Assembly of the algal CO ₂ -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 subcompartments—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 found that pyrenoid proteins share a sequence motif. We show that the motif is necessary and 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 subcompartments together. Our findings advance our understanding of pyrenoid biogenesis and illustrate how a single protein motif can underlie the architecture of a complex multilayered phase-separated organelle.

Liquid-Liquid Phase Separation: Unraveling the Enigma of Biomolecular Condensates in Microbial Cells

“…In eukaryotic microalgae, liquid-like Rubisco-EPYC1 (Essential Pyrenoid Component 1) condensates display functional similarity to Rubisco-CcmM condensates, they are found compartmentalized in an analogous chloroplastlike CCM compartment called the pyrenoid (Figure 3B; The pyrenoid matrix is predominantly composed of Rubisco-EPYC1 complexes, forming by the multivalent interactions of EPYC1 peptide (orange) and Rubisco (green and yellow) (Freeman Rosenzweig et al, 2017;Wunder et al, 2018;Meyer et al, 2020;Barrett et al, 2021). Cryo-EM supported a structural model (7jsx, He et al, 2020), showing that EPYC1 binds close to the equator of the Rubisco cylinder and forms a codependent network of the specific low-affinity bonds (Mackinder et al, 2016;He et al, 2020).…”

“…Cryo-ET also showed that both the algal EPYC1 and cyanobacterial CcmM bind close to the equator of the Rubisco cylinder ( Wang et al, 2019 ; He et al, 2020 ), although the binding sites are different. Specifically, EPYC1 binds uniquely to the Rubisco small subunit (RbcS) via electrostatic and hydrophobic interactions ( Figure 3B ; He et al, 2020 ; Meyer et al, 2020 ), while CcmM contacts both Rubisco large subunit (RbcL, Wang et al, 2019 ) and RbcS via salt bridges and van der Waals contacts ( Figure 3A ). Both EPYC1 and CcmM have repeat regions and intrinsically disordered proteins, and as has been side, the Rubisco condensation events appear to be regulated in a similar multivalent mechanism.…”

Condensation of Rubisco into a proto-pyrenoid in higher plant chloroplasts

“…Recent work in the model green alga Chlamydomonas reinhardtii (hereafter Chlamydomonas) has shown that the pyrenoid is a liquid-like condensate that undergoes liquid −liquid phase separation when the CCM is active 14 . Phase separation is facilitated by multivalent interactions between the intrinsically disordered Rubisco-binding protein EPYC1 and the small subunit of Rubisco (SSU) [15][16][17][18][19][20] , of which residues on the α-helices of the native SSU isoforms are critical for binding to EPYC1. Previously we have shown that Arabidopsis can assemble a functional plant-algal hybrid Rubisco complex, with the native large subunit of Rubisco (LSU) and an SSU from Chlamydomonas, which has similar kinetic properties to wild-type (WT) Arabidopsis Rubisco 21 .…”

Chloroplast transit peptides often require downstream unstructured sequence in Chlamydomonas reinhardtii

“…1I,J), strikingly visible as fluorescence from inside the pyrenoid, a proteinaceous structure within the chloroplast that generates a visible dip in chlorophyll fluorescence (Mackinder et al, 2017). Note that the RBCA-cTP+23 construct contains a pyrenoid-localisation motif (Meyer et al, 2020). By contrast, mitochondrial targeting peptides (mTPs) from carbonic anhydrase 4 (CAH4) and γ-carbonic anhydrase 2 (CAG2) show mitochondrial targeting, evident as co-localisation of Venus and MitoTracker fluorescence, in the absence of any post-cleavage site residues (Fig.…”