Abstract
Stresses increasing the load of unfolded proteins that enter the endoplasmic reticulum (ER) trigger a protective response termed the unfolded protein response (UPR). Stromal cell-derived factor2 (SDF2)-type proteins are highly conserved throughout the plant and animal kingdoms. In this study we have characterized AtSDF2 as crucial component of the UPR in Arabidopsis thaliana. Using a combination of biochemical and cell biological methods, we demonstrate that SDF2 is induced in response to ER stress conditions causing the accumulation of unfolded proteins. Transgenic reporter plants confirmed induction of SDF2 during ER stress. Under normal growth conditions SDF2 is highly expressed in fast growing, differentiating cells and meristematic tissues. The increased production of SDF2 due to ER stress and in tissues that require enhanced protein biosynthesis and secretion, and its association with the ER membrane qualifies SDF2 as a downstream target of the UPR. Determination of the SDF2 three-dimensional crystal structure at 1.95 A resolution revealed the typical beta-trefoil fold with potential carbohydrate binding sites. Hence, SDF2 might be involved in the quality control of glycoproteins. Arabidopsis sdf2 mutants display strong defects and morphological phenotypes during seedling development specifically under ER stress conditions, thus establishing that SDF2-type proteins play a key role in the UPR.
Highlights
In all eukaryotes, nascent secretory and membrane proteins synthesized at the rough endoplasmic reticulum (ER)6 are translocated, potentially glycosylated, and soon begin to fold with the assistance of molecular chaperones and other folding factors
Under normal growth conditions Stromal cell-derived factor2 (SDF2) is highly expressed in fast growing, differentiating cells and meristematic tissues
To analyze whether SDF2-type proteins play a functional role in the unfolded protein response (UPR), Arabidopsis is an ideal model system because the principal mechanisms of the UPR are conserved between metazoa and plants [2], and Arabidopsis contains only one gene of the SDF2-type (AtSDF2; At2g25110)
Summary
Plant Material and Growth Conditions—Arabidopsis ecotype Columbia-0 was used as wild-type line. The PCR product was cut with NcoI/XbaI and cloned into pAmy. Plasmid pAS66: the SDF2 coding sequence was amplified by PCR using primers 877 and 878. Subcellular Localization of SDF2—Microsomal membranes of plant materials were prepared as described [13] using extraction buffer (50 mM HEPES, 10 mM KCl, 0.5% polyvinylpyrolidone 40, 10% sucrose, and a mix of protease inhibitors (Sigma)) containing 5 mM MgCl2 or 5 mM EDTA. Preparation of cytosol (S1), 16,000 ϫ g supernatant containing microsomal contents (S2), and a 16,000 ϫ g total membrane pellet (P) was performed as described in Ref. 15. Structure Determination and Refinement of SDF2—Cloning, expression, purification, and crystallization of SDF2 as well as x-ray data collection were performed as described elsewhere [16]. Atomic coordinates and structure factors have been deposited in the Protein Data Bank under the accession code 3MAL
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