Structure and Assembly of the Cyanobacterial Carboxysome Matrix

Intracellular microcompartments are potentially useful tools for metabolic regulation and optimization. The cyanobacterial β-carboxysome is a natural microcompartment used to concentrate bicarbonate and carbon dioxide, while excluding oxygen, in the vicinity of the carbon fixing enzymes ribulose-1,5- bisphosphate carboxylase (RuBisCO) and carbonic anhydrase (CA). Our lab has previously shown that in Synechococcus elongatus β-carboxysomes assemble in an inside-to-outside manner, with RuBisCo aggregations budding off existing carboxysomes prior to encapsulation by an outer protein shell. Based on
previous observations and known protein structures, it is hypothesized that this initial protein aggregation (termed the carboxysome matrix) has an ordered lattice-like structure and is a key aspect of β-carboxysome assembly, guiding
both the final size and number of carboxysomes within the cell. Here we describe our current efforts to elucidate the spatial and temporal pattern of internal carboxysome matrix assembly using super resolution microscopy (SIM) and in vitro techniques. To this end we have established a β-cyanobacterial line expressing a fluorescently tagged copy of the inner matrix structural protein CcmM for in vivo imaging alongside fluorescently tagged RuBisCo and carboxysome shell proteins. We have also purified the short isoform of CcmM (M35), RuBisCo small and large subunits, and shell components for characterization with electron microscopy. We hope that by developing an improved picture of carboxysome self-assembly using these techniques, we will provide a useful foundation for producing synthetic protein-based microcompartments and engineering improved photosynthetic efficiency in plants and cyanobacteria.