Point mutations in the catalytic domain disrupt cellulose synthase (CESA6) vesicle trafficking and protein dynamics

Abstract

Cellulose, as the main component of the plant cell wall, is synthesized by a multimeric protein complex named the cellulose synthase (CESA) complex or CSC. In plant cells, CSCs are transported through the endomembrane system to the PM, but how catalytic activity or conserved motifs around the catalytic core domain influence vesicle trafficking or protein dynamics is not well understood. Here, we used a functional YFP-tagged AtCESA6 and site-directed mutagenesis to create 18 single amino acid replacement mutants in key motifs of the catalytic domain including DDG, DXD, TED and QXXRW, to comprehensively analyze how catalytic activity affects plant growth, cellulose biosynthesis, as well as CSC dynamics and trafficking. Plant growth and cellulose content were reduced by nearly all mutations. Moreover, mutations in most conserved motifs reduced the velocity of CSC movement in the PM as well as delivery of CSCs to the PM. Interestingly, the abundance of YFP-CESA6 in the Golgi apparatus was increased or reduced by mutations in DDG and QXXRW motifs, respectively. Post-Golgi trafficking of CSCs was also differentially perturbed by these mutations and, based on these phenotypes, the 18 mutants could be divided into two major groups. Group I comprises mutations causing significantly increased Golgi fluorescence intensity with either an increase or no change in the abundance of cortical SmaCCs compared with wild-type. In contrast, Group II represents mutations with significantly decreased Golgi fluorescence intensity and/or reduced SmaCC density. We propose that Group I mutations cause CSC trafficking defects whereas Group II mutations affect normal CSC rosette formation and hence interfere with subsequent CSC trafficking. Collectively, our results demonstrate that the catalytic domain of CESA6 is essential not only for cellulose biosynthesis, but also complex formation, protein folding and dynamics, vesicle trafficking, or all of the above.

The TRAPPIII subunit, Trs85, has a dual role in the trafficking of cellulose synthase complexes in Arabidopsis

“…We also found that after photobleaching, YFP-CESA6 particles with an atypical pause phase of over 110 sec were almost 50% more abundant in prc1-1 trs85-1 than prc1-1 (Figure 4d). CESA6 particles that remain static at the plasma membrane have also been reported in exocytosis mutants (Zhu et al, 2018), endocytotic mutants (Bashline et al, 2015) and transgenic lines that have point mutations in the catalytic domain of YFP-CESA6 (Huang, 2022). These static particles likely represent unsuccessful insertion events, delayed endocytotic events, or the insertion of functionally inactive CSCs.…”

Litchi aspartic protease LcAP1 enhances plant resistance via suppressing cell death triggered by the pectate lyase PlPeL8 from Peronophythora litchii

“…The inability of PlPeL8 mutants to induce cell death might stem from the crucial roles of New Phytologist certain residues in mediating protein interactions, dimer formation, structure alterations, or folding modifications. A recent study has revealed the involvement of several aspartic acid residues within the catalytic domain of cellulose synthase CESA6 involved in complex formation, protein folding, and dynamics (Huang et al, 2023). Additionally, amino acids at positions C18 and C44 are crucial for Botrytis HR-inducing protein 1 (Hip1)induced cell death and might be associated with native conformation (Jeblick et al, 2023).…”