Biochemical analysis of IMMUTANS, a potential plastoquinol oxidase of the thylakoid membrane

Abstract

Analysis of the variegated immutans mutant of Arabidopsis thaliana has led to the recent identification of a chloroplast-located protein with strong similarities to the mitochondrial alternative oxidase [1,2]. This sequence similarity suggests a role for IMMUTANS as a plastoquinol oxidase and thus a possible component of a respiratory electron transfer chain within the chloroplast. To investigate the biochemical properties of IMMUTANS further we have generated monospecific antibodies following overexpression of the IMMUTANS gene in E. coli. These antibodies have been used to analyse the location of IMMUTANS within the chloroplast of higher plants and to estimate the level of expression in various plastid types. In order to obtain direct evidence for a role as a quinol oxidase, we also describe attempts to refold the enzyme from the insoluble inclusion bodies obtained after over-expression in E. coli.