Tom-1 Mutants Research Articles

Hect E3 ubiquitin ligase Tom1 controls Dia2 degradation during the cell cycle

The ubiquitin proteasome system plays a pivotal role in controlling the cell cycle. The budding yeast F-box protein Dia2 is required for genomic stability and is targeted for ubiquitin-dependent degradation in a cell cycle-dependent manner, but the identity of the ubiquitination pathway is unknown. We demonstrate that the Hect domain E3 ubiquitin ligase Tom1 is required for Dia2 protein degradation. Deletion of DIA2 partially suppresses the temperature-sensitive phenotype of tom1 mutants. Tom1 is required for Dia2 ubiquitination and degradation during G1 and G2/M phases of the cell cycle, whereas the Dia2 protein is stabilized during S phase. We find that Tom1 binding to Dia2 is enhanced in G1 and reduced in S phase, suggesting a mechanism for this proteolytic switch. Tom1 recognizes specific, positively charged residues in a Dia2 degradation/NLS domain. Loss of these residues blocks Tom1-mediated turnover of Dia2 and causes a delay in G1-to-S phase progression. Deletion of DIA2 rescues a delay in the G1-to-S phase transition in the tom1Δ mutant. Together our results suggest that Tom1 targets Dia2 for degradation during the cell cycle by recognizing positively charged residues in the Dia2 degradation/NLS domain and that Dia2 protein degradation contributes to G1-to-S phase progression.

In Vivo Analysis of Conserved C. elegans Tomosyn Domains

Neurosecretion is critically dependent on the assembly of a macromolecular complex between the SNARE proteins syntaxin, SNAP-25 and synaptobrevin. Evidence indicates that the binding of tomosyn to syntaxin and SNAP-25 interferes with this assembly, thereby negatively regulating both synaptic transmission and peptide release. Tomosyn has two conserved domains: an N-terminal encompassing multiple WD40 repeats predicted to form two β-propeller structures and a C-terminal SNARE-binding motif. To assess the function of each domain, we performed an in vivo analysis of the N- and C- terminal domains of C. elegans tomosyn (TOM-1) in a tom-1 mutant background. We verified that both truncated TOM-1 constructs were transcribed at levels comparable to rescuing full-length TOM-1, were of the predicted size, and localized to synapses. Unlike full-length TOM-1, expression of the N- or C-terminal domains alone was unable to restore inhibitory control of synaptic transmission in tom-1 mutants. Similarly, co-expression of both domains failed to restore TOM-1 function. In addition, neither the N- nor C-terminal domain inhibited release when expressed in a wild-type background. Based on these results, we conclude that the ability of tomosyn to regulate neurotransmitter release in vivo depends on the physical integrity of the protein, indicating that both N- and C-terminal domains are necessary but not sufficient for effective inhibition of release in vivo.

Ubiquitin-mediated mRNP dynamics and surveillance prior to budding yeast mRNA export

The evolutionarily conserved mRNA export receptor Mex67/NXF1 associates with mRNAs through its adaptor, Yra1/REF, allowing mRNA ribonucleoprotein (mRNP) exit through nuclear pores. However, alternate adaptors should exist, since Yra1 is dispensable for mRNA export in Drosophila and Caenorhabditis elegans. Here we report that Mex67 interacts directly with Nab2, an essential shuttling mRNA-binding protein required for export. We further show that Yra1 enhances the interaction between Nab2 and Mex67, and becomes dispensable in cells overexpressing Nab2 or Mex67. These observations appoint Nab2 as a potential adaptor for Mex67, and define Yra1/REF as a cofactor stabilizing the adaptor-receptor interaction. Importantly, Yra1 ubiquitination by the E3 ligase Tom1 promotes its dissociation from mRNP before export. Finally, loss of perinuclear Mlp proteins suppresses the growth defects of Tom1 and Yra1 ubiquitination mutants, suggesting that Tom1-mediated dissociation of Yra1 from Nab2-bound mRNAs is part of a surveillance mechanism at the pore, ensuring export of mature mRNPs only.

Differential Regulation of Synaptic Vesicle Tethering and Docking by UNC-18 and TOM-1

The assembly of SNARE complexes between syntaxin, SNAP-25 and synaptobrevin is required to prime synaptic vesicles for fusion. Since Munc18 and tomosyn compete for syntaxin interactions, the interplay between these proteins is predicted to be important in regulating synaptic transmission. We explored this possibility, by examining genetic interactions between C. elegans unc-18(Munc18), unc-64(syntaxin) and tom-1(tomosyn). We have previously demonstrated that unc-18 mutants have reduced synaptic transmission, whereas tom-1 mutants exhibit enhanced release. Here we show that the unc-18 mutant release defect is associated with loss of two morphologically distinct vesicle pools; those tethered within 25 nm of the plasma membrane and those docked with the plasma membrane. In contrast, priming defective unc-13 mutants accumulate tethered vesicles, while docked vesicles are greatly reduced, indicating tethering is UNC-18-dependent and occurs in the absence of priming. C. elegans unc-64 mutants phenocopy unc-18 mutants, losing both tethered and docked vesicles, whereas overexpression of open syntaxin preferentially increases vesicle docking, suggesting UNC-18/closed syntaxin interactions are responsible for vesicle tethering. Given the competition between vertebrate tomosyn and Munc18, for syntaxin binding, we hypothesized that C. elegans TOM-1 may inhibit both UNC-18-dependent vesicle targeting steps. Consistent with this hypothesis, tom-1 mutants exhibit enhanced UNC-18 plasma membrane localization and a concomitant increase in both tethered and docked synaptic vesicles. Furthermore, in tom-1;unc-18 double mutants the docked, primed vesicle pool is preferentially rescued relative to unc-18 single mutants. Together these data provide evidence for the differential regulation of two vesicle targeting steps by UNC-18 and TOM-1 through competitive interactions with syntaxin.

Tomosyn negatively regulates both synaptic transmitter and neuropeptide release at the C. elegans neuromuscular junction

The SNARE proteins, syntaxin, SNAP-25 and synaptobrevin form a tertiary complex essential for vesicle fusion. Proteins that influence SNARE complex assembly are therefore likely to be important regulators of fusion events. In this study we have focused on tomosyn, a highly conserved, neuronally enriched, syntaxin-binding protein that has been implicated in the regulation of vesicle exocytosis. To directly test the role of tomosyn in neurosecretion we analysed loss-of-function mutants in the single Caenorhabditis elegans tomosyn gene, tom-1. These mutants exhibit enhanced synaptic transmission based on electrophysiological analysis of neuromuscular junction activity. This phenotype is the result of increased synaptic vesicle priming. In addition, we present evidence that tom-1 mutants also exhibit enhanced peptide release from dense core vesicles. These results indicate that tomosyn negatively regulates secretion for both vesicle types, possibly through a common mechanism, interfering with SNARE complex formation, thereby inhibiting vesicle fusion.

Tomosyn Negatively Regulates CAPS-Dependent Peptide Release atCaenorhabditis elegansSynapses

The syntaxin-interacting protein tomosyn is thought to be a key regulator of exocytosis, although its precise mechanism of action has yet to be elucidated. Here we examined the role of tomosyn in peptide secretion in Caenorhabditis elegans tomosyn (tom-1) mutants. Ultrastructural analysis of tom-1 mutants revealed a 50% reduction in presynaptic dense-core vesicles (DCVs) corresponding to enhanced neuropeptide release. Conversely, overexpression of TOM-1 led to an accumulation of DCVs. Together, these data provide the first in vivo evidence that TOM-1 negatively regulates DCV exocytosis. In C. elegans, neuropeptide release is promoted by the calcium-dependent activator protein for secretion (CAPS) homolog UNC-31. To test for a genetic interaction between tomosyn and CAPS, we generated tom-1;unc-31 double mutants. Loss of TOM-1 suppressed the behavioral, electrophysiological, and DCV ultrastructural phenotypes of unc-31 mutants, indicating that TOM-1 antagonizes UNC-31-dependent DCV release. Because unc-31 mutants exhibit synaptic transmission defects, we postulated that loss of DCV release in these mutants and the subsequent suppression by tom-1 mutants could simply reflect alterations in synaptic activity, rather than direct regulation of DCV release. To distinguish between these two possibilities, we analyzed C. elegans Rim mutants (unc-10), which have a comparable reduction in synaptic transmission to unc-31 mutants, specifically attributed to defects in synaptic vesicle (SV) exocytosis. Based on this analysis, we conclude that the changes in DCV release in tom-1 and unc-31 mutants reflect direct effects of TOM-1 and UNC-31 on DCV exocytosis, rather than altered SV release.

Keeping Things under Control at the Neuromuscular Junction

In vertebrates, neurons signal muscles to contract by releasing a neurotransmitter, acetylcholine, into the intercellular space. They do so by fusing spherical acetylcholine-filled packages with the plasma membrane. But what controls when—and how many—such packages, known as synaptic vesicles, release their contents? A protein called tomosyn is thought to play a key role in keeping the process under control. But until now its mode of action was unknown. To clarify tomosyn’s role in regulating acetylcholine release at the neuromuscular junction, Elena O. Gracheva, Janet E. Richmond, and colleagues enlisted the help of Caenorhabditis elegans, a salt-grain-sized nematode that, because of its simple nervous system and tractable genetics, is widely used to study how neurons work. C. elegans has a gene, tom-1, that makes a class of proteins, TOM-1, similar to vertebrate tomosyn. After discovering that C. elegans TOM-1 is biochemically very similar to tomosyn in vertebrates, the researchers decided to use the nematode as a model for exploring vertebrate tomosyn function. They began by measuring traits of the nerve-to-muscle signal in tom-1 mutants that are unable to properly produce TOM-1. They found that the evoked release of acetylcholine onto the muscle was enhanced in tom-1 mutants relative to non-mutant (wild-type) organisms, due to a prolonged evoked response. Other studies have shown that tomosyn is able to block the formation of a protein complex called the SNARE complex, known to mediate vesicle release. Could this be how tomosyn regulates synaptic transmission? Before they could conclude so, the researchers had some alternative explanations to discount. By comparing post-synaptic electrophysiological activity in wild-type and mutant strains, they showed that the effects observed in the tom-1 mutants were not due to changes in post-synaptic receptor kinetics. By comparing the morphology of neurons in wild-type and mutant strains, they also showed the effects were not due to altered neuronal connectivity. And by comparing the number and distribution of neuromuscular synapses in the two (using a technique called immunolabeling), they demonstrated the moderating effect of tomosyn is not due to changes in how synapses develop, either. Next they tested whether the tom-1 mutant trait could be reversed by selectively expressing TOM-1 in cholinergic neurons (acetylcholine-producing neurons). They found the nerve-to-muscle signal to be less prominent in this situation than in either wild-type or tom-1 strains, while the duration of the response evoked in the muscle was like that of wild-type worms. The conclusion: TOM-1 regulates synaptic transmission on the nerve side, rather than on the muscle side, of the synapse. With the hypothesis that tomosyn acts on the vesicle release part of signal transmission strengthened by these findings, the researchers decided to look more closely at the vesicles themselves. Electron microscopic examination of synapses in wild-type and mutant strains showed the number of vesicles did not differ but that vesicle distribution did, with tom-1 mutants having more vesicles in contact with the plasma membrane and a broader distribution along the membrane. In addition, the mutant worms whose cholinergic function had been restored through selective TOM-1 expression had fewer contacting vesicles and less distributed vesicles in their cholinergic synapses. An important part of the vesicle-signaling process at the neuromuscular junction is the preparation of vesicles for release, a process known as “priming.” Previous research has shown that synapses in a C. elegans mutant known as unc-13(s69), which has priming problems, have few vesicles in contact with the plasma membrane and do not release acetylcholine. This suggests that the vesicles lined up on the membrane in the tom-1 mutants represent a superabundance of primed vesicles. To test this hypothesis, the researchers produced tom-1 unc-13(s69) double mutants. As they predicted, these double mutants had more contacting vesicles than unc-13(s69) alone and restored acetylcholine release. The researchers concluded that TOM-1’s role in moderating signal transmission at the neuromuscular junction occurs by limiting the number of vesicles primed to release acetylcholine into the intercellular space.

Tomosyn Inhibits Synaptic Vesicle Priming in Caenorhabditis elegans

Caenorhabditis elegans TOM-1 is orthologous to vertebrate tomosyn, a cytosolic syntaxin-binding protein implicated in the modulation of both constitutive and regulated exocytosis. To investigate how TOM-1 regulates exocytosis of synaptic vesicles in vivo, we analyzed C. elegans tom-1 mutants. Our electrophysiological analysis indicates that evoked postsynaptic responses at tom-1 mutant synapses are prolonged leading to a two-fold increase in total charge transfer. The enhanced response in tom-1 mutants is not associated with any detectable changes in postsynaptic response kinetics, neuronal outgrowth, or synaptogenesis. However, at the ultrastructural level, we observe a concomitant increase in the number of plasma membrane-contacting vesicles in tom-1 mutant synapses, a phenotype reversed by neuronal expression of TOM-1. Priming defective unc-13 mutants show a dramatic reduction in plasma membrane-contacting vesicles, suggesting these vesicles largely represent the primed vesicle pool at the C. elegans neuromuscular junction. Consistent with this conclusion, hyperosmotic responses in tom-1 mutants are enhanced, indicating the primed vesicle pool is enhanced. Furthermore, the synaptic defects of unc-13 mutants are partially suppressed in tom-1 unc-13 double mutants. These data indicate that in the intact nervous system, TOM-1 negatively regulates synaptic vesicle priming.

Using Microarrays to Facilitate Positional Cloning: Identification of Tomosyn as an Inhibitor of Neurosecretion

Forward genetic screens have been used as a powerful strategy to dissect complex biological pathways in many model systems. A significant limitation of this approach has been the time-consuming and costly process of positional cloning and molecular characterization of the mutations isolated in these screens. Here, the authors describe a strategy using microarray hybridizations to facilitate positional cloning. This method relies on the fact that premature stop codons (i.e., nonsense mutations) constitute a frequent class of mutations isolated in screens and that nonsense mutant messenger RNAs are efficiently degraded by the conserved nonsense-mediated decay pathway. They validate this strategy by identifying two previously uncharacterized mutations: (1) tom-1, a mutation found in a forward genetic screen for enhanced acetylcholine secretion in Caenorhabditis elegans, and (2) an apparently spontaneous mutation in the hif-1 transcription factor gene. They further demonstrate the broad applicability of this strategy using other known mutants in C. elegans, Arabidopsis, and mouse. Characterization of tom-1 mutants suggests that TOM-1, the C. elegans ortholog of mammalian tomosyn, functions as an endogenous inhibitor of neurotransmitter secretion. These results also suggest that microarray hybridizations have the potential to significantly reduce the time and effort required for positional cloning.

Genes encoding ribosomal proteins Rps0A/B of Saccharomyces cerevisiae interact with TOM1 mutants defective in ribosome synthesis.

The Saccharomyces cerevisiae RPS0A/B genes encode proteins of the 40S ribosomal subunit that are required for the maturation of 18S rRNA. We show here that the RPS0 genes interact genetically with TOM1. TOM1 encodes a member of the hect-domain-containing E3 ubiquitin-protein ligase family that is required for growth at elevated temperatures. Mutant alleles of the RPS0 and TOM1 genes have synergistic effects on cell growth at temperatures permissive for TOM1 mutants. Moreover, the growth arrest of TOM1 mutants at elevated temperatures is partially suppressed by overexpression of RPS0A/B. Strains with mutant alleles of TOM1 are defective in multiple steps in rRNA processing, and interactions between RPS0A/B and TOM1 stem, in part, from their roles in the maturation of ribosomal subunits. Ribosome synthesis is therefore included among the cellular processes governed by members of the hect-domain-containing E3 ubiquitin-protein ligase family.

Yeast Pdr13p and Zuo1p molecular chaperones are new functional Hsp70 and Hsp40 partners

The deletion of the TOM1 gene encoding a putative ubiquitin ligase causes a temperature sensitive cellular growth in Saccharomyces cerevisiae. The arrested cells exhibit pleiotropic defects in nuclear division, maintenance of nuclear structure and heat stress responses. We previously identified a zuo1 mutation as an extragenic suppressor of the tom1 mutant. ZUO1 encodes a DnaJ-related Hsp40. Here we show that a recessive cold sensitive mutation in PDR13 coding for an Hsp70 suppressed the tom1 mutation. The pdr13 deletion mutant was sensitive to high osmolarity, just like the zuo1 deletion mutant. A zuo1 pdr13 double deletion mutant did not show additive phenotypes. Furthermore, a tagged-Zuo1p was co-immunoprecipitated with a tagged-Pdr13p. Taken together, we propose that Pdr13p and Zuo1p are a new pair of Hsp70:Hsp40 molecular chaperones. In addition, Pdr13p co-sedimented with translating ribosomes and this association was independent of the presence of Zuo1p.

Extragenic suppressors that rescue defects in the heat stress response of the budding yeast mutant tom1.

The TOM1 gene codes for a so-called HECT protein, a putative ubiquitin ligase, in Saccharomyces cerevisiae. Deletion of the entire gene (tom1-10) or the sequence encoding the HECT domain (tom1-2) causes temperature sensitivity for growth. Here we report the isolation of extragenic, recessive suppressors of tom1-2, which were designated tmr (for tom1 revertant) mutations. These were classified into eight complementation groups and six of the genes were identified: tmr1/cyr1, tmnr2/sch9, tmr3/zuo1, tmr4, tmr5/mot1, tmr6/sse1, tmr7 and tmr8/kre6. These results suggested that the tom1 phenotype can be rescued by down-regulating the cAMP/PKA pathway. It was found that the temperature sensitivity of the tom1-2 mutant is indeed suppressed by multiple copies of PDE2 or BCY1, which encode negative regulators of the cAMP/PKA pathway. The MSN2 gene, which encodes a zinc-finger transcription factor involved in the general stress response is also a multicopy suppressor of tom1. It was found that induction levels of both STRE-mediated (general stress response) and HSE-mediated gene expression (heat shock response) upon shift to high temperature are reduced by more than half in the tom1 mutant. Most of the isolated tmr mutations rescued one of the defects seen in both types of heat stress response in the tom1 mutant.

Yeast tom1 mutant exhibits pleiotropic defects in nuclear division, maintenance of nuclear structure and nucleocytoplasmic transport at high temperatures

A tom1-1 mutant was isolated from Saccharomyces cerevisiae. At high temperatures, 60% of the cells were arrested as dumbbell forms with a single large nucleus containing duplicated DNA and a short spindle. Electron-microscopy showed electron-dense structures scattered within the nucleus. Indirect immunofluorescent microscopy revealed these structures to be fragmented nucleoli since the dotted structures were stained with anti-Nop1(fibrillarin) antibody in large regions of the nuclei. Fluorescent in situ hybridization analysis using oligo(dT) revealed nuclear accumulation of poly(A)+RNA. We cloned TOM1 which encodes a large protein (380kDa) with a hect (homologous to E6-AP C terminus)-domain at its C terminus. Deletions of either this hect-region or the entire gene made cellular growth temperature-sensitive. Site-directed mutagenesis of the conserved cysteine residue (tom1C3235A) in the hect-domain, supposed to be necessary for thioester-bond formation with ubiquitin, abolished the gene function. When a functional glutathione S-transferase (GST)-tagged hect protein was overproduced, it facilitated the protein conjugation with a myc-tagged ubiquitinRA, while this was not seen when GST-hectC3235A was overproduced. The protein conjugation with a hemagglutinin-tagged Smt3 was not affected by the overproduction of GST-hect. Taken together, we suggest that Tom1 is a ubiquitin ligase. As a multi-copy suppressor of tom1, we isolated STM3/NPI46/FPR3 which encodes a nucleolar nucleolin-like protein. We discuss possible functions of Tom1 with respect to the pleiotropic defects of nuclear division, maintenance of nuclear structure, and nucleocytoplasmic transport.

A high dose of the STM1 gene suppresses the temperature sensitivity of the tom1 and htr1 mutants in Saccharomyces cerevisiae

A new gene ( STM1;_suppressor of_to_ml) Saccharomyces cerevisiae was isolated by the ability to suppress the temperature sensitivity of a tom1 mutant, by increasing its gene dosage. The gene could also suppress the temperature sensitivity of the htr1 disruptant (Kikuchi et al. (1994) Mol. Gen. Genet. 245, 107–116) and was physically mapped in the region near PEP3 on chromosome XII R. The predicted gene product (29999 Da) is basic and partially homologous to various histone H1. The level of the gene expression increased 2-fold when exposed to mating pheromone.