Functional and Evolutionary Analysis of the CASPARIAN STRIP MEMBRANE DOMAIN PROTEIN Family1[C][W]

Functional and Evolutionary Analysis of the CASPARIAN STRIP MEMBRANE DOMAIN PROTEIN Family1[C][W] Daniele Roppolo *, Brigitte Boeckmann, Alexandre Pfister, Emmanuel Boutet, Maria C. Rubio, Valérie Dénervaud-Tendon, Joop E.M. Vermeer, Jacqueline Gheyselinck, Ioannis Xenarios, and Niko Geldner Department of Plant Molecular Biology, University of Lausanne, Quartier Sorge, Lausanne 1015, Switzerland (D.R., A.P., M.C.R., V.D.-T., J.E.M.V., N.G.); Swiss Institute of Bioinformatics, Centre Médical Universitaire, 1211 Geneva 4, Switzerland (B.B., E.B., I.X.); Departamento de Nutrición Vegetal, Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas, 50080 Zaragoza, Spain (M.C.R.); Vital-IT Group and University of Lausanne, Quartier Sorge, Bâtiment Génopode, Lausanne 1015, Switzerland (I.X.); and Institute of Plant Sciences, University of Bern, 3013 Bern, Switzerland (D.R., J.G.) 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 struc- the micrometer range). The question of how membrane tures that may be generated in vitro according to simple proteins are retained locally and prevented from diffusing physicochemical principles. In vivo, however, membranes freely is of high importance to cell biology. Polarly local- are highly complex and host a plethora of proteins that ized proteins may be retained in their respective domains mediate the transfer of molecules and communication by membrane fences; in such a situation, polarly localized across the membrane. Proteins may be trapped in mem- proteins are mobile in their domains but cannot diffuse brane by their transmembrane domains, anchored by lipid through tightly packed scaffold proteins forming a mo- tails, or attach to membrane-integral proteins. A further lecular fence within the membrane. Membrane fences level of complexity is seen when membrane proteins are delimiting polar domains have been described in different not equally distributed but occupy only a limited fraction organisms. For example, diffusion between membrane of the available surface (i.e. when they are polarly local- compartments is prevented in budding yeast (Saccharo- ized or when they form small membrane subdomains in myces cerevisiae)atthelevelofthebudneck(Barraletal., 2000; Takizawa et al., 2000); in ciliated vertebrate cells, between ciliary and periciliary membranes (Hu et al., 1 This work was supported by the Swiss National Science Foun- 2010); in epithelial cells, between apical and basolateral dation (to D.R. and N.G.) and the European Research Council (to membranes (van Meer and Simons, 1986); in neurons, N.G.). The Swiss-Prot group (B.B., E.B., and I.X.) is part of the Swiss between axon and soma (Kobayashi et al., 1992; Winckler Institute of Bioinformatics, and its activities are supported by the et al., 1999; Nakada et al., 2003); and in spermatozoa, at Swiss Federal Government through the State Secretariat for Educa- the level of the annulus (Myles et al., 1984; Nehme et al., tion, Research, and Innovation. 1993). The existence of membrane scaffolds that prevent * Address correspondence to [email protected]. free protein diffusion has also been described in bacteria The author responsible for distribution of materials integral to the (Baldi and Barral, 2012; Schlimpert et al., 2012). In plants, findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: we have shown the existence of a strict membrane fence in Daniele Roppolo ([email protected]). the root endodermis, where a median domain splits the [C] Some figures in this article are displayed in color online but in cell in two lateral halves occupied by different sets of black and white in the print edition. proteins (Alassimone et al., 2010). The situation in the [W] The online version of this article contains Web-only data. plant endodermis is analogous to the separation of animal www.plantphysiol.org/cgi/doi/10.1104/pp.114.239137 epithelia into apical and basolateral domains; indeed, a Ò Plant Physiology , August 2014, Vol. 165, pp. 1709–1722, www.plantphysiol.org Ó 2014 American Society of Plant Biologists. All Rights Reserved. 1709 Downloaded from www.plantphysiol.org on October 14, 2014 - Published by www.plant.org Copyright © 2014 American Society of Plant Biologists. All rights reserved. Roppolo et al. parallel between epithelia and endodermal cells has been number of CASP-like (CASPL) proteins in the plant drawn, despite the different origin of multicellularity in kingdom. Our aim was to provide the molecular basis plants and animals (Grebe, 2011). for the discovery of additional membrane domains in The protein complexes responsible for the formation of plants and for the identification of proteins involved in membrane fences have been identified. Septins are a local cell wall modifications. We extended our phylo- family of proteins able to oligomerize and form filaments genetic analysis outside of the plant kingdom and (Saarikangas and Barral, 2011); their role in the formation found conservation between CASPLs and the MARVEL of membrane fences has been demonstrated in several protein family. Conserved residues are located in organisms and cellular situations, including the yeast transmembrane domains, and we provide evidence bud neck (Barral et al., 2000; Takizawa et al., 2000), animal suggesting that these domains are involved in CASP cilia (Hu et al., 2010), and mammalian spermatozoa (Ihara localization. We explored the potential use of the et al., 2005; Kissel et al., 2005; Kwitny et al., 2010). At the CASPL module in plants by investigating CASPL ex- axonal initial segment of neurons, AnkyrinG is necessary pression patterns and their ability to form membrane to establish and maintain a membrane scaffold where domains in the endodermis. Moreover, we related the different membrane proteins are immobilized and stabi- appearance of the Casparian strips in the plant kingdom to lized (Hedstrom et al., 2008; Sobotzik et al., 2009). In Cau- the emergence of a CASP-specific signature that was not lobacter crescentus, the stalk protein Stp forms a complex found in the genomes of plants lacking Casparian strips. 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 RESULTS et al., 1993, 1998). Occludins are four-membrane-span pro- CASPLs Belong to the MARVEL Protein Family teins and belong to the MARVEL protein family (Sánchez- Pulido et al., 2002), as do Tricellulin and MARVELD3, In an attempt to understand the evolutionary history of which are also tight junction-associated proteins (Furuse the CASPs, we analyzed sequenced plant genomes and et al., 1993; Ikenouchi et al., 2005; Steed et al., 2009). EST databases for their repertoire of CASP homologs that In Arabidopsis (Arabidopsis thaliana), our group identi- we termed CASPLs. In all, we annotated over 350 proteins fied a family of proteins that form a membrane fence in from more than 50 plant species. Arabidopsis CASPs the endodermis (Roppolo et al., 2011). These CASPARIAN (AtCASPs) have four predicted transmembrane domains, STRIP MEMBRANE DOMAIN PROTEINS (CASP1 to with cytoplasmic N and C termini, variable N terminus CASP5) are four-transmembrane proteins that form a length, short C terminus, and short intracellular loop. median domain referred to as the Casparian strip mem- Homologous plant proteins are conserved in the trans- brane domain (CSD). CASPs are initially targeted to the membrane domains, particularly the first (TM1) and the whole plasma membrane, then they are quickly removed third (TM3): an Arg in TM1 and an Asp in TM3 are pre- from lateral plasma membranes and remain localized ex- sent in the vast majority of CASPLs (Fig. 1A; Supplemental clusively at the CSD; there, they show an extremely low Fig. S1). Six proteins with sequence similarity were iden- turnover, although they are eventually removed (Roppolo tified in green algae: Chlorokybus atmophyticus (charophyte), et al., 2011). The membrane proteins NOD26-LIKE IN- Ostreococcus tauri, Ostreococcus lucimarinus,andMicromonas TRINSIC PROTEIN5;1 and BORON TRANSPORTER1 pusilla (chlorophyte),

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