Lateral organization of cytochrome b6f in thylakoid membranes controls photosynthetic electron transfer efficiency

Photosynthetic electron transfer relies on the coordinated function and spatial organization of large protein complexes within the thylakoid membrane. The cytochrome b6f complex (b6f) functionally interconnects photosystem (PS) II and PSI in photosynthetic electron transfer and is equally distributed between appressed and non-appressed thylakoid membranes. Here, we investigate the functional link between the lateral distribution of b6f and efficient photosynthetic electron flow in Chlamydomonas reinhardtii. We engineered strains with stromal fusions between PetA of b6f and two fluorescent proteins (FPs) of different molecular mass: Clover and ATeam. Under oxic conditions, these strains exhibited significantly slower electron transfer rates (ETR), lower PSII quantum yields, and increased donor-side limitation of PSI. State transitions were diminished in the fusion strains, accompanied by a strong impairment of STT7-dependent function, suggesting that the presence of fused FPs at b6f interfere with STT7 function. Yet, ETR phenotypes were STT7-independent and FP fusion did not impact intrinsic b6f function. In situ cryogenic electron tomography revealed a significant depletion of b6f from appressed thylakoid membranes in the ATeam strains, while the overall membrane protein concentration remained unchanged. Overall, our data indicate that a balanced distribution of b6f between appressed and non-appressed thylakoid membranes is essential for regulating photosynthetic electron transfer, highlighting the functional importance of thylakoid molecular architecture in vivo.