Molecular architecture of the plant callose synthase complex
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The biosynthesis of plant cell wall polysaccharide, callose, is facilitated by a large 200 kDa integral membrane enzyme, CALLOSE SYNTHASE (CALS). Despite being crucial for a number of plant developmental processes, including cell division, defense against pathogens, vascular tissue formation or cell-to-cell communication, there is a significant knowledge gap in its molecular architecture. Similar to the CELLULOSE SYNTHASE complex, the ability to form an oligomeric assembly, called the CALS complex, has been proposed for CALS. We used tandem affinity purification (TAP) and proximity biotinylation coupled with mass spectrometry to isolate the AtCALS1 protein from Arabidopsis cell culture with its putative stable and transient interactors. The obtained AtCALS1 interactome gives an unprecedented insight into the regulatory network of CALS folding, trafficking, activity and degradation, showcasing the enigmatic life cycle of the CALS complex in the plant cell.
Furthermore, we obtained a structural model of AtCALS1 by combining artificial intelligence-based protein structure prediction with data from TAP coupled with on-beads chemical cross-linking and subsequent mass spectrometry. We show that CALS1 forms a higher-order assembly with other CALS homologs. The interaction of novel plant-specific domains mediates the assembly of this CALS complex. In addition, coarse-grained molecular dynamics (MD) simulations revealed specific lipid fingerprints formed by different signaling anionic phospholipids. In connection with the membrane lipid-modifying enzymes enriched within the AtCALS1 interactome, we analyze the effect of lipid composition on CALS stability and activity. The CALS structural model is further validated by all-atom MD simulations of callose subunit laminaribiose within the presumed CALS catalytic core, based on which a mechanism of callose translocation into the extracellular space is inferred. In summary, we provide the first look into the molecular architecture of the plant CALS complex, the regulation of its assembly, trafficking and activity, with profound implications on the callose synthesis in the cell wall.
Equity and Inclusion: This proposal describes proceedings from a predominantly female research subgroup from an underrepresented Eastern European country. The equal participation opportunity on the project was promoted for all team members.
