Assembly of a sub-cellular protein complex generates asymmetry in the bacterium Caulobacter crescentus [electronic resource]
Keywords: Biology.

The subcellular localization of different cell fate determinants at the cell poles serves to enable asymmetric cell division in many diverse cell types. The bacterium Caulobacter crescentus carries out an asymmetric cell division in each cell cycle. Key to the generation and maintenance of asymmetry in Caulobacter is the localization of two distinct sets of two-component signaling proteins to opposing cell poles. The asymmetric localization of these proteins ensures differential transcriptional readouts of the genome to produce two daughter cells with different cell fates upon the completion of cell division. The molecular mechanisms that are utilized to recruit these proteins to the pole remain ill defined. In this thesis work, I present evidence detailing the assembly of a polar protein complex that drives the asymmetric cell cycle of Caulobacter. Key to the recruitment of multiple polar proteins is the PopZ protein that forms a polymeric matrix at the cell pole. As it is one of the first proteins known to localize to the Caulobacter cell pole and is required for localization of at least 10 known polar proteins, it is critical to know how PopZ functions as a polar organizer. To understand how polar organizing centers are established by PopZ, I generated a set of mutated PopZ proteins and investigated the mutant strains for defects in sub-cellular localization and recruitment activity. I identified a domain within the C-terminal 76 amino acids of PopZ that is necessary and sufficient for accumulation as a single subcellular focus, and a 23 amino acid domain at the N-terminus that is necessary for bipolar targeting. Mutations in either domain caused defects in the recruitment of other factors to the cell poles, indicating a role for dynamic PopZ localization in polar organization. Mutations in the C-terminal domain also blocked discrete steps in the PopZ matrix assembly pathway. Biophysical analysis of purified wildtype and assembly-defective mutant proteins revealed that the PopZ self-associates into an elongated trimer, which readily forms a dimer of trimers through lateral contact. The final six amino acids of PopZ are necessary for connecting the hexamers into long filaments, which are important for subcellular localization. Thus, PopZ undergoes multiple orders of self-assembly, and the formation of an interconnected superstructure is a key feature of polar organization in Caulobacter. Downstream of PopZ, localization of the histidine kinase DivJ specifically to the stalked cell pole is critical for defining the stalked cell fate. As the Caulobacter cell cycle progresses, both the flagellum-bearing pole and the new stalked pole have a PopZ polymeric matrix, but the new stalked pole acquires the SpmX protein and the DivJ histidine kinase. Using both a heterologous in vivo expression system and binding assays with purified proteins, I determined the pathway of stalked pole complex formation. I demonstrate that SpmX, which is synthesized only at the swarmer to stalked cell transition, binds directly to PopZ via its lysozyme-like domain. Subsequently, newly synthesized DivJ binds directly to SpmX. The positioning of DivJ at one cell pole spatially restricts this histidine kinase so that upon division it is sequestered to the progeny stalked cell where it mediates stalked cell fate determination. Analysis of SpmX truncations and amino acid substitutions revealed that the additional regions of the SpmX protein, including a proline rich domain and the transmembrane domains, contribute to the asymmetric localization of SpmX, and consequently DivJ. Mistimed overproduction of SpmX resulted in the initiation of lateral growth zones providing insight into the function of the SpmX protein as a mediator of the three dimensional organization of the cell and, via DivJ, the maintenance of asymmetry. This thesis work utilizes a combination of biochemistry, cell biology, and molecular biology to detail the assembly of a protein complex that serves to maintain and generate cellular asymmetry in Caulobacter. The insights gained into the mechanisms that drive formation of this complex have broad implications in the understanding of subcellular protein localization in other bacteria, as well as in the understanding of cellular asymmetry in all kingdoms of life.