Proteomic analysis of the pyrenoid‐traversing membranes of <i>Chlamydomonas reinhardtii</i> reveals novel components

Pyrenoids are algal CO₂-fixing organelles that mediate approximately one-third of global carbon fixation. Most pyrenoids are traversed by membranes that are thought to supply them with concentrated CO₂. Despite the critical nature of these membranes for pyrenoid function, they are poorly understood, with few protein components known in any species. Here, we identify protein components of the pyrenoid-traversing membranes from the leading model alga, Chlamydomonas reinhardtii, by affinity purification and mass spectrometry of membrane fragments. Our proteome includes previously known proteins as well as novel candidates. We further characterize two of the novel pyrenoid-traversing membrane-resident proteins: Cre10.g452250, which we name Pyrenoid Membrane Enriched 1 (PME1), and Cre02.g143550, also known as Low-CO₂-Induced 16 (LCI16). We confirm the pyrenoid-traversing membrane localization of LCI16 and observe that PME1 and LCI16 physically interact. We find that neither protein is required for normal membrane morphology or growth under CO₂-limiting conditions, but that both mutants show a similar proteomic profile to those of established pyrenoid mutants. Taken together, our study identifies the proteome of the pyrenoid-traversing membranes and initiates the characterization of a novel pyrenoid-traversing membrane complex, building toward a mechanistic understanding of the pyrenoid.