title: Structural and functional analysis of tomosyn identifies domains important in exocytotic regulation.
abstract: Tomosyn is a 130-kDa cytosolic R-SNARE protein that associates with Q-SNAREs and reduces exocytotic activity. Two paralogous genes, tomosyn-1 and -2, occur in mammals and produce seven different isoforms via alternative splicing. Here, we map the structural differences between the yeast homologue of m-tomosyn-1, Sro7, and tomosyn genes/isoforms to identify domains critical to the regulation of exocytotic activity to tomosyn that are outside the soluble N-ethylmaleimide-sensitive attachment receptor 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 three additional loop domains that emanate from a β-propeller core. Notably, deletion of loops 1 and 3 eliminates tomosyn inhibitory activity on secretion without altering its soluble N-ethylmaleimide-sensitive attachment receptor pairing with syntaxin1A. By comparison, deletion of loop 2, which contains the hypervariable splice region, did not reduce the ability of tomosyn to inhibit regulated secretion. However, exon variation within the hypervariable splice region 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 small ubiquitin-related modifier target site (Lys-730) 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 inhibition by tomosyn on exocytotic secretion.
authors.affiliation: Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan 48109, USA.
authors.forename: Antionette L
authors.initials: AL
authors.lastname: Williams
authors.forename: Noa
authors.initials: N
authors.lastname: Bielopolski
authors.forename: Daphna
authors.initials: D
authors.lastname: Meroz
authors.forename: Alice D
authors.initials: AD
authors.lastname: Lam
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Passmore
authors.forename: Nir
authors.initials: N
authors.lastname: Ben-Tal
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Ernst
authors.forename: Uri
authors.initials: U
authors.lastname: Ashery
authors.forename: Edward L
authors.initials: EL
authors.lastname: Stuenkel
mesh_terms: D017398:Alternative Splicing; D020816:Amino Acid Motifs; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D005089:Exocytosis; D019382:Human Growth Hormone / Q000378:metabolism; D006801:Humans; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D016716:PC12 Cells; D017434:Protein Structure, Tertiary; D050683:R-SNARE Proteins / Q000737:chemistry*; D051381:Rats; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism; D050827:Syntaxin 1 / Q000737:chemistry
chemical_list: D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; C484767:STXBP5 protein, human; C529669:SUMO2 protein, rat; D025841:Small Ubiquitin-Related Modifier Proteins; C484769:Stxbp5 protein, rat; D050827:Syntaxin 1; D019382:Human Growth Hormone