Assembly of the algal CO₂-fixing organelle, the pyrenoid, is guided by a Rubisco-binding motif

Abstract

Approximately one-third of the Earth’s photosynthetic CO2 assimilation occurs in a pyrenoid, an organelle containing the CO2-fixing enzyme Rubisco. How constituent proteins are recruited to the pyrenoid, and how the organelle’s sub-compartments - membrane tubules, a surrounding phase-separated Rubisco matrix, and a peripheral starch sheath - are held together is unknown. Using the model alga Chlamydomonas reinhardtii, we discovered that pyrenoid proteins share a sequence motif. We show that the motif is sufficient to target proteins to the pyrenoid and that the motif binds to Rubisco, suggesting a mechanism for targeting. The presence of the Rubisco-binding motif on proteins that localize to the tubules and on proteins that localize to the matrix-starch sheath interface suggests that the motif holds the pyrenoid’s three sub-compartments together. Our findings advance our understanding of pyrenoid biogenesis and illustrate how a single protein motif can underlie the architecture of a complex multi-layered phase-separated organelle.One Sentence SummaryA ubiquitous Rubisco-binding motif targets proteins to the pyrenoid and holds together the pyrenoid’s three sub-compartments.

Discovery of a carbonic anhydrase-Rubisco complex within the alpha-carboxysome

“…An emerging theme of CCM self-assembly is that Rubisco interacts with various CCM proteins via Short Linear Motifs (SLiMs) found in intrinsically disordered proteins/regions (IDP/Rs) (27)(28)(29). Since CsoSCA's N48-peptide appears to bind Rubisco (Fig.…”

“…In contrast to these bacterial systems, in the model algae Chlamydomonas reinhardtii a repeat SLiM in the disordered scaffolding protein EPYC is essential for pyrenoid formation (42,43) and binds on top of the small subunit via salt bridges and a hydrophobic interface (28). The sequence motif is shared among many pyrenoid proteins, suggesting a mechanism for protein targeting as well as more broadly organizing pyrenoid ultrastructure (29). The versatility in binding motif and binding site, and the convergent function of diverse Rubisco as a hub of interaction implies this might be a general feature, which raises a final question.…”

Diffusion barriers and adaptive carbon uptake strategies enhance the modeled performance of the algal CO2-concentrating mechanism

“…Then, the newly formed matrix needs to be positioned proximal to low-pH thylakoids, and the thylakoid CA needs to be localized to the pyrenoid-proximal thylakoid lumen. Recent work in Chlamydomonas has shown that a Rubisco-binding motif targets proteins to the pyrenoid, and appears to link the matrix to the intra-pyrenoid tubules and starch sheath (70), thus providing a framework for manipulating the organization of an engineered pyrenoid. For example, constructing a fusion protein of the plant thylakoid CA and a membrane protein containing the Rubisco-binding motif may promote the desired colocalization of the pyrenoid matrix, thylakoids, and thylakoid CA.…”

Synthetic Biology Approaches To Enhance Microalgal Productivity

“…Pyrenoidsthe microalgal subcompartments in which RuBisCO is often located to facilitate higher local CO 2 concentrationsare another complication facing the expression of heterologous RuBisCO in microalgae. It was recently determined that the pyrenoid compartment assembles by specific binding of pyrenoid-associated proteins to RuBisCO [43]. This assembly is likely disrupted when replacing a native RuBisCO by a heterologous candidate.…”