The machinery for trafficking proteins through the
secretory pathway is well conserved in eukaryotes, but
remains poorly characterized in filamentous fungi.
Secretion is believed to be highly polarized in fungal
hyphae and to mainly occur from hyphal tips. We describe
the isolation of the snc1 and sso1 genes encoding
exocytic SNARE proteins from Trichoderma reesei. The
encoded SNCI protein can complement Snc protein depletion
in S. cerevisiae whilst the T. reesei SSOI protein was
unable to complement depletion of its yeast homologues.
The localization and interactions of the T. reesei SNARE
proteins were studied with advanced fluorescence imaging
methods using fluorescent fusions of the SNARE proteins.
The SSOI and SNCI proteins co-localized in
sterol-independent clusters on the plasma membrane in
sub-apical but not apical hyphal regions. The v-SNARE
SNCI (but not the t-SNARE SSOI) localized to the apical
vesicle cluster within the Spitzenkörper of the growing
hyphal tips when expressed from the homologous T. reesei
cbh1 promoter. Using fluorescence lifetime imaging
microscopy (FLIM) and fluorescence energy transfer (FRET)
analysis, we quantified the interactions between these
proteins with high spatial resolution in living cells.
Our data showed that the site of SNARE complex formation
between these proteins is on the plasma membrane of
non-growing hyphae in old sub-peripheral regions of the
colony, but that there is no interaction between the
proteins in growing hyphal tips in at the colony margin.
These findings suggest spatially distinct sites of
exocytosis within filamentous fungi and the existence of
multiple exocytic SNAREs which are functionally and
|Conference||3rd European Federation of Biotechnology Conference |
|Period||13/06/07 → 16/06/07|