THE MECHANISM, FUNCTION, AND UTILITY OF CTP SYNTHETASE POLYMERS IN ESCHERICHIA COLI, A NOVEL FORM OF CELLULAR METABOLIC REGULATION

Abstract

All organisms require CTP to maintain their genome, produce RNA, and build phospholipids. CTP Synthetase (CtpS) is the enzyme responsible for catalyzing the conversion of UTP to CTP. CtpS is one of a growing list of metabolic enzymes now shown to form large-scale ordered structures such as filaments. CtpS abundance and enzymatic activity is regulated at transcriptional and post-translational levels. Many of these strategies have been well characterized such as reiterative transcriptional control of CtpS mRNA expression and allosteric control of protein activity. Recently, several groups including our own observed CtpS assembly into large filaments in prokaryotic and eukaryotic cells. Here, I describe how polymerization into filaments regulates CtpS activity in E. coli cells and represents yet another potent regulatory mechanism for this complex enzyme. CtpS polymerizes in response to an accumulation of its product, CTP. The repeating unit of these polymers is the otherwise enzymatically active CtpS tetramer. Paradoxically, the CtpS tetramer is in an active conformation within the polymer, but CtpS polymers are catalytically inactive and must dissociate to resume CTP production. We hypothesize that conformational flexibility is central to the catalytic mechanism of CtpS, explaining why incorporation into a sterically hindered polymer abolishes activity. Disruption of normal polymerization renders cells unable to maintain normal, balanced nucleotide pools and causes a growth defect. Using a functional fluorescent fusion to CtpS, we can directly observe CtpS polymerization in cells. These CtpS polymers respond to changes in cellular metabolic state in a manner consistent with their predicted use as a protein sequestration and storage device. CtpS polymer dynamics are also utilized during developmental transitions where metabolic needs change over time. Because individual CtpS concentrations and abundance of CtpS polymers vary from cell to cell, we suggest that CtpS polymerization can be used as marker of single-cell metabolic state. Together, this work characterizes a molecular mechanism for the regulation of CtpS activity by polymerization and demonstrates its utility as a sensitive regulator of nucleotide pools as well as a tool to probe physiology at the single-cell level.