Browse
Submit Data
Databases
API
Help

Dataset Information

13 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

The TOG protein Stu2/XMAP215 interacts covalently and noncovalently with SUMO.

ABSTRACT: Stu2p is the yeast member of the XMAP215/Dis1/ch-TOG family of microtubule-associated proteins that promote microtubule polymerization. However, the factors that regulate its activity are not clearly understood. Here we report that Stu2p in the budding yeast Saccharomyces cerevisiae interacts with SUMO by covalent and noncovalent mechanisms. Stu2p interacted by two-hybrid analysis with the yeast SUMO Smt3p, its E2 Ubc9p, and the E3 Nfi1p. A region of Stu2p containing the dimerization domain was both necessary and sufficient for interaction with SUMO and Ubc9p. Stu2p was found to be sumoylated both in vitro and in vivo. Stu2p copurified with SUMO in a pull-down assay and vice versa. Stu2p also bound to a nonconjugatable form of SUMO, suggesting that Stu2p can interact noncovalently with SUMO. In addition, Stu2p interacted with the STUbL enzyme Ris1p. Stu2p also copurified with ubiquitin in a pull-down assay, suggesting that it can be modified by both SUMO and ubiquitin. Tubulin, a major binding partner of Stu2p, also interacted noncovalently with SUMO. By two-hybrid analysis, the beta-tubulin Tub2p interacted with SUMO independently of the microtubule stressor, benomyl. Together, these findings raise the possibility that the microtubule polymerization activities mediated by Stu2p are regulated through sumoylation pathways.

SUBMITTER: Greenlee M

PROVIDER: S-EPMC6712953 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Regulation of microtubule dynamics by TOG-domain proteins XMAP215/Dis1 and CLASP.

Project description:The molecular mechanisms by which microtubule-associated proteins (MAPs) regulate the dynamic properties of microtubules (MTs) are still poorly understood. We review recent advances in our understanding of two conserved families of MAPs, the XMAP215/Dis1 and CLASP family of proteins. In vivo and in vitro studies show that XMAP215 proteins act as microtubule polymerases at MT plus ends to accelerate MT assembly, and CLASP proteins promote MT rescue and suppress MT catastrophe events. These are structurally related proteins that use conserved TOG domains to recruit tubulin dimers to MTs. We discuss models for how these proteins might use these individual tubulin dimers to regulate dynamic behavior of MT plus ends.

| S-EPMC3202638 | biostudies-literature

MCAK-independent functions of ch-Tog/XMAP215 in microtubule plus-end dynamics.

Project description:The formation of a functional bipolar mitotic spindle is essential for genetic integrity. In human cells, the microtubule polymerase XMAP215/ch-Tog ensures spindle bipolarity by counteracting the activity of the microtubule-depolymerizing kinesin XKCM1/MCAK. Their antagonistic effects on microtubule polymerization confer dynamic instability on microtubules assembled in cell-free systems. It is, however, unclear if a similar interplay governs microtubule behavior in mammalian cells in vivo. Using real-time analysis of spindle assembly, we found that ch-Tog is required to produce or maintain long centrosomal microtubules after nuclear-envelope breakdown. In the absence of ch-Tog, microtubule assembly at centrosomes was impaired and microtubules were nondynamic. Interkinetochore distances and the lengths of kinetochore fibers were also reduced in these cells. Codepleting MCAK with ch-Tog improved kinetochore fiber length and interkinetochore separation but, surprisingly, did not rescue centrosomal microtubule assembly and microtubule dynamics. Our data therefore suggest that ch-Tog has at least two distinct roles in spindle formation. First, it protects kinetochore microtubules from depolymerization by MCAK. Second, ch-Tog plays an essential role in centrosomal microtubule assembly, a function independent of MCAK activity. Thus, the notion that the antagonistic activities of MCAK and ch-Tog determine overall microtubule stability is too simplistic to apply to human cells.

| S-EPMC2593372 | biostudies-literature

The XMAP215 family drives microtubule polymerization using a structurally diverse TOG array.

Project description:XMAP215 family members are potent microtubule (MT) polymerases, with mutants displaying reduced MT growth rates and aberrant spindle morphologies. XMAP215 proteins contain arrayed tumor overexpressed gene (TOG) domains that bind tubulin. Whether these TOG domains are architecturally equivalent is unknown. Here we present crystal structures of TOG4 from Drosophila Msps and human ch-TOG. These TOG4 structures architecturally depart from the structures of TOG domains 1 and 2, revealing a conserved domain bend that predicts a novel engagement with α-tubulin. In vitro assays show differential tubulin-binding affinities across the TOG array, as well as differential effects on MT polymerization. We used Drosophila S2 cells depleted of endogenous Msps to assess the importance of individual TOG domains. Whereas a TOG1-4 array largely rescues MT polymerization rates, mutating tubulin-binding determinants in any single TOG domain dramatically reduces rescue activity. Our work highlights the structurally diverse yet positionally conserved TOG array that drives MT polymerization.

| S-EPMC4142611 | biostudies-literature

TUBA1A tubulinopathy mutants disrupt neuron morphogenesis and override XMAP215/Stu2 regulation of microtubule dynamics.

Project description:Heterozygous, missense mutations in α- or β-tubulin genes are associated with a wide range of human brain malformations, known as tubulinopathies. We seek to understand whether a mutation's impact at the molecular and cellular levels scale with the severity of brain malformation. Here, we focus on two mutations at the valine 409 residue of TUBA1A, V409I, and V409A, identified in patients with pachygyria or lissencephaly, respectively. We find that ectopic expression of TUBA1A-V409I/A mutants disrupt neuronal migration in mice and promote excessive neurite branching and a decrease in the number of neurite retraction events in primary rat neuronal cultures. These neuronal phenotypes are accompanied by increased microtubule acetylation and polymerization rates. To determine the molecular mechanisms, we modeled the V409I/A mutants in budding yeast and found that they promote intrinsically faster microtubule polymerization rates in cells and in reconstitution experiments with purified tubulin. In addition, V409I/A mutants decrease the recruitment of XMAP215/Stu2 to plus ends in budding yeast and ablate tubulin binding to TOG (tumor overexpressed gene) domains. In each assay tested, the TUBA1A-V409I mutant exhibits an intermediate phenotype between wild type and the more severe TUBA1A-V409A, reflecting the severity observed in brain malformations. Together, our data support a model in which the V409I/A mutations disrupt microtubule regulation typically conferred by XMAP215 proteins during neuronal morphogenesis and migration, and this impact on tubulin activity at the molecular level scales with the impact at the cellular and tissue levels.

| S-EPMC9236607 | biostudies-literature

The Dis1/Stu2/XMAP215 Family Gene FgStu2 Is Involved in Vegetative Growth, Morphology, Sexual and Asexual Reproduction, Pathogenicity and DON Production of Fusarium graminearum.

Project description:The conserved Dis1/Stu2/XMAP215 microtubule association proteins (MAPs) family plays an important role in microtubule dynamics, nucleation, and kinetochore-microtubule attachments. However, function of Dis1/Stu2/XMAP215 homolog in plant pathogenic fungi has not been determined. Here, we identified and investigated the Dis1/Stu2/XMAP215 homolog (FGSG_10528) in Fusarium graminearum (FgStu2p). Co-localization experiment and co-immunoprecipitation (Co-IP) assay demonstrated that FgStu2p is a microtubule associated protein. Besides, FgStu2 could also interact with Fgγ-tubulin and presumed FgNdc80, which suggested that the FgStu2 gene might associate with microtubule nucleation and kinetochore-microtubule attachments like Dis1/Stu2/XMAP215 homologs in other species. Moreover, the FgStu2 promoter replacement mutants (FgStu2-Si mutants) produced twisted hyphae and decreased growth rate. Microscope examination further showed that the microtubule polymerization was reduced in FgStu2-Si mutants, which could account for the aberrant morphology. Although the microtubule polymerization was affected in FgStu2-Si mutants, the FgStu2-Si mutants didn't show highly increased sensitivity to anti-microtubule fungicide carbendazim (methyl benzimidazol-2-ylcarbamate [MBC]). In addition, the FgStu2-Si mutants exhibited curved conidia, decreased number of conidial production, blocked ability of perithecia production, decreased pathogenicity and deoxynivalenol (DON) production. Taken together, these results indicate that the FgStu2 gene plays a crucial role in vegetative growth, morphology, sexual reproduction, asexual reproduction, virulence and deoxynivalenol (DON) production of F. graminearum, which brings new insights into the functions of Dis1/Stu2/XMAP215 homolog in plant pathogenic fungi.

| S-EPMC7714731 | biostudies-literature

Fission yeast Dis1 is an unconventional TOG/XMAP215 that induces microtubule catastrophe to drive chromosome pulling.

Project description:The shortening of microtubules attached to kinetochores is the driving force of chromosome movement during cell division. Specific kinesins are believed to shorten microtubules but are dispensable for viability in yeast, implying the existence of additional factors responsible for microtubule shortening. Here, we demonstrate that Dis1, a TOG/XMAP215 ortholog in fission yeast, promotes microtubule shortening to carry chromosomes. Although TOG/XMAP215 orthologs are generally accepted as microtubule polymerases, Dis1 promoted microtubule catastrophe in vitro and in vivo. Notably, microtubule catastrophe was promoted when the tip was attached to kinetochores, as they steadily anchored Dis1 at the kinetochore-microtubule interface. Engineered Dis1 oligomers artificially tethered at a chromosome arm region induced the shortening of microtubules in contact, frequently pulling the chromosome arm towards spindle poles. This effect was not brought by oligomerised Alp14. Thus, unlike Alp14 and other TOG/XMAP215 orthologs, Dis1 plays an unconventional role in promoting microtubule catastrophe, thereby driving chromosome movement.

| S-EPMC9701203 | biostudies-literature

Tomosyn interacts with the SUMO E3 ligase PIASγ.

Project description:Protein modification by Small Ubiquitin-like MOdifier (SUMO) entities is involved in a number of neuronal functions, including synaptogenesis and synaptic plasticity. Tomosyn-1 (syntaxin-binding protein 5; STXPB5) binds to t-SNARE (Soluble NSF Attachment Protein Receptor) proteins to regulate neurotransmission and is one of the few neuronal SUMO substrate proteins identified. Here we used yeast two-hybrid screening to show that tomosyn-1 interacts with the SUMO E3 ligase PIASγ (Protein Inhibitor of Activated STAT; PIAS4 or ZMIZ6). This novel interaction involved the C-terminus of tomosyn-1 and the N-terminus of PIASγ. It was confirmed by two-way immunoprecipitation experiments using the full-length proteins expressed in HEK293T cells. Tomosyn-1 was preferentially modified by the SUMO-2/3 isoform. PIASγ-dependent modification of tomosyn-1 with SUMO-2/3 presents a novel mechanism to adapt secretory strength to the dynamic synaptic environment.

| S-EPMC3948876 | biostudies-literature

Fission yeast ch-TOG/XMAP215 homologue Alp14 connects mitotic spindles with the kinetochore and is a component of the Mad2-dependent spindle checkpoint.

Project description:The TOG/XMAP215-related proteins play a role in microtubule dynamics at its plus end. Fission yeast Alp14, a newly identified TOG/XMAP215 family protein, is essential for proper chromosome segregation in concert with a second homologue Dis1. We show that the alp14 mutant fails to progress towards normal bipolar spindle formation. Intriguingly, Alp14 itself is a component of the Mad2-dependent spindle checkpoint cascade, as upon addition of microtubule-destabilizing drugs the alp14 mutant is incapable of maintaining high H1 kinase activity, which results in securin destruction and premature chromosome separation. Live imaging of Alp14-green fluorescent protein shows that during mitosis, Alp14 is associated with the peripheral region of the kinetochores as well as with the spindle poles. This is supported by ChIP (chromatin immunoprecipitation) and overlapping localization with the kinetochore marker Mis6. An intact spindle is required for Alp14 localization to the kinetochore periphery, but not to the poles. These results indicate that the TOG/XMAP215 family may play a central role as a bridge between the kinetochores and the plus end of pole to chromosome microtubules.

| S-EPMC125509 | biostudies-literature

Nucleolar and spindle-associated protein 1 (NUSAP1) interacts with a SUMO E3 ligase complex during chromosome segregation.

Project description:The mitotic spindle is composed of dynamic microtubules and associated proteins that together direct chromosome movement during mitosis. The spindle plays a vital role in accurate chromosome segregation fidelity and is a therapeutic target in cancer. Nevertheless, the molecular mechanisms by which many spindle-associated proteins function remains unknown. The nucleolar and spindle-associated protein NUSAP1 is a microtubule-binding protein implicated in spindle stability and chromosome segregation. We show here that NUSAP1 localizes to dynamic spindle microtubules in a unique chromosome-centric pattern, in the vicinity of overlapping microtubules, during metaphase and anaphase of mitosis. Mass spectrometry-based analysis of endogenous NUSAP1 interacting proteins uncovered a cell cycle-regulated interaction between the RanBP2-RanGAP1-UBC9 SUMO E3 ligase complex and NUSAP1. Like NUSAP1 depletion, RanBP2 depletion impaired the response of cells to the microtubule poison Taxol. NUSAP1 contains a conserved SAP domain (SAF-A/B, Acinus, and PIAS). SAP domains are common among many other SUMO E3s, and are implicated in substrate recognition and ligase activity. We speculate that NUSAP1 contributes to accurate chromosome segregation by acting as a co-factor for RanBP2-RanGAP1-UBC9 during cell division.

| S-EPMC5655498 | biostudies-literature

Ndc80 internal loop interacts with Dis1/TOG to ensure proper kinetochore-spindle attachment in fission yeast.

Project description:The Ndc80 complex, a conserved outer kinetochore complex, comprising four components (Ndc80/Hec1, Nuf2, Spc24, and Spc25), constitutes one of the core microtubule-binding sites within the kinetochore. Despite this knowledge, molecular mechanisms by which this complex contributes to establishment of correct bipolar attachment of the kinetochore to the spindle microtubule remain largely elusive. Here we show that the conserved internal loop of fission yeast Ndc80 directly binds the Dis1/TOG microtubule-associated protein, thereby coupling spindle microtubule dynamics with kinetochore capture. Ndc80 loop mutant proteins fail to recruit Dis1 to kinetochores, imposing unstable attachment and frequent spindle collapse. In these mutants, mitotic progression is halted attributable to spindle assembly checkpoint activation, and chromosomes remain in the vicinity of the spindle poles without congression. dis1 deletion precisely phenocopies the loop mutants. Intriguingly, forced targeting of Dis1 to the Ndc80 complex rescues loop mutant's defects. We propose that Ndc80 comprises two microtubule-interacting interfaces: the N-terminal region directly binds the microtubule lattice, while the internal loop interacts with the plus end of microtubules via Dis1/TOG. Therefore, our results provide a crucial insight into how the Ndc80 complex establishes stable bipolar attachment to the spindle microtubule.

| S-EPMC3049873 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data