The C-terminal domain of SEC-10 is fundamental for exocyst function, apical organization, and cell morphogenesis in Neurospora crassa.

Abstract

The exocyst complex is crucial for vesicles secretion. In Neurospora crassa, apical growth is determined by the Spitzenkörper (SPK), where secretory vesicles accumulate before fusing with the plasma membrane (PM). Exocyst subunits SEC-3, -5, -6, -8, and -15 localize to the PM of hyphal tips, while EXO-70 and EXO-84 are found at the SPK. The localization of SEC-10 had remained elusive. This study used SEC-10 tagged with green fluorescent protein (GFP) at its N- or C-terminus to investigate its function. Endogenous GFP-tagging of SEC-10 at the N-terminus preserved exocyst function, whereas C-terminal tagging caused growth and polarity defects, including absence of an SPK, indicative of exocyst dysfunction. Nanoscopic deconvolution elucidated discrete exocytic sites at the PM. A sec-10 knockout mutant was only viable in a heterokaryotic state, confirming SEC-10's essential role in hyphal morphogenesis. Mass spectrometry showed fewer exocyst subunits interacting with SEC-10-GFP compared with GFP-SEC-10, highlighting the importance of the SEC-10 C-terminus in exocyst assembly and stability. SEC-10 sequence analysis revealed a disordered glycine-rich loop at the C-terminal region, conserved in some filamentous fungi, that could provide flexibility to this domain. These findings suggest that an unobstructed SEC-10 C-terminus is indispensable for exocyst-mediated vesicle tethering and fusion at the PM.