Identification of SUMO related proteins in planta

Hee Jin Park, Purdue University

Abstract

Reversible conjugation of the small ubiquitin modifier (SUMO) peptide to proteins (sumoylation) plays important roles in many cellular processes including DNA repair, subcellular localization, transcriptional regulation, cell cycle progression, ubiquitination antagonism in animal and yeast. SUMO binding itself seems to affect biological function. In plants, sumoylation is involved in abiotic stress responses (phosphate deficiency, heat, low temperature, and drought) and biotic stress (defense reaction to pathogen infection), and flowering time. In animal and yeast more than 200 SUMO target proteins have been characterized, but only a few plant SUMO targets are known through null loss of function AtSIZ1, SUMO E3 ligase, atsiz1 mutant phenotypes: AtMYB30, PHR1, ICE1, FLD, GTEs. Amending this deficiency, two independent proteomic-type approaches were developed. Ultimately, my goal is to characterize biological functions of genuine plant targets of sumoylation. The first system includes overexpression of AtSUMO1/AtSUM1 fused to 6His-3Flag tag in Arabidopsis. Total protein extracts were applied to Ni-NTA affinity chromatography followed by 2D SDS-PAGE. Gel-excised putative SUMO-binding candidate proteins were identified by MALDI-TOP mass spectrometry. System two was designed for affinity chromatography with His-GST-AtSUM1 and total protein extracts from Arabidopsis. SUMO-binding protein complexes were then purified and identified by LC-MS/MS. More than 20 SUMO binding proteins were identified from these two individual means. Several of these candidate proteins have been analyzed by a split ubiquitination assay in yeast and additionally by split-luciferase complement assays in planta to confirm binding activity to AtSUM1, and productive sumoylation through in vivo assays in E.coli. Among the proteins that were found reliably sumoylated are AtMYB31 (transcription factor), ACS7 (1-Amino-cyclopropane-1-carboxylate synthase 7), and Rho GDI1 (Rho GDP-dissociation inhibitor 1). The precise function of sumoylation and the rationale in terms of altered activity that sumoylation confers on these proteins is presently unknown. SIZs, members of PIAS [protein inhibitor of activated signal transducers and activators of transcription (STAT)] and SAP (scaffold attachment factor A/B/acinus/PIAS)/MIZ (SIZ) proteins exhibit SUMO E3-ligase activity that facilitates the conjugation of SUMO proteins to target substrates. There are two homologous SIZs, SIZ1 and SIZ2 in rice (Oryza sativa). OsSIZ proteins are localized to the nucleus and showed sumoylation activities in a tobacco system. Increased amounts of SUMO conjugates associated with environmental stresses such as high and low temperature, NaCl and abscisic acid (ABA) in rice plants. The expression of OsSIZ1 and OsSIZ2 in siz1-2 Arabidopsis plants partially complemented the morphological mutant phenotype and enhanced levels of SUMO conjugates under heat shock conditions. In addition, ABA hypersensitivity of atsiz1-2 seed germination was partially suppressed by OsSIZ1 and OsSIZ2. The results suggest that rice SIZ1 and SIZ2 are able to functionally complement Arabidopsis SIZ1 in the SUMO conjugation pathway. Their effects on the Arabidopsis mutant suggest a function for these genes related to stress responses and stress adaptation. This study expands the list of Arabidopsis putative SUMO targets and helps in understanding SUMO conjugation in monocot plants.

Degree

Ph.D.

Advisors

Bressan, Purdue University.

Subject Area

Plant biology|Plant sciences

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