Abstract
The machinery for trafficking proteins through the secretory pathway is well conserved in eukaryotes, from fungi to mammals. We describe the isolation of the snc1, sso1, and sso2 genes encoding exocytic SNARE proteins from the filamentous fungus Trichoderma reesei. The localization and interactions of the T. reesei SNARE proteins were studied with advanced fluorescence imaging methods. The SSOI and SNCI proteins co-localized in sterol-independent clusters on the plasma membrane in subapical but not apical hyphal regions. The vesicle SNARE SNCI also localized to the apical vesicle cluster within the Spitzenkörper of the growing hyphal tips. Using fluorescence lifetime imaging microscopy and Foerster resonance energy transfer analysis, we quantified the interactions between these proteins with high spatial resolution in living cells. Our data showed that the site of ternary SNARE complex formation between SNCI and SSOI or SSOII, respectively, is spatially segregated. SNARE complex formation could be detected between SNCI and SSOI in subapical hyphal compartments along the plasma membrane, but surprisingly, not in growing hyphal tips, previously thought to be the main site of exocytosis. In contrast, SNCI.SSOII complexes were found exclusively in growing apical hyphal compartments. These findings demonstrate spatially distinct sites of plasma membrane SNARE complex formation in fungi and the existence of multiple exocytic SNAREs, which are functionally and spatially segregated. This is the first demonstration of spatially regulated SNARE interactions within the same membrane.
Highlights
SNARE complex formation in the yeast secretory pathway has been shown to begin with the formation of binary complexes between two t-SNAREs, Ssop and Sec9 [7], that are capable of binding the v-SNARE Sncp
As the v-SNARE interacts with t-SNAREs after formation of the fungal SSO-Sec9 binary complex [7], by quantifying the interaction of a vesicular v-SNARE with two plasma membrane t-SNAREs using fluorescence lifetime imaging microscopy (FLIM)/ FRET analysis, we have identified different subcellular sites for exocytic SNARE ternary complex formation in living fungal hyphae
We have described three T. reesei SNARE genes, snc1, sso1, and sso2, that encode a v-SNARE and two t-SNARE proteins, respectively
Summary
The interactions between a sole v-SNARE and two alternative t-SNAREs are spatially segregated This is the first time that SNARE protein interactions have been demonstrated directly in living fungal cells; these data reveal contrasts with mammalian SNARE cluster regulation and suggest an alternative exocytic pathway to the constitutive secretion known to occur via the Spitzenkorper at hyphal tips. This is the first time that spatially regulated SNARE interactions have been found on the plasma membrane
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
