Preassembly and independent trafficking of the exocyst complex in <i>Arabidopsis</i>.

Abstract

Membrane fusion, the culmination of eukaryotic membrane trafficking, is orchestrated by the exocyst complex (a conserved octamer comprising SC1 and SC2 heterotetramers) and sensitive factor attachment protein receptor (SNARE) complexes. Although <i>trans</i>-SNARE complex formation is essential for function, a similar <i>trans</i>-interaction mechanism for the exocyst remains uncertain. We employed advanced live-cell imaging combined with genetic and pharmacological techniques to dissect the spatiotemporal dynamics of exocyst subunit interactions and cargo vesicle association in <i>Arabidopsis thaliana</i> hypocotyl cells. Our results demonstrate that subunits from SC1 and SC2 colocalize and undergo actin-dependent transport to the plasma membrane (PM). Disruption of either SEC6 (SC1) or EXO70A1 (SC2) prevented PM association of both subcomplexes, indicating cytoplasmic preassembly of the <i>cis</i>-exocyst complex before PM recruitment. Critically, we found that the exocyst does not directly bind vesicles carrying cellulose synthase complexes (CSCs). Instead, exocyst subunits first migrate on CSC-negative vesicles, which then coalesce with CSC-carrying vesicles at the cell cortex via heterotypic fusion. Together, our findings reveal a parallel mechanism for exocyst assembly and cargo loading. This coordinated process may represent a broadly conserved strategy to ensure efficient membrane trafficking in eukaryotic cells.