UC Berkeley

Many bacteria employ a protein organelle, the carboxysome, to concentrate carbon dioxide

and catalyze the initial fixation reaction. Only 10 genes from Halothiobacillus

neapolitanus are sufficient for heterologous expression of carboxysomes in Escherichia coli,

opening the door to mechanistic analysis of the assembly process of this 200 MDa+

complex. One of these genes, csoS2, produces two highly repetitive intrinsically-disordered

protein isoforms and has been shown to be indispensable in carboxysome assembly.

Detailed functional characterization of csoS2, however, has been hindered by the lack of

understanding of how this single gene yields expression of two gene products. In this work,

we set out to develop a deeper understanding of CsoS2's biogenesis and its function in α-

carboxysome assembly. Using tandem mass spectrometry and biochemical assays, we have

revealed that -1 programmed ribosomal frameshifting (- 1 PRF) is responsible for the

generation of a truncated protein with C-terminus translated from the -1 frame, CsoS2A, in

addition to the full-length protein, CsoS2B. We show for the first time that CsoS2B can be

independently produced by mutations of -1 PRF elements and only CsoS2B is necessary for

the assembly of H. neapolitanus carboxysomes in native and heterologous hosts. With the

knowledge of the identity of CsoS2 isoforms, we next investigate the ability of individual

CsoS2 domains to participate in protein-protein interaction with RuBisCO, the primary

enzymatic component of the carboxysome. Here, we demonstrate that the 259-aa N-

terminal domain of CsoS2 multivalently binds RuBisCO, potentially via its short

amphiphatic helices. Finally, based on our findings, we propose a hypothetical model that

describes the formation and maturation of the α-carboxysome. This work illustrates, for

the first time, the simultaneous involvement of cotranslational regulation and an

intrinsically-disordered protein in the assembly of a prokaryotic organelle.