Significance: Eukaryotic algae, which play a fundamental role in global CO ₂ fixation, enhance the performance of the carbon-fixing enzyme Rubisco by placing it into an organelle called the pyrenoid. Despite the ubiquitous presence and biogeochemical importance of this organelle, how Rubisco assembles to form the pyrenoid remains a long-standing mystery. Our discovery of an abundant repeat protein that binds Rubisco in the pyrenoid represents a critical advance in our understanding of pyrenoid biogenesis. The repeat sequence of this protein suggests elegant models to explain the structural arrangement of Rubisco enzymes in the pyrenoid. Beyond advances in basic understanding, our findings open doors to the engineering of algal pyrenoids into crops to enhance yields.

Significance: Many cellular structures are assembled via phase separation, forming liquid-like droplets in a manner analogous to oil in water. How can the cell control whether there is 1 droplet or many? Here, we provide insights into this question by studying the pyrenoid, an algal liquid-like organelle that mediates a substantial portion of global CO ₂ fixation. Whereas most algae have just 1 pyrenoid, we characterize a mutant that has approximately 10 pyrenoids per cell. Our results suggest a model where imposing an increased surface area on the liquid-like pyrenoid matrix favors the formation of multiple pyrenoids, with a concomitant loss of photosynthetic efficiency. Our findings advance our basic understanding of the biogenesis of the pyrenoid and of phase-separated organelles in general.

The pyrenoid of the unicellular green alga Chlamydomonas reinhardtii is a microcompartment situated in the centre of the cup-shaped chloroplast, containing up to 90% of cellular Rubisco. Traversed by a network of dense, knotted thylakoid tubules, the pyrenoid has been proposed to influence thylakoid biogenesis and ultrastructure. Mutants that are unable to assemble a pyrenoid matrix, due to expressing a vascular plant version of the Rubisco small subunit, exhibit severe growth and photosynthetic defects and have an ineffective carbon-concentrating mechanism (CCM). The present study set out to determine the cause of photosynthetic limitation in these pyrenoid-less lines. We tested whether electron transport and light use were compromised as a direct structural consequence of pyrenoid loss or as a metabolic effect downstream of lower CCM activity and resulting CO₂ limitation. Thylakoid organization was unchanged in the mutants, including the retention of intrapyrenoid-type thylakoid tubules, and photosynthetic limitations associated with the absence of the pyrenoid were rescued by exposing cells to elevated CO₂ levels. These results demonstrate that Rubisco aggregation in the pyrenoid functions as an essential element for CO₂ delivery as part of the CCM, and does not play other roles in maintenance of photosynthetic membrane energetics.

Mitochondria and chloroplasts emerged from primary endosymbiosis. Most proteins of the endosymbiont were subsequently expressed in the nucleo-cytosol of the host and organelle-targeted via the acquisition of N-terminal presequences, whose evolutionary origin remains enigmatic. Using a quantitative assessment of their physico-chemical properties, we show that organelle targeting peptides, which are distinct from signal peptides targeting other subcellular compartments, group with a subset of antimicrobial peptides. We demonstrate that extant antimicrobial peptides target a fluorescent reporter to either the mitochondria or the chloroplast in the green alga Chlamydomonas reinhardtii and, conversely, that extant targeting peptides still display antimicrobial activity. Thus, we provide strong computational and functional evidence for an evolutionary link between organelle-targeting and antimicrobial peptides. Our results support the view that resistance of bacterial progenitors of organelles to the attack of host antimicrobial peptides has been instrumental in eukaryogenesis and in the emergence of photosynthetic eukaryotes.

The N-terminal sequence stretch that defines subcellular targeting for most nuclear encoded chloroplast proteins is usually considered identical to the sequence that is cleaved upon import. Yet here this study shows that for eight out of ten tested Chlamydomonas chloroplast transit peptides, significant additional sequence stretches past the cleavage site are required to enable efficient chloroplast import of heterologous cargo proteins. Analysis of Chlamydomonas cTPs with known cleavage sites and replacements of native post-cleavage residues with alternative sequences points to a role for unstructured sequence at mature protein N-termini.