Polymerization in the actin ATPase clan regulates hexokinase activity in yeast.

The actin fold is found in cytoskeletal polymers, chaperones, and various metabolic enzymes. Many actin-fold proteins, such as the carbohydrate kinases, do not polymerize. We found that Glk1, a Saccharomyces cerevisiae glucokinase, forms two-stranded filaments with ultrastructure that is distinct from that of cytoskeletal polymers. In cells, Glk1 polymerized upon sugar addition and depolymerized upon sugar withdrawal. Polymerization inhibits enzymatic activity; the Glk1 monomer-polymer equilibrium sets a maximum rate of glucose phosphorylation regardless of Glk1 concentration. A mutation that eliminated Glk1 polymerization alleviated concentration-dependent enzyme inhibition. Yeast containing nonpolymerizing Glk1 were less fit when growing on sugars and more likely to die when refed glucose. Glk1 polymerization arose independently from other actin-related filaments and may allow yeast to rapidly modulate glucokinase activity as nutrient availability changes.

Evolution of polymer formation within the actin superfamily.

While many are familiar with actin as a well-conserved component of the eukaryotic cytoskeleton, it is less often appreciated that actin is a member of a large superfamily of structurally related protein families found throughout the tree of life. Actin-related proteins include chaperones, carbohydrate kinases, and other enzymes, as well as a staggeringly diverse set of proteins that use the energy from ATP hydrolysis to form dynamic, linear polymers. Despite differing widely from one another in filament structure and dynamics, these polymers play important roles in ordering cell space in bacteria, archaea, and eukaryotes. It is not known whether these polymers descended from a single ancestral polymer or arose multiple times by convergent evolution from monomeric actin-like proteins. In this work, we provide an overview of the structures, dynamics, and functions of this diverse set. Then, using a phylogenetic analysis to examine actin evolution, we show that the actin-related protein families that form polymers are more closely related to one another than they are to other nonpolymerizing members of the actin superfamily. Thus all the known actin-like polymers are likely to be the descendants of a single, ancestral, polymer-forming actin-like protein.

Identification of ClpP substrates in Caulobacter crescentus reveals a role for regulated proteolysis in bacterial development.

Energy-dependent proteases ensure the timely removal of unwanted proteins in a highly selective fashion. In Caulobacter crescentus, protein degradation by the ClpXP protease is critical for cell cycle progression; however, only a handful of substrates are currently known. Here, we use a trapping approach to identify putative substrates of the ClpP associated proteases in C. crescentus. Biochemical validation of several of these targets reveals specific protease recognition motifs and suggests a need for ClpXP-specific degradation beyond degradation of known cell cycle regulators. We focus on a particular instance of regulated proteolysis in Caulobacter by exploring the role of ClpXP in degrading the stalk synthesis transcription factor TacA. We show that TacA degradation is controlled during the cell cycle dependent on the ClpXP regulator CpdR and that stabilization of TacA increases degradation of another ClpXP substrate, CtrA, while restoring deficiencies associated with prolific CpdR activity. Together, our work reveals a number of new validated ClpXP substrates, clarifies rules of protease substrate selection, and demonstrates how regulated protein degradation is critical for Caulobacter development and cell cycle progression.

Adaptor-dependent degradation of a cell-cycle regulator uses a unique substrate architecture.

In Caulobacter crescentus, the ClpXP protease degrades several crucial cell-cycle regulators, including the phosphodiesterase PdeA. Degradation of PdeA requires the response regulator CpdR and signals a morphological transition in concert with initiation of DNA replication. Here, we report the structure of a Per-Arnt-Sim (PAS) domain of PdeA and show that it is necessary for CpdR-dependent degradation in vivo and in vitro. CpdR acts as an adaptor, tethering the amino-terminal PAS domain to ClpXP and promoting recognition of the weak carboxyl-terminal degron of PdeA, a combination that ensures processive proteolysis. We identify sites on the PAS domain needed for CpdR recognition and find that one subunit of the PdeA dimer can be delivered to ClpXP by its partner. Finally, we show that improper stabilization of PdeA in vivo alters cellular behavior. These results introduce an adaptor/substrate pair for ClpXP and reveal broad diversity in adaptor-mediated proteolysis.