Determination of the oligomeric state of cAAP and cGEP, two chloroplast-localized serine proteases

Plant proteases function in numerous cellular and physiological processes. A number of plant peptidases are chloroplast-localized. Arabidopsis thaliana genes At2g47390 and At5g36210 encode chloroplast-localized glutamyl e ndopeptidase (cGEP) and chloroplast-localized acyl aminoacyl peptidase (cAAP), respectively. Although little is known about the function of cGEP and cAAP, cGEP has been implicated in senescence, and both cGEP and cAAP may function in response to carbon starvation under etiolated conditions. Both cGEP and cAAP are S9 serine peptidases. Many serine peptidases form protein complexes. The ability of cGEP and cAAP to homo- or heterodimerize was first examined using the yeast two-hybrid system. Results suggested that cGEP and cAAP do not homo- or heterodimerize. As an additional means of examining potential protein-protein interactions, his-tagged cGEP and cAAP lines were generated for immunoblot analysis following native PAGE. Although his-tagged cGEP lines were not recovered, native PAGE coupled to immunoblot analysis revealed that his-tagged cAAP is monomeric.