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J. Biol. Chem., Vol. 282, Issue 31, 22775-22785, August 3, 2007

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Spatially Segregated SNARE Protein Interactions in Living Fungal Cells^*

Mari Valkonen¹, Eric R. Kalkman, Markku Saloheimo, Merja Penttilä, Nick D. Read, and Rory R. Duncan^¶

From the VTT Biotechnology, P. O. Box 1000, FIN-02044 VTT, Finland, the Fungal Cell Biology Group, Institute of Cell Biology, University of Edinburgh, Edinburgh EH9 3JH, United Kingdom, and the ^¶Membrane Biology Group, University of Edinburgh, George Square, EH8 9XD, United Kingdom

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.

Received for publication, January 31, 2007 , and in revised form, May 14, 2007.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY676605, EF121546, and EF583658.

^* The work was supported by a grant from the Academy of Finland (Grant Number 106685) in the frame of the Academy of Finland research program "VTT Industrial Biotechnology" (Finnish Centre of Excellence Program 2000-2005, Project 64330) (to M. V.) and a Wellcome Trust RCDF Fellowship (to R. R. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Tel.: 358-20-7225825; Fax: 358-20-7227071; E-mail: mari.valkonen{at}vtt.fi.

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