Increased size exlusion limit 2(ISE2) DEVH-box RNA helicase localizes in chloroplasts

OROURKE GRETA, WIRTH SONIA, KOBAYASHI KEN

Puerto Madryn

Congreso; XLVI Reunión Anual de la Sociedad Argentina de Investigaciones Bioquímicas.; 2010

SAIB

Intercellular communication in plants is mediated by plasmodesmata (PD), cytoplasmic channels that span cell walls connecting directly the cytoplasm of juxtaposed adjacent cells, and play key roles in coordinating development programs. Arabidopsis thaliana ise2 gene encodes a DEVH-box type RNA helicase that is described to regulate PDs function and architecture. Our firsts evidences localized ISE2-GFP fusion protein in cytoplasmic granules resembling RNA containing stress granules. However, recent proteomic database localized the endogenous ISE2 in chloroplast. In order elucidate this controversy; we produced specific antiserum to ISE2. The carboxy-terminal region of ISE2 fused to His tag was produced in E. coli. The protein purification was then performed in nickel-agarose resins, followed by a mouse immunization protocol. Finally, the antiserum specificity and titer were determined by western blot analysis. We also purified chloroplasts from transgenic Arabidopsis thaliana plant overexpressing ISE2-GFP and detected it using anti-GFP antibody in western blot. We detected ISE2-GFP in the organella preparation, suggesting that endogenous ISE2 localizes in chloroplast. Endogenous ISE2 subcellular localization was analyzed by our ISE2 antiserum. Our results suggest that lack of ISE2 might produce chloroplast alteration leading to deregulation of PD function.