The plastoglobule-localized protein AtABC1K6 is a Mn2+-dependent kinase necessary for timely transition to reproductive growth

Abstract

ABSTRACT The A bsence of bc 1 Complex (ABC1) is an ancient, atypical protein kinase family that emerged prior to the archaeal-eubacterial divergence. Loss-of-function mutants in ABC1 genes are linked to respiratory defects in microbes and humans, and to compromised photosynthetic performance and stress tolerance in plants. However, demonstration of protein kinase activity remains elusive, hampering their study. Here, we investigate a homolog from Arabidopsis thaliana , AtABC1K6, and demonstrate in vitro protein kinase activity as autophosphorylation, which we replicate with a human ABC1 ortholog. We show that AtABC1K6 protein kinase activity requires an atypical buffer composition, including Mn 2+ as divalent cation co-factor and a low salt concentration. AtABC1K6 associates with plastoglobule lipid droplets of A. thaliana chloroplasts, along with five paralogs. Protein kinase activity associated with isolated A. thaliana plastoglobules was inhibited at higher salt concentrations, but could accommodate Mg 2+ as well as Mn 2+ , indicating salt sensitivity, but not the requirement for Mn 2+ , may be a general characteristic of ABC1s. Loss of functional AtABC1K6 impairs the developmental transition from vegetative to reproductive growth. This phenotype is complemented by the wild-type sequence of AtABC1K6 but not by a kinase-dead point mutant in the unique Ala-triad of the ATP-binding pocket, demonstrating the physiological relevance of the protein’s kinase activity. We suggest that ABC1s are bona fide protein kinases with a unique regulatory mechanism. Our results open the door to detailed functional and mechanistic studies of ABC1s and plastoglobules. SIGNIFICANCE STATEMENT The A bsence of bc 1 Complex (ABC1) is an ancient, atypical protein kinase family with enigmatic physiological roles in a wide range of species including plants, humans and microbes. While mutants demonstrate their critical role for organismal survival, their study has been severely hampered by the previous inability to determine catalytic function. Here, we demonstrate in vitro protein kinase activity with an A. thaliana homolog, AtABC1K6. Loss of functional AtABC1K6 impairs the developmental transition from vegetative to reproductive growth. The lack of phenotypic complementation with a kinase-dead point mutant demonstrates the physiological relevance of the protein’s kinase activity. Our results present the experimental means to investigate the targets, functions, and regulation of ABC1s.