Structural and functional analysis of tomosyn identifies domains important in exocytotic regulation

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.     

Phosphorylation and Degradation of Tomosyn-2 De-represses Insulin Secretion

The abundance and functional activity of proteins involved in the formation of the SNARE complex are tightly regulated for efficient exocytosis. Tomosyn proteins are negative regulators of exocytosis. Tomosyn causes an attenuation of insulin secretion by limiting the formation of the SNARE complex. We hypothesized that glucose-dependent stimulation of insulin secretion from β-cells must involve reversing the inhibitory action of tomosyn. Here, we show that glucose increases tomosyn protein turnover. Within 1 h of exposure to 15 mm glucose, ∼50% of tomosyn was degraded. The degradation of tomosyn in response to high glucose was blocked by inhibitors of the proteasomal pathway. Using ³²P labeling and mass spectrometry, we showed that tomosyn-2 is phosphorylated in response to high glucose, phorbol esters, and analogs of cAMP, all key insulin secretagogues. We identified 11 phosphorylation sites in tomosyn-2. Site-directed mutagenesis was used to generate phosphomimetic (Ser → Asp) and loss-of-function (Ser → Ala) mutants. The Ser → Asp mutant had enhanced protein turnover compared with the Ser → Ala mutant and wild type tomosyn-2. Additionally, the Ser → Asp tomosyn-2 mutant was ineffective at inhibiting insulin secretion. Using a proteomic screen for tomosyn-2-binding proteins, we identified Hrd-1, an E3-ubiquitin ligase. We showed that tomosyn-2 ubiquitination is increased by Hrd-1, and knockdown of Hrd-1 by short hairpin RNA resulted in increased abundance in tomosyn-2 protein levels. Taken together, our results reveal a mechanism by which enhanced phosphorylation of a negative regulator of secretion, tomosyn-2, in response to insulin secretagogues targets it to degradation by the Hrd-1 E3-ubiquitin ligase.     

A Highlights from MBoC Selection: Structural basis for recognition of the Sec4 Rab GTPase by its effector,the Lgl/tomosyn homologue,Sro7

Members of the tomosyn/Lgl/Sro7 family play important roles in vesicle trafficking and cell polarity in eukaryotic cells. The yeast homologue, Sro7, is believed to act as a downstream effector of the Sec4 Rab GTPase to promote soluble N-ethylmaleimide–sensitive factor adaptor protein receptor (SNARE) assembly during Golgi-to–cell surface vesicle transport. Here we describe the identification of a Sec4 binding site on the surface of Sro7 that is contained within a cleft created by the junction of two adjacent β-propellers that form the core structure of Sro7. Computational docking experiments suggested four models for interaction of GTP-Sec4 with the Sro7 binding cleft. Further mutational and biochemical analyses confirmed that only one of the four docking arrangements is perfectly consistent with our genetic and biochemical interaction data. Close examination of this docking model suggests a structural basis for the high substrate and nucleotide selectivity in effector binding by Sro7. Finally, analysis of the surface variation within the homologous interaction site on tomosyn-1 and Lgl-1 structural models suggests a possible conserved Rab GTPase effector function in tomosyn vertebrate homologues.     

Positional cloning of a type 2 diabetes quantitative trait locus; tomosyn-2, a negative regulator of insulin secretion

We previously mapped a type 2 diabetes (T2D) locus on chromosome 16 (Chr 16) in an F2 intercross from the BTBR T (+) tf (BTBR) Lep(ob/ob) and C57BL/6 (B6) Lep(ob/ob) mouse strains. Introgression of BTBR Chr 16 into B6 mice resulted in a consomic mouse with reduced fasting plasma insulin and elevated glucose levels. We derived a panel of sub-congenic mice and narrowed the diabetes susceptibility locus to a 1.6 Mb region. Introgression of this 1.6 Mb fragment of the BTBR Chr 16 into lean B6 mice (B6.16(BT36-38)) replicated the phenotypes of the consomic mice. Pancreatic islets from the B6.16(BT36-38) mice were defective in the second phase of the insulin secretion, suggesting that the 1.6 Mb region encodes a regulator of insulin secretion. Within this region, syntaxin-binding protein 5-like (Stxbp5l) or tomosyn-2 was the only gene with an expression difference and a non-synonymous coding single nucleotide polymorphism (SNP) between the B6 and BTBR alleles. Overexpression of the b-tomosyn-2 isoform in the pancreatic β-cell line, INS1 (832/13), resulted in an inhibition of insulin secretion in response to 3 mM 8-bromo cAMP at 7 mM glucose. In vitro binding experiments showed that tomosyn-2 binds recombinant syntaxin-1A and syntaxin-4, key proteins that are involved in insulin secretion via formation of the SNARE complex. The B6 form of tomosyn-2 is more susceptible to proteasomal degradation than the BTBR form, establishing a functional role for the coding SNP in tomosyn-2. We conclude that tomosyn-2 is the major gene responsible for the T2D Chr 16 quantitative trait locus (QTL) we mapped in our mouse cross. Our findings suggest that tomosyn-2 is a key negative regulator of insulin secretion.     

Three splicing variants of tomosyn and identification of their syntaxin-binding region

We have recently isolated a neural tissue-specific syntaxin-1-binding protein, named tomosyn, which is capable of dissociating Munc18/n-Sec1/rbSec1 from syntaxin-1 to form a 10S tomosyn complex, an intermediate complex converted to the 7S SNARE complex. We isolated here two splicing variants of tomosyn: one had 36 amino acids (aa) insertion and another had 17 aa deletion. We named original one m-tomosyn, big one b-tomosyn, and small one s-tomosyn. s-Tomosyn as well as m-tomosyn was mainly expressed in brain whereas b-tomosyn was ubiquitously expressed. All the isoforms bound to syntaxin-1, but not to syntaxin-2, -3, or -4, and had a region highly homologous to VAMP, another syntaxin-binding protein. This region was necessary but not sufficient for high-affinity binding of tomosyn to syntaxin-1.     

Age and Visual Experience-dependent Expression of NMDAR1 Splice Variants in Rat Retina

The N-methyl-D-aspartate receptor (NMDAR) is a key molecule mediating brain plasticity related processes. Knowing that alternative splicing of the NMDAR1 (NR1) subunit offers molecular diversity to NMDAR, controls the forward trafficking of the NR1 protein and is important for placing NMDA receptors at synapses, we investigated herein the postnatal developmental expression and the influence of visual deprivation on NR1 subunit splice variants in rat retina. Real-time PCR was performed using oligonucleotide primers specific for N- terminal (NR1a, NR1b) and C-terminal splice variants (NR1-1, NR1-2, NR1-3, NR1-4). The developmental profiles of mRNA expression levels of all NR1 isoforms peaked at the end of the third week. Dark rearing led to reductions in both N- and C-terminal NR1 variants in several developmental ages and a significant interaction between age and visual experience was observed for NR1a, NR1-2 and NR1-4 expression. Our results have demonstrated a developmental and visual experience-dependent regulation of NR1 splicing in rat retina.     

Developmentally regulated alternative splicing of densin modulates protein-protein interaction and subcellular localization

Densin is a member of the leucine-rich repeat (LRR) and PDZ domain (LAP) protein family that binds several signaling molecules via its C-terminal domains, including calcium/calmodulin-dependent protein kinase II (CaMKII). In this study, we identify several novel mRNA splice variants of densin that are differentially expressed during development. The novel variants share the LRR domain but are either prematurely truncated or contain internal deletions relative to mature variants of the protein (180 kDa), thus removing key protein–protein interaction domains. For example, CaMKIIα coimmunoprecipitates with densin splice variants containing an intact C-terminal domain from lysates of transfected HEK293 cells, but not with variants that only contain N-terminal domains. Immunoblot analyses using antibodies to peptide epitopes in the N- and C- terminal domains of densin are consistent with developmental regulation of splice variant expression in brain. Moreover, putative splice variants display different subcellular fractionation patterns in brain extracts. Expression of green fluorescent protein (GFP)-fused densin splice variants in HEK293 cells shows that the LRR domain can target densin to a plasma membrane-associated compartment, but that the splice variants are differentially localized and have potentially distinct effects on cell morphology. In combination, these data show that densin splice variants have distinct functional characteristics suggesting multiple roles during neuronal development.     

Different expression patterns for ubiquitous calpains and Capn3 splice variants in monkey ocular tissues

The purpose of the present investigation was to compare the expression of ubiquitous and tissue-specific calpains in ocular tissues from the Macaca fascicularis monkey. Calpain isoforms in retina and corneal epithelium from adult M. fascicularis monkeys were characterized by RT-PCR, cDNA cloning and sequencing. Calpain isoform activities in ocular tissues were investigated by fractionation on DEAE-HPLC, immunoblotting, and casein zymography. Capn3 splice variants in the ocular tissues from rat, rabbit and monkey were compared after RT-PCR. RT-PCR analysis revealed that numerous splice variants of Capn3 were expressed in the epithelium from monkey cornea. The variants contained deletions or insertions in or around the IS1, IS2, and NS regions. The cDNAs for Capn3 variants were highly conserved, yet the expression patterns of the Capn3 isoforms were widely different among the mammalian species. In contrast, the expression patterns of ubiquitous calpains in ocular tissues were conserved among the mammalian species, and similarities between monkey and human cDNAs for Capn1 (mu-calpain) and Capn2 (m-calpain) were 98 and 99%, respectively. These results suggested that differences in expression patterns of Capn3 variants might be related to the function of each variant in a particular tissue or species.     

Expression pattern of an evolutionarily conserved splice variant in the rat Tacc2 gene

The transforming acidic coiled‐coil containing protein 2 (Tacc2) gene and its paralogs, Tacc1 and Tacc3 encode proteins that are associated with the centrosome and involved in microtubule assembly during the cell cycle. Tacc2 produces several splice variants, which are poorly characterized, especially in the rat. Characterization of the temporal/spatial expression patterns of these isoforms would be useful in understanding their distinct and overlapping functions. By comparative sequence analyses of Tacc2 in multiple species, we identified a third splice variant in rat, which is much shorter in size (1,021 aa) than the longest isoform (2,834 aa). This newly identified Tacc2 splice variant (isoform 3) uses a distinct first exon and generates a different open reading frame. Although Isoform 3 is expressed predominantly during developmental stages, the long Tacc2 isoform (isoform 1) is distributed mainly in adult tissues. Multiple protein sequence analyses revealed that Tacc2 Isoform 3 could be the ancient form, as it is conserved in mammals, birds, and amphibians; whereas the long Tacc2 isoforms may have evolved in the mammalian lineage by adding exons toward the 5′ region of the ancient isoform. genesis 52:378–386, 2014. © 2014 Wiley Periodicals, Inc.     

VAMP-1 has a highly variable C-terminus generated by alternative splicing.

VAMP-1 (synaptobrevin1) is one of the key proteins in the SNARE complex which is involved in regulated exocytosis. Recently, Isenmann et al. (1998, Mol. Biol. Cell 9, 1649-1660) showed the extreme C-terminal region of VAMP-1A and 1B to be involved in subcellular targeting of the isoforms. Four new splice variants (VAMP-1C to F) were identified in addition to the previously published variants VAMP-1A and VAMP-1B. Interestingly, the four new isoforms also have variable sequences only at the extreme C-terminus. This suggests that the C-terminal region has an important function for VAMP-1 and vesicle targeting. All six variants were a result of alternative splicing that linked exons 1-4 which encode the conserved region of VAMP-1 with one of the exons 5A to 5F that encodes the highly variable extreme C-terminus. Exon (5A-E) encode C-termini of two to five amino acid residues, whereas exon 5F encoded a long C-terminal amino acid extension. The splice variants were differentially expressed in human brain, kidney, and inflammatory cells.     

Structural basis for the inhibitory role of tomosyn in exocytosis

Upon Ca2+ influx synaptic vesicles fuse with the plasma membrane and release their neurotransmitter cargo into the synaptic cleft. Key players during this process are the Q-SNAREs syntaxin 1a and SNAP-25 and the R-SNARE synaptobrevin 2. It is thought that these membrane proteins gradually assemble into a tight trans-SNARE complex between vesicular and plasma membrane, ultimately leading to membrane fusion. Tomosyn is a soluble protein of 130 kDa that contains a COOH-terminal R-SNARE motif but lacks a transmembrane anchor. Its R-SNARE motif forms a stable core SNARE complex with syntaxin 1a and SNAP-25. Here we present the crystal structure of this core tomosyn SNARE complex at 2.0-A resolution. It consists of a four-helical bundle very similar to that of the SNARE complex containing synaptobrevin. Most differences are found on the surface, where they prevented tight binding of complexin. Both complexes form with similar rates as assessed by CD spectroscopy. In addition, synaptobrevin cannot displace the tomosyn helix from the tight complex and vice versa, indicating that both SNARE complexes represent end products. Moreover, data bank searches revealed that the R-SNARE motif of tomosyn is highly conserved throughout all eukaryotic kingdoms. This suggests that the formation of a tight SNARE complex is important for the function of tomosyn.     

PKA-catalyzed phosphorylation of tomosyn and its implication in Ca2+-dependent exocytosis of neurotransmitter

Neurotransmitter is released from nerve terminals by Ca2+-dependent exocytosis through many steps. SNARE proteins are key components at the priming and fusion steps, and the priming step is modulated by cAMP-dependent protein kinase (PKA), which causes synaptic plasticity. We show that the SNARE regulatory protein tomosyn is directly phosphorylated by PKA, which reduces its interaction with syntaxin-1 (a component of SNAREs) and enhances the formation of the SNARE complex. Electrophysiological studies using cultured superior cervical ganglion (SCG) neurons revealed that this enhanced formation of the SNARE complex by the PKA-catalyzed phosphorylation of tomosyn increased the fusion-competent readily releasable pool of synaptic vesicles and, thereby, enhanced neurotransmitter release. This mechanism was indeed involved in the facilitation of neurotransmitter release that was induced by a potent biological mediator, the pituitary adenylate cyclase-activating polypeptide, in SCG neurons. We describe the roles and modes of action of PKA and tomosyn in Ca2+-dependent neurotransmitter release.     

Disruption of the developmentally-regulated Col2a1 pre-mRNA alternative splicing switch in a transgenic knock-in mouse model

The present study describes the generation of a knock-in mouse model to address the role of type II procollagen (Col2a1) alternative splicing in skeletal development and maintenance. Alternative splicing of Col2a1 precursor mRNA is a developmentally-regulated event that only occurs in chondrogenic tissue. Normally, chondroprogenitor cells synthesize predominantly exon 2-containing mRNA isoforms (type IIA and IID) while Col2a1 mRNA devoid of exon 2 (type IIB) is the major isoform produced by differentiated chondrocytes. Another isoform, IIC, has also been identified that contains a truncated exon 2 and is not translated into protein. The biological significance of this IIA/IID to IIB splicing switch is not known. Utilizing a splice site targeting knock-in approach, a 4 nucleotide mutation was created to convert the 5' splice site of Col2a1 exon 2 from a weak, non-consensus sequence to a strong, consensus splice site. This resulted in apparent expression of only the IIA mRNA isoform, as confirmed in vitro by splicing of a type II procollagen mini-gene containing the 5' splice site mutation. To test the splice site targeting approach in vivo, homozygote mice engineered to retain IIA exon 2 (Col2a1(+ex2)) were generated. Chondrocytes from hindlimb epiphyseal cartilage of homozygote mice were shown to express only IIA mRNA and protein at all pre- and post-natal developmental stages analyzed (E12.5, E16.5, P0, P3, P7, P14, P28 and P70). As expected, type IIB procollagen was the major isoform produced in wild type cartilage at all post-natal time points. Col2a1(+ex2) homozygote mice are viable, appear healthy and display no overt phenotype to date. However, research is currently underway to investigate the biological consequence of persistent expression of the exon 2-encoded conserved cysteine-rich domain in post-natal skeletal tissues.     

Isoform and splice-variant specific functions of dynamin-2 revealed by analysis of conditional knock-out cells

Dynamin (Dyn) is a multifunctional GTPase implicated in several cellular events, including endocytosis, intracellular trafficking, cell signaling, and cytokinesis. The mammalian genome encodes three isoforms, Dyn1, Dyn2, and Dyn3, and several splice variants of each, leading to the suggestion that distinct isoforms and/or distinct splice variants might mediate distinct cellular functions. We generated a conditional Dyn2 KO cell line and performed knockout and reconstitution experiments to explore the isoform- and splice variant specific cellular functions of ubiquitously expressed Dyn2. We find that Dyn2 is required for clathrin-mediated endocytosis (CME), p75 export from the Golgi, and PDGF-stimulated macropinocytosis and cytokinesis, but not for other endocytic pathways. Surprisingly, CME and p75 exocytosis were efficiently rescued by reintroduction of Dyn2, but not Dyn1, suggesting that these two isoforms function differentially in vesicular trafficking in nonneuronal cells. Both isoforms rescued macropinocytosis and cytokinesis, suggesting that dynamin function in these processes might be mechanistically distinct from its role in CME. Although all four Dyn2 splice variants could equally restore CME, Dyn2ba and -bb were more effective at restoring p75 exocytosis. This splice variant specificity correlated with their differential targeting to the Golgi. These studies reveal isoform and splice-variant specific functions for Dyn2.