The Structural Basis of Rubisco Phase Separation in the Pyrenoid

Abstract

Approximately one-third of global CO2 fixation occurs in a phase separated algal organelle called the pyrenoid. Existing data suggest that the pyrenoid forms by the phase-separation of the CO2-fixing enzyme Rubisco with a linker protein; however, the molecular interactions underlying this phase-separation remain unknown. Here we present the structural basis of the interactions between Rubisco and its intrinsically disordered linker protein EPYC1 (Essential Pyrenoid Component 1) in the model alga Chlamydomonas reinhardtii. We find that EPYC1 consists of five evenly-spaced Rubisco-binding regions that share sequence similarity. Single-particle cryo-electron microscopy of one of these regions in complex with Rubisco indicates that each Rubisco holoenzyme has eight binding sites for EPYC1, one on each Rubisco small subunit. Interface mutations disrupt binding, phase separation, and pyrenoid formation. Cryo-electron tomography supports a model where EPYC1 and Rubisco form a co-dependent multivalent network of specific low-affinity bonds, giving the matrix liquid-like properties. Our results advance the structural and functional understanding of the phase separation underlying the pyrenoid, an organelle that plays a fundamental role in the global carbon cycle.

Assembly of the algal CO 2 -fixing organelle, the pyrenoid, is guided by a Rubisco-binding motif

“…Two observations led us to hypothesize that proteins bearing the motif are recruited to the pyrenoid via binding to Rubisco. First, the motif is present in each of the regions that were found to mediate EPYC1's binding to Rubisco in yeast two-hybrid experiments (20) and as short peptides in vitro (21). Second, the other five motifcontaining proteins were also previously found to bind to Rubisco: (14), and SAGA2, RBMP1, RBMP2, and CSP41A by affinity purification-mass spectrometry (18).…”

“…4B). In this model, multiple copies of the motif on EPYC1 mediate cohesion of the matrix by bringing Rubisco holoenzymes together (21). Moreover, the presence of the same motif on the pyrenoid tubule-localized transmembrane proteins, RBMP1 and RBMP2, recruits Rubisco to the tubules and favors assembly of the matrix around them.…”

“…4C). Second, point mutations that disrupt the EPYC1 binding site on Rubisco not only eliminate the matrix but additionally disrupt the association of the starch sheath with the pyrenoid (21). The additional disruption of the starch sheath can be explained by the same Rubisco-binding site being required not only for binding to EPYC1 but also for binding the motif on other proteins that connect the starch and tubules to Rubisco (Fig.…”

“…S9, A and B to G). The amino acids of the Rubisco small subunit that are essential for binding the motif (21,34) are also broadly present in the Volvocales ( fig. S9H).…”

“…(B and C) Arrays of 18-amino acid peptides tiling across the sequences of SAGA2 (B) and RBMP2 (C) were synthesized and probed with Rubisco. The binding signal in (B) and (C) is normalized to a control EPYC1 peptide known to bind to Rubisco (one unit of binding)(21). The positions of motifs are indicated below each graph (to scale).on November 24, 2020 http://advances.sciencemag.org/ Downloaded from…”