Proteomic analysis of the pyrenoid-traversing membranes of Chlamydomonas reinhardtii reveals novel components

Summary Pyrenoids are algal CO 2 -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 CO 2 . 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-CO 2 -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 CO 2 -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.