Reference: Williams AL, et al. (2011) Structural and functional analysis of tomosyn identifies domains important in exocytotic regulation. J Biol Chem 286(16):14542-53

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Abstract

Tomosyn is a 130kDa cytosolic R-SNARE protein that associates with Q-SNAREs and reduces exocytotic activity. Two paralogous genes, tomosyn-1 and -2, occur in mammals and via alternative splicing produce 7 different isoforms. Here, we map structural differences between the yeast homologue of m-tomosyn-1, Sro7, and tomosyn genes/isoforms to identify domains critical to tomosyn regulation of exocytotic activity that are outside the SNARE motif. Homology modeling of m-tomosyn-1 based on the known structure of yeast Sro7 revealed a highly conserved functional conformation, but with tomosyn containing 3 additional loop domains that emanate from a beta-propeller core. Notably, deletion of loops 1 and 3 eliminate tomosyn inhibitory activity on secretion without altering its SNARE pairing with syntaxin1A. By comparison, deletion of loop 2, which contains the hypervariable splice region (HVR), did not reduce the ability of tomosyn to inhibit regulated secretion. However, exon variation within the HVR resulted in significant differences in protein accumulation of tomosyn-2 isoforms. Functional analysis of s-tomosyn-1, m-tomosyn-1, m-tomosyn-2 and xb-tomosyn-2 demonstrated that they exert similar inhibitory effects on elevated K+-induced secretion in PC12 cells, although m-tomosyn-2 was novel in strongly augmenting basal secretion. Finally, we report that m-tomosyn-1 is a target substrate for SUMO 2/3 conjugation and that mutation of this SUMO target site (K730) enhances m-tomosyn-1 inhibition of secretion without altering interaction with syntaxin1A. Together these results suggest that multiple domains outside the R-SNARE of tomosyn are critical to the efficacy of tomosyn inhibition of exocytotic secretion.

Reference Type
Journal Article
Authors
Williams AL, Bielopolski N, Meroz D, Lam AD, Passmore DR, Ben-Tal N, Ernst SA, Ashery U, Stuenkel EL
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