A Tip-High Gradient of a Putative Plasma Membrane SNARE Approximates the Exocytotic Gradient in Hyphal Apices of the Fungus Neurospora crassa

Abstract

Gupta, G. D., and Heath, I. B. 2000. A tip-high gradient of a putative plasma membrane SNARE approximates the exocytotic gradient in hyphal apices of the fungus Neurospora crassa. Antibodies to the Saccharomyces cereviseae plasma membrane t-SNARE Sso2p identify a putative 39-kDa homologue in Neurospora crassa. The 39-kDa protein is enriched in plasma membrane (PM) and occurred with other membranes. It is extractable by detergent, but not chaotropic or alkali agents, suggesting membrane insertion. Immunoprecipitation with anti-Sso2p coprecipitated a ∼100-kDa, Mg+-ATP-sensitive band with the 39-kDa protein, suggesting a ternary SNARE complex. Affinity-purified anti-Sso2p gave hyphal staining patterns most consistent with protein localization on both the PM and intracellular exocytotic apical wall vesicles. The PM staining in hyphal apices formed a tip-high gradient, not as steep as that predicted by the “hyphoid equation,” but closer to published gradients of cell wall matrix deposition. We conclude that the t-SNAREs are transported to the PM on the apical vesicles, but their tip-high gradient alone is insufficient to explain the vesicle fusion gradient in growing tips.