Methods to study stromal-cell derived factor 2 in the context of ER stress and the unfolded protein response in Arabidopsis thaliana.
The accumulation of misfolded or unfolded polypeptides in the endoplasmic reticulum (ER) provokes ER stress and triggers protective signaling pathways termed the unfolded protein response (UPR). Stromal cell-derived factor 2 (SDF2)-type proteins are conserved throughout the animal and plant kingdoms. Upon UPR activation transcription of SDF2-type genes is significantly enhanced in metazoan and plants, suggesting an evolutionarily conserved role. However, the precise molecular function of SDF2-type proteins still needs to be established. Most eukaryotes have two SDF2 homologous, whereas the model plant Arabidopsis thaliana has a single SDF2, thus representing an ideal model system to study the functional role of SDF2-type proteins. This chapter provides techniques to study SDF2 in the context of ER stress in Arabidopsis. We describe available sdf2 mutants, and methods to evaluate ER stress sensitivity of seedlings. Further, we summarize tools and methods that are helpful to monitor UPR induction in general (e.g., SDF2 promoter-reporter fusion constructs and SDF2-specific antibodies). In Section 6, we provide protocols for the expression and purification of recombinant SDF2 protein that can be used for further biochemical studies.
Department of Cell Chemistry, Centre for Organismal Studies (COS), University of Heidelberg, Heidelberg, Germany.
Andrea
A
Schott
Sabine
S
Strahl
D017360:Arabidopsis / Q000033:anatomy & histology / Q000502:physiology*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000502:physiology*; D006801:Humans; D018517:Plant Roots / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D036226:Seedlings / Q000235:genetics / Q000378:metabolism; D013312:Stress, Physiological; D014026:Nicotiana / Q000033:anatomy & histology / Q000502:physiology; D056811:Unfolded Protein Response / Q000502:physiology*
D029681:Arabidopsis Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; C102467:SDF2 protein, human