The Role of MITH1 as a Membrane-Condensate Tether in Pyrenoid Tubule Biogenesis in Chlamydomonas reinhardtii

Abstract

The pyrenoid is a key organelle in the algal carbon-concentrating mechanism (CCM) which is responsible for one-third of global carbon dioxide fixation; it optimizes photosynthesis by channeling concentrated CO2 through membrane tubules into a matrix containing the CO2-fixing enzyme, Rubisco, and the linker protein EPYC1. Engineering a pyrenoid into crop plants lacking native CCMs would increase yields and heat resilience while minimizing water usage, providing a reliable means of food production in the face of climate change. Previous work identified MITH1 as a protein required for proper pyrenoid tubule formation in the model alga Chlamydomonas reinhardtii (Chlamydomonas), but the mechanisms underlying MITH1 function remain uncharacterized. This thesis aims to determine which domains of MITH1 are responsible for its role in tubule biogenesis. In vitro liposome flotation assays demonstrate that the MITH1-VIPP1-like domain is sufficient to bind and shape liposomes, suggesting that it is critical for membrane interactions. By transforming MITH1 constructs lacking different domains into wildtype and mith1 mutant Chlamydomonas, I discovered that MITH1’s long coiled-coil (CC) domain is necessary for binding Rubisco, and that halving the CC domain creates disorganized tubules that retain pyrenoid function. These findings suggest that MITH1 functions as a multivalent adaptor protein, with its N-terminal domain mediating membrane interaction and its coiled-coil region facilitating Rubisco matrix integration, providing critical insights for bioengineering efforts to introduce pyrenoid-based CCMs into C3 crop plants and increasing our understanding of this important organelle.