Journal of Experimental Botany, Vol. 51, No. 351, pp. 1761-1762,
October 2000
© 2000 Oxford University Press
Gene Note |
CEF, a Sec24 homologue of Arabidopsis thaliana, enhances the survival of yeast under oxidative stress conditions1
Vakgroep Moleculaire Genetica & Departement Plantengenetica, Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium
Received 2 May 2000; Accepted 16 May 2000
Abstract
Budding yeast strains that produced the Arabidopsis thaliana protein CEF or its amino-terminal proline-rich domain were more tolerant to hydroperoxides. CEF is homologous to animal and yeast Sec24 proteins. These data suggest that CEF plays a protective role through protein transport during growth under pro-oxidant conditions.
Key words: Arabidopsis, oxidative stress, Sec24.
Reactive oxygen species (ROS) are oxygen-derived molecules that can severely damage cellular components (Halliwell and Gutteridge, 1989
). To neutralize ROS excess, cells have evolved molecular defences, including small antioxidants and scavenging enzymes (Noctor and Foyer, 1998). To identify new components of this defence system, plant cDNAs were overexpressed at random in the oxidative stress-sensitive yap1-deficient strain of Saccharomyces cerevisiae (Kuge and Jones, 1994). The Sec24 homologue of Arabidopsis thaliana (L.) Heynh., designated CEF, enhanced the resistance of yeast to oxidative damage.
The yap1-deficient yeast strain WYT (Kuge and Jones, 1994) was transformed (Dohmen et al., 1991) with an A. thaliana cDNA library constructed in the yeast overexpressing vector pFL61 (Minet et al., 1992). Approximately 100 cells per colony from 105 primary transformants were plated onto minimal SD medium containing the drug tert-butyl hydroperoxide (tBuOOH), a compound that causes lipid peroxidation. Putatively tolerant clones were grown in liquid medium, serially diluted, and spotted onto 0.2 mM tBuOOH SD medium. Thus, the resistant clone (C84) was identified and its plasmid, named pC84, isolated. To confirm that the resistant phenotype was associated with a plant protein, the WYT and the isogenic YAP1+DY yeast strains (Kuge and Jones, 1994) were transformed with the plasmid pC84. When assayed in a semi-quantitative drug resistance test, the WYT(pC84) and DY(pC84) transformants were consistently more tolerant to tBuOOH (data not shown). The resistance phenotype was therefore independent of the YAP1 function.
Because the 1.2 kb cDNA C84, present in pC84, was partial, a 3.6 kb full-length cDNA of clone eighty-four (CEF) was isolated by screening an A. thaliana cDNA library in 
Zap. The overproduction of the full-length CEF was tested for biological effects. Although C84 (coding for the CEF1–310 polypeptide) conferred slightly higher levels of resistance against 0.2 mM tBuOOH than the full-length CEF (coding for the full-length CEF1–1097 protein), both cDNAs increased the yeast tolerance against tBuOOH and hydrogen peroxide, but not against diamide (Fig. 1).
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Amino acid sequence comparison suggested that CEF belongs to the superfamily of Sec24 proteins, which participate in protein transport through the endoplasmic reticulum (Pagano et al., 1999
; Roberg et al., 1999). On average, CEF has 34.7% amino acid identity with four Sec24 human proteins (GenBank AJ131244, AJ131245, 1723050, and 3882231) and 30.9% with three Sec24 yeast proteins (SWISS-PROT P53953, P38810, and P40482). Furthermore, CEF has a similar domain organization, including (i) a conserved carboxyl-terminal part and a variable 1/3 amino-terminal part, (ii) a proline-rich N-terminal part and (iii) a conserved zinc finger-like motif (Fig. 2). In Arabidopsis, CEF belongs to a small gene family, because a second highly similar gene (Accession no. 3063706) was identified by the BLAST-X search of DNA sequences.
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The CEF gene had a relatively constitutive expression. A hybridizing 3.6 kb transcript, which could be detected in all Arabidopsis plant parts, was most abundant in inflorescence stems (data not shown). CEF transcript levels did not change when plants were challenged with experimental oxidative conditions. A translational fusion of CEF1–1097 to the green-fluorescent protein (GFP) was localized in the cytosol of stably transformed tobacco BY2 cells (data not shown). Similarly, human Sec24B and Sec24C are cytosolic proteins (Pagano et al., 1999
). Two explanations are proposed for the CEF-mediated hydroperoxide resistance of yeast. Because the production of CEF1–310 is sufficient to confer hydroperoxide resistance, the production of CEF1–310 and CEF1–1097 could facilitate protein export, resulting in an improvement of protection, reparation, and renewal of cellular components damaged upon hydroperoxide exposure. Alternatively, compared with yeast Sec24 proteins, both plant and animal homologues have an extended N-terminal domain, rich in proline (P) and glutamine (Q) residues, which resembles PQ-rich trans-activating domains. Therefore, the 34 kDa large CEF1–310 can passively diffuse through the nuclear envelope and have pleiotropic effects on gene expression, resulting in an increased hydroperoxide resistance. This hypothesis may account for the fact that yeast producing CEF1–310 is more resistant than that producing CEF1–1097.
Acknowledgments
Dr M Minet is acknowledged for the Arabidopsis cDNA library, Dr J Jones for the yeast strains, and M De Cock for help in preparing the manuscript. This research was supported by a grant from the Fund for Scientific Research (Flanders) (G.0047.96). EB-B is the recipient of a predoctoral Human Capital and Mobility fellowship from the European Union (41SF6694).
Notes
1 CEF was deposited in the EMBL databank with the accession number AJ251579. 
2 To whom correspondence should be addressed. Fax: +32 9 264 5349. E-mail: diinz{at}gengenp.rug.ac.be
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