A Conserved Domain of the Arabidopsis GNOM Protein Mediates Subunit Interaction and Cyclophilin 5 Binding

Abstract

The Arabidopsis GNOM protein, a guanine nucleotide exchange factor (GEF) that acts on ADP ribosylation factor (ARF)-type G proteins, is required for coordination of cell polarity along the apical-basal embryo axis. Interallelic complementation of gnom mutants suggested that dimerization is involved in GNOM function. Here, direct interaction between GNOM molecules is demonstrated in vitro and by using a yeast two-hybrid system. Interaction was confined to an N-terminal domain conserved within a subgroup of large ARF GEFs. The same domain mediated in vitro binding to cyclophilin 5 (Cyp5), which was identified as a GNOM interactor in two-hybrid screening. Cyp5 displayed peptidylprolyl cis / trans -isomerase and protein refolding activities that were sensitive to cyclosporin A. Cyp5 protein accumulated in several plant organs and, like GNOM, was partitioned between cytosolic and membrane fractions. Cyp5 protein was also expressed in the developing embryo. Our results suggest that Cyp5 may regulate the ARF GEF function of the GNOM protein during embryogenesis. INTRODUCTIONThe GNOM gene was identified by multiple mutant alleles in a search for mutations affecting body organization of the Arabidopsis embryo . All gnom alleles are defective in establishing the apical-basal axis of embryo polarity. The earliest defect observed in the gnom embryo, which is a perturbed division of the zygote, is followed by irregular cell division and elongation patterns at subsequent stages (Mayer et al., 1993). gnom seedlings lack a root, are defective in coordinated alignment of vascular cells, and display variably reduced apical structures (Mayer et al., 1993). These alterations have been attributed to defects in the establishment of coordinated cell polarity along the apical-basal axis in early embryos (Steinmann et al., 1999). Cloning of the GNOM gene (also called EMB30 ) revealed sequence similarity to the yeast vesicle trafficking protein Sec7p, including a central region called the Sec7 domain (Shevell et al., 1994;Busch et al., 1996). Proteins with Sec7 domains catalyze guanine nucleotide exchange on small GTP binding proteins of the ADP ribosylation factor (ARF) family required for vesicle coating in membrane trafficking (Chardin et al., 1996;Sata et al., 1998;Springer et al., 1999).Guanine nucleotide exchange factors (GEFs) that act on ARFs (ARF GEFs) group into small and large family members, and large ARF GEFs include the yeast proteins Gea1p, Gea2p, and Sec7p (Chardin et al., 1996;Peyroche et al., 1996;Sata et al., 1998). GNOM is a membrane-associated, functional ARF GEF of the Gea type, suggesting its involvement in vesicular trafficking (Steinmann et al., 1999). Apart from the Sec7 domain, large ARF GEFs contain as yet functionally uncharacterized N-and C-terminal regions . In the case of GNOM, however, there is genetic evidence for subunit interaction, because gnom alleles with different mutations in the Sec7 domain exhibit full complementation (Busch et al., 1996). In this study, we demonstrate a physical interaction of...

The Arabidopsis SERK1 protein interacts with the AAA-ATPase AtCDC48, the 14-3-3 protein GF14λ and the PP2C phosphatase KAPP

“…The AtSERK1 kinase domain interacts with AtCDC48 and GF14k in yeast To identify proteins that physically interact with At-SERK1 (Hecht et al 2001), a yeast two-hybrid cDNA library from Arabidopsis young silique tissue (Grebe et al 2000) was screened with the kinase domain of AtSERK1 (a.a. 266-625, AtSERK1 kinase ). Two clones were found to contain the coding sequence for the Cterminal 62 and 65 residues of the AtCDC48A protein (At3g09840).…”

“…An Arabidopsis (ecotype Landsberg erecta) cDNA library was prepared as a fusion to the activation domain in vector pJG4-5 (Origene Technologies Inc.) using mRNA from young silique tissue (Grebe et al 2000). The primary library contained approximately 2·10 6 cDNA clones.…”

The diageotropica gene of tomato encodes a cyclophilin: a novel player in auxin signaling

“…The AtCYP19-4/CYP5 protein has an Nterminal endoplasmic reticulum transport signal and interacts with the N-terminal region of the GNOM protein, a guanine nucleotide exchange factor (GEF), i.e., required for the proper polar localization of the auxin-efflux carrier PIN1 (Geldner et al 2003). The Arabidopsis gnom mutant exhibits defective embryo development and since AtCyp19-4/Cyp5 is also expressed in developing embryos, CYP5 may regulate the GEF function of the GNOM protein during embryogenesis (Grebe et al 2000). The Arabidopsis Squint (Sqn) gene encodes AtCYP40 and sqn mutants have reduced numbers of juvenile leaves (Berardini et al 2001), providing the first direct evidence of a role for CYPs in regulating vegetative development.…”

“…The Arabidopsis CYP, AtCYP18-3/ROC1 (Grebe et al 2000) in expression vector pGEX-4X-1 (Amersham Biosciences, Piscataway, NJ), was used as a positive control. A 5 ml overnight culture of E. coli strain BL21 Gold (Stratagene) carrying each fusion protein construct was inoculated into 500 ml of 2X YT medium containing ampicillin (100 lg/ml) and incubated at 37°C with vigorous shaking.…”

cDNA microarray analysis of gene expression changes during pollination, pollen-tube elongation, fertilization, and early embryogenesis in rice pistils

“…The high diversity of the cyclophilin isoforms suggests that plant cyclophilins are involved in a wide variety of cellular processes. The rice cyclophilin homolog (Os1752) identified in this study is 60% identical to AtCYP19-4 (Cyp5) (Grebe et al 2000;Romano et al 2004). Cyp5 has been shown to interact with GNOM, which is involved in the coordination of cell polarity along the apical-basal embryo axis in Arabidopsis (Grebe et al 2000).…”