of proteins to the plasma membrane or their metabolic stability in the presence or absence of cell-cell contact. Likewise, immunofluorescence analysis of desmosome formation demonstrated an apparently normal desmosome assembly in the presence of nocodazole or colchicine upon induction of cell-cell contact. These results indicate that an intact microtubular network is not necessary for the processing or transport of the desmosomal membrane core glycoproteins to the plasma membrane in the absence or presence of cell-cell contact. Furthermore, the integration of the cytoplasmic plaque and membrane core domains induced by cell-cell contact at the plasma membranes of adjacent cells does not require the presence of functional microtubules.     

Striatin, a calmodulin-dependent scaffolding protein, directly binds caveolin-1.

Caveolins are scaffolding proteins able to collect on caveolae a large number of signalling proteins bearing a caveolin-binding motif. The proteins of the striatin family, striatin, SG2NA, and zinedin, are composed of several conserved, collinearly aligned, protein-protein association domains, among which a putative caveolin-binding domain [Castets et al. (2000) J. Biol. Chem. 275, 19970-19977]. They are associated in part with membranes. These proteins are mainly expressed within neurons and thought to act both as scaffolds and as Ca(2+)-dependent signalling proteins [Bartoli et al. (1999) J. Neurobiol. 40, 234-243]. Here, we show that (1) rat brain striatin, SG2NA and zinedin co-immunoprecipitate with caveolin-1; (2) all are pulled down by glutathione-S-transferase (GST)-caveolin-1; (3) a fragment of recombinant striatin containing the putative caveolin-binding domain binds GST-caveolin-1. Hence, it is likely that the proteins of the striatin family are addressed to membrane microdomains by their binding to caveolin, in accordance with their putative role in membrane trafficking [Baillat et al. (2001) Mol. Biol. Cell 12, 663-673].     

It takes two to Get3

Tail-anchored (TA) membrane proteins perform essential cellular functions. They are posttranslationally inserted into the endoplasmic reticulum (ER) membrane by interaction of the Get3 chaperone with the Get1/2 receptor. Two independent structural and functional analyses of the Get3/receptor complex by Stefer et?al. and Mariappan et?al. now provide insights into TA protein insertion.     

Arhgef16, a novel Elmo1 binding partner,promotes clearance of apoptotic cells via RhoG-dependent Rac1 activation

Elmo is an evolutionarily conserved mammalian ortholog of Caenorhabditis elegans CED-12 with proposed roles during the removal of apoptotic cells, cell migration, neurite outgrowth, and myoblast fusion (Katoh and Negishi (2003) [1], Park and Tosello (2007) [2], Grimsley et al. (2004) [3], Hamoud et al. (2014) [4]). Elmo mediates these cellular processes by interacting with various proteins located in the plasma membrane, cytoplasm and nucleus, and by modulating their activities although it has no intrinsic catalytic activity (Park and Tosello (2007) [2], Hamoud et al. (2014) [4], Li et al. (2013) [5], Margaron, Fradet and Cote (2013) [6], and Mauldin et al. (2013)[7]). Because there are a limited number of proteins known to interact with Elmo, we performed a yeast two-hybrid screen using Elmo1 as bait to identify Elmo1-interacting proteins and to evaluate their mode of regulation. Arhgef16 was one of the proteins identified through the screen and subsequent analyses revealed that Arhgef16 interacted with Elmo1 in mammalian cells as well. Expression of Arhgef16 in phagocytes promoted engulfment of apoptotic cells, and engulfment mediated by Arhgef16 increased synergistically in the presence of Elmo1 but was abrogated in the absence of Elmo1. In addition, Arhgef16-mediated removal of apoptotic cells was dependent on RhoG, but independent of Dock1. Taken together, this study suggests that the newly identified Elmo1-interacting protein, Arhgef16, functions synergistically with Elmo1 to promote clearance of apoptotic cells in a RhoG-dependent and Dock1-independent manner.