Drosophila melanogaster Mini Spindles TOG3 Utilizes Unique Structural Elements to Promote Domain Stability and Maintain a TOG1- and TOG2-like Tubulin-binding Surface

Howard, Amy E.; Fox, Jaime C.; Slep, Kevin C.

doi:10.1074/jbc.m114.633826

“…Not requiring a linked array of TOGs to obtain activities promoted by the intact protein may have implications for other TOG-containing regulatory factors. Indeed, recent structures of TOG3, TOG4, and TOG5 from XMAP215-family microtubule polymerases ( Howard et al. , 2015 ; Byrnes and Slep, 2017 ), and of TOG domains from cilium-localized regulatory factors ( Das et al.…”

Section: Discussionmentioning

confidence: 99%

An isolated CLASP TOG domain suppresses microtubule catastrophe and promotes rescue

Majumdar

¹

,

Kim

²

,

Chen

³

et al. 2018

MBoC

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Microtubules are heavily regulated dynamic polymers of αβ-tubulin that are required for proper chromosome segregation and organization of the cytoplasm. Polymerases in the XMAP215 family use arrayed TOG domains to promote faster microtubule elongation. Regulatory factors in the cytoplasmic linker associated protein (CLASP) family that reduce catastrophe and/or increase rescue also contain arrayed TOGs, but how CLASP TOGs contribute to activity is poorly understood. Here, using Saccharomyces cerevisiae Stu1 as a model CLASP, we report structural, biochemical, and reconstitution studies that clarify functional properties of CLASP TOGs. The two TOGs in Stu1 have very different tubulin-binding properties: TOG2 binds to both unpolymerized and polymerized tubulin, and TOG1 binds very weakly to either. The structure of Stu1-TOG2 reveals a CLASP-specific residue that likely confers distinctive tubulin-binding properties. The isolated TOG2 domain strongly suppresses microtubule catastrophe and increases microtubule rescue in vitro, contradicting the expectation that regulatory activity requires an array of TOGs. Single point mutations on the tubulin-binding surface of TOG2 ablate its anti-catastrophe and rescue activity in vitro, and Stu1 function in cells. Revealing that an isolated CLASP TOG can regulate polymerization dynamics without being part of an array provides insight into the mechanism of CLASPs and diversifies the understanding of TOG function.

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“…The structure of TOG domains is highly conserved for microtubule binding, where the intra-HEAT loop in the discontinuous TOG domain binds tubulin (63)(64)(65)(66). TOG domains are thought to regulate microtubule growth and dynamics (67).…”

Section: Role Of the Armc9-togaram1 Complex In Ciliary Length And Stamentioning

confidence: 99%

Dysfunction of the ciliary ARMC9/TOGARAM1 protein module causes Joubert syndrome

Latour¹,

Weghe²,

Rusterholz³

et al. 2020

Journal of Clinical Investigation

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Joubert syndrome (JBTS) is a recessive neurodevelopmental ciliopathy, characterized by a pathognomonic hindbrain malformation. All known JBTS-genes encode proteins involved in the structure or function of primary cilia, ubiquitous antenna-like organelles essential for cellular signal transduction. Here, we use the recently identified JBTS-associated protein ARMC9 in tandem-affinity purification and yeast two-hybrid screens to identify a novel ciliary module whose dysfunction underlies JBTS. In addition to known JBTS-associated proteins CEP104 and CSPP1, we identify CCDC66 and TOGARAM1 as ARMC9 interaction partners. We show that TOGARAM1 variants cause JBTS and disrupt TOGARAM1 interaction with ARMC9. Using a combination of protein interaction analyses and characterization of patient-derived fibroblasts, CRISPR/Cas9-engineered zebrafish and hTERT-RPE1 cells, we demonstrate that dysfunction of ARMC9 or TOGARAM1 results in short cilia with decreased axonemal acetylation and polyglutamylation, but relatively intact transition zone function. Aberrant cold-and serum-induced ciliary loss in both ARMC9 and TOGARAM1 patient cell lines suggests a role for this new JBTSassociated protein module in ciliary stability.

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“…The structure of TOG domains is highly conserved for microtubule binding, where the intra-HEAT loop within the discontinuous TOG domain binds tubulin (52)(53)(54)(55). TOG domains are thought to function to positively regulate microtubule growth and dynamics (56).…”

Section: Role Of the Armc9-togaram1 Complex In Ciliary Length And Stamentioning

confidence: 99%

ARMC9 and TOGARAM1 define a Joubert syndrome-associated protein module that regulates axonemal post-translational modifications and cilium stability

Latour

¹

,

Weghe

²

,

Rusterholz

³

et al. 2019

Preprint

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Joubert syndrome (JBTS) is a recessive neurodevelopmental ciliopathy, characterized by a pathognomonic hindbrain malformation. All known JBTS-genes encode proteins involved in the structure or function of primary cilia, ubiquitous antenna-like organelles essential for cellular signal transduction. Here, we use the recently identified JBTS-associated protein ARMC9 in tandem-affinity purification and yeast twohybrid screens to identify a novel ciliary module composed of ARMC9-TOGARAM1-CCDC66-CEP104-CSPP1. TOGARAM1-variants cause JBTS and disrupt its interaction with ARMC9. Using a combination of protein interaction analyses and characterization of patient-derived fibroblasts, CRISPR/Cas9-engineered zebrafish and hTERT-RPE1 cells, we demonstrate that dysfunction of ARMC9 or TOGARAM1 results in short cilia with decreased axonemal acetylation and glutamylation, but relatively intact transition zone function. Aberrant serum-induced ciliary resorption and cold-induced depolymerization in both ARMC9 and TOGARAM1 patient cells lines suggest a role for this new JBTS-associated protein complex in ciliary stability.part by the transition zone (TZ) that connects the axoneme to the membrane and acts as a partition.Approximately half of the known JBTS proteins, including RPGRIP1L (JBTS7) (14) and CC2D2A (JBTS9) (15), assemble into multi-protein complexes at the ciliary TZ where they are thought to organize the molecular gate that regulates ciliary protein entry and exit (16); and dysfunction of TZ organization is thought to play a key role in JBTS. Another subset of JBTS-associated proteins, including ARL13B (JBTS8) and INPP5E (JBTS1) (6), associate with the ciliary membrane beyond the TZ. These proteins are thought to play a role in regulation of signaling pathways such as Hh signaling via maintenance of ciliary lipid composition (17, 18). Different JBTS-associated proteins have been found to function at the basal body or distal segment/tip (4). CSPP1 (JBTS 21) (19) and CEP104 (JBTS25) (20) were mainly detected at the centrosomes and ciliary basal bodies, however their exact molecular function, and how defects in these proteins lead to JBTS, are less well understood. CEP104 localizes to the ciliary tip during ciliogenesis, where it is required for structural integrity in the motile cilia of Chlamydomonas and Tetrahymena (21, 22). Mutations in the gene encoding the ciliary tip kinesin KIF7 (JBTS12) cause JBTS, which were linked to defects in tubulin acetylation and Hedgehog signaling (23).Recently, we identified mutations in the gene encoding armadillo repeat motif containing 9 (ARMC9) in individuals with JBTS (JBTS30). ARMC9 localizes to centrioles (27) and the proximal portion of the cilium (28) in mammalian cilia. ARMC9 transcript levels are upregulated with induction of ciliogenesis and armc9 dysfunction in zebrafish yields typical ciliopathy phenotypes (27). As ARMC9 has not been identified as a component of the ciliary JS-associated protein modules mentioned above, we here use ARMC9 as a bait in protein interaction screen...

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Drosophila melanogaster Mini Spindles TOG3 Utilizes Unique Structural Elements to Promote Domain Stability and Maintain a TOG1- and TOG2-like Tubulin-binding Surface

Cited by 11 publications

References 71 publications

An isolated CLASP TOG domain suppresses microtubule catastrophe and promotes rescue

An isolated CLASP TOG domain suppresses microtubule catastrophe and promotes rescue

Dysfunction of the ciliary ARMC9/TOGARAM1 protein module causes Joubert syndrome

ARMC9 and TOGARAM1 define a Joubert syndrome-associated protein module that regulates axonemal post-translational modifications and cilium stability

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