Molecular analysis and subcellular localization of peptidase silpepsin 2 from Silybum marianum

MARIA LAURA COLOMBO; AGUSTINA FERNÁNDEZ; CONSTANZA LIGGIERI; SANDRA ELIZABETH VAIRO CAVALLI

Córdoba

Congreso; LII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2016

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Typical plant aspartic peptidases (APs)are comprised of both pre- and pro-regions as well as a C-terminal region containing a plant-specific insertion (PSI). APs from thistle flowers precursors have highly conserved signal peptides that direct the proteins to the secretory pathway.The aims of this work were to perform a molecular analysis of silpepsin 2, an AP from flowers of Silybum marianum, and to analyse its subcellular localization by confocal laser scanning microscopy. A structural model of the zymogen was built using MODELLER v9.14 based on the crystal structure of prophytepsin from barley as template (PDB: 1QDM), found with HHPred fold assignment method. The best model was assessed for quality using both energetic and structural criteria. Typical AP folding patternwas observed. The subcellular localization of silpepsin 2 was examinedby transient expression of a red fluorescent protein (RFP) fusion protein in leaf epidermis of Nicotiana benthamiana accomplished by GATEWAY technology ( vector pB7RWG2) and Agrobacterium- mediated DNA transfer. 1, 2, and 3 days post-infiltration, cells were imaged by confocal laser scanning microscopy to assess the intracellular localization of the protein. Results obtained indicate that silpesin 2 directs RFP to the vacuole in accordance with the expected localization in the secretory pathway.