Elsevier

Gene

Volume 230, Issue 1, 1 April 1999, Pages 33-40
Gene

Isolation and characterization of a cDNA clone from Arabidopsis thaliana with partial sequence similarity to integrins

https://doi.org/10.1016/S0378-1119(99)00068-2Get rights and content

Abstract

An Arabidopsis thaliana cDNA, called At14a, was isolated by immunoscreening an expression library with an anti-integrin antibody. The At14a cDNA is 1459 nucleotides and has an open reading frame encoding a protein of 385 amino acids and a predicted molecular weight of 43 kDa. At14a has a small domain that has sequence similarities to integrins from fungi, insects and humans. Transcripts of At14a are found in all Arabidopsis tissues examined, and when expressed as an epitope tagged fusion protein in transgenic plants, At14a localizes partly to the plasma membrane.

Introduction

Integrins are a family of proteins that act as cell surface receptors in animal cells and play both structural and signal transducing roles. These proteins are involved in diverse biological processes including cell proliferation, cell differentiation, cell migration, cell–cell adhesion and cell–matrix adhesion (Clark and Brugge, 1995, Hynes, 1992, Schwartz et al., 1995). Integrins are heterodimeric and are composed of non-homologous α and β subunits that span the plasma membrane. Each subunit has a large extracellular domain followed by a transmembrane segment and a short cytoplasmic tail at the C-terminus. In animal cells, there are at least 14 different α and eight different β subunits that heterodimerize in different combinations to give about 20 different cell surface receptors.

Immunological experiments performed about a decade ago using an antibody against the highly conserved cytoplasmic domain of β1 integrin had suggested the presence of integrins in a variety of vertebrates, invertebrates and yeasts (Marcantonio and Hynes, 1988). Genes encoding integrin subunits have been isolated from Drosophila (Bogaert et al., 1987, Yee and Hynes, 1993), Caenorhabditis elegans (Gettner et al., 1995), sponges (Brower et al., 1997, Pancer et al., 1997) and a coral (Brower et al., 1997). An integrin-like gene, uso1, has also been isolated from Saccharomyces cerevisiae by screening an expression library with two antibodies against leukocyte integrins αX and αM (Hostetter et al., 1995). Another integrin-like gene, αint1, from Candida albicans has limited similarity to vertebrate integrins (Gale et al., 1996).

Integrins have also been suggested to be present in algae and plants. Protein extracts from the brown alga Fucus show cross-reactivity to the anti-integrin antibody (Quatrano et al., 1991). In Saprolegnia, another alga, a protein that is expected to function in cytoplasm-cell wall attachment is a possible integrin (Kaminskyj and Heath, 1995). Another protein that has been shown to be involved in gravity perception and localizes to the cell–extracellular matrix junction in Chara corallina is also reported to be integrin-like (Wayne et al., 1992). Animal integrins recognize the sequence arg–gly–asp (RGD), which is a distinct motif conserved in several proteins that comprise the extracellular matrix in animal cells (Hynes, 1992). RGD-binding proteins have also been reported to be present in soybean root-cell cultures (Schindler et al., 1989), tobacco suspension cells adapted to grow in high NaCl concentration (Zhu et al., 1993), and the plasma membrane of Arabidopsis (Canut et al., 1998). Using an anti-integrin antibody, Gens et al. (1996) reported the presence of cross-reacting proteins in fine puncta on the surface of onion protoplasts. A similar punctate localization pattern was observed by Katembe et al. (1997) in the columella and peripheral cells of Arabidopsis root tips. Coinjection of integrin and spectrin antibodies into epidermal cells of onion bulb scales has also indicated the presence of integrin-like proteins on the inner side of plasma membrane and on the endoplasmic reticulum and all the major organelles in the cytoplasm (Reuzeau et al., 1997). However, no gene from plants has been reported that shares a sequence homology with integrins from other organisms.

In this paper, we report the full-length sequence of a cDNA clone (At14a) from Arabidopsis thaliana that was isolated using an antibody against a peptide corresponding to a highly conserved region in the cytoplasmic domain of a vertebrate β1 integrin. At14a possesses a transmembrane domain and a small region that has sequence similarities to integrins from fungi, insects and humans, and is localized partly in the plasma membranes of Arabidopsis.

Section snippets

Library and antibodies

The A. thaliana expression library (in lambda ZapII vector) used in this study was obtained from the Arabidopsis Biological Resource Center at Ohio State University. The anti-integrin antibody was raised in rabbit against the peptide VTTVVNPKYEGK. An anti-c-Myc monoclonal (9E10) culture supernatant was obtained from the Tissue Culture Facility at UNC Lineberger Comprehensive Cancer Center. Anti-RD28 antibody was a gift from Maarten Chrispeels.

cDNA library screen

An appropriate dilution of the library was plated on

Isolation of cDNA clone by immunoscreening

We screened an A. thaliana cDNA expression library with a polyclonal antibody made against a highly conserved peptide in the cytoplasmic domain of vertebrate β1 integrins. We obtained seven different genes in this screen. One of these, At14a, had a sequence similarity to integrins, although it lacked obvious homology to the peptide used to raise the antibody. The nucleotide sequence of At14a and its deduced amino acid sequence are presented in Fig. 1. The insert size of At14a is 1459

Discussion

The cDNA clone At14a was isolated in a screen of an A. thaliana expression library with an anti-integrin antibody. The deduced amino acid sequence for this clone has restricted similarity to integrin-like proteins isolated from C. albicans (Gale et al., 1996), Drosophila (Yee and Hynes, 1993), humans (Argraves et al., 1987), and S. cerevisiae (Hostetter et al., 1995).

The integrin-like protein αInt1 from C. albicans has limited sequence similarity to β integrins. However, it localizes to the

Acknowledgements

This project was supported by grants from the Office of Naval Research (N00014-93-1-0888) and the National Science Foundation (IBN-96-04672) to R.S.Q. We thank Marteen Chrispeels for the anti-RD28 antibody, Susan Whitfield for scanning and printing the figures, Natacha Bies for help in making the sequence figure and Jason Reed for comments on the manuscript. We also thank Mary Anderson at the Nottingham Arabidopsis Stock Center for analysis of segregation data and mapping of At14a.

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