scholarly journals Septin ring size scaling and dynamics require the coiled-coil region of Shs1p

2012 ◽  
Vol 23 (17) ◽  
pp. 3391-3406 ◽  
Author(s):  
Rebecca A. Meseroll ◽  
Louisa Howard ◽  
Amy S. Gladfelter
Keyword(s):  
Coiled Coil ◽  
Higher Order ◽  
Membrane Curvature ◽  
Negative Regulator ◽  
Ring Size ◽  
Septin Ring ◽  
Ring Assembly ◽  
Coiled Coil Domain ◽  
C Terminus ◽  
Important Negative Regulator

Septins are conserved GTP-binding proteins that assemble into heteromeric complexes that form filaments and higher-order structures in cells. What directs filament assembly, determines the size of higher-order septin structures, and governs septin dynamics is still not well understood. We previously identified two kinases essential for septin ring assembly in the filamentous fungus Ashbya gossypii and demonstrate here that the septin Shs1p is multiphosphorylated at the C-terminus of the protein near the predicted coiled-coil domain. Expression of the nonphosphorylatable allele shs1-9A does not mimic the loss of the kinase nor does complete truncation of the Shs1p C-terminus. Surprisingly, however, loss of the C-terminus or the predicted coiled-coil domain of Shs1p generates expanded zones of septin assemblies and ectopic septin fibers, as well as aberrant cell morphology. The expanded structures form coincident with ring assembly and are heteromeric. Interestingly, while septin recruitment to convex membranes is increased, septin localization is diminished at concave membranes in these mutants. Additionally, the loss of the coiled-coil leads to increased mobility of Shs1p. These data indicate the coiled-coil of Shs1p is an important negative regulator of septin ring size and mobility, and its absence may make septin assembly sensitive to local membrane curvature.

scholarly journals Drosophila CLIP-190 and mammalian CLIP-170 display reduced microtubule plus end association in the nervous system

2015 ◽  
Vol 26 (8) ◽  
pp. 1491-1508 ◽  
Author(s):  
Robin Beaven ◽  
Nikola S. Dzhindzhev ◽  
Yue Qu ◽  
Ines Hahn ◽  
Federico Dajas-Bailador ◽  
...  
Keyword(s):  
Nervous System ◽  
Current Knowledge ◽  
Coiled Coil ◽  
Growth Dynamics ◽  
Loss Of Function ◽  
Axon Extension ◽  
Coiled Coil Domain ◽  
C Terminus

Axons act like cables, electrically wiring the nervous system. Polar bundles of microtubules (MTs) form their backbones and drive their growth. Plus end–tracking proteins (+TIPs) regulate MT growth dynamics and directionality at their plus ends. However, current knowledge about +TIP functions, mostly derived from work in vitro and in nonneuronal cells, may not necessarily apply to the very different context of axonal MTs. For example, the CLIP family of +TIPs are known MT polymerization promoters in nonneuronal cells. However, we show here that neither Drosophila CLIP-190 nor mammalian CLIP-170 is a prominent MT plus end tracker in neurons, which we propose is due to low plus end affinity of the CAP-Gly domain–containing N-terminus and intramolecular inhibition through the C-terminus. Instead, both CLIP-190 and CLIP-170 form F-actin–dependent patches in growth cones, mediated by binding of the coiled-coil domain to myosin-VI. Because our loss-of-function analyses in vivo and in culture failed to reveal axonal roles for CLIP-190, even in double-mutant combinations with four other +TIPs, we propose that CLIP-190 and -170 are not essential axon extension regulators. Our findings demonstrate that +TIP functions known from nonneuronal cells do not necessarily apply to the regulation of the very distinct MT networks in axons.

scholarly journals Functional Evolution of the Photolyase/Cryptochrome Protein Family: Importance of the C Terminus of Mammalian CRY1 for Circadian Core Oscillator Performance

2006 ◽  
Vol 26 (5) ◽  
pp. 1743-1753 ◽  
Author(s):  
Inês Chaves ◽  
Kazuhiro Yagita ◽  
Sander Barnhoorn ◽  
Hitoshi Okamura ◽  
Gijsbertus T. J. van der Horst ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Coiled Coil ◽  
Structural Similarity ◽  
Light Signaling ◽  
Core Domain ◽  
Coiled Coil Domain ◽  
C Terminus ◽  
Localization Signal ◽  
Species Specific

ABSTRACT Cryptochromes (CRYs) are composed of a core domain with structural similarity to photolyase and a distinguishing C-terminal extension. While plant and fly CRYs act as circadian photoreceptors, using the C terminus for light signaling, mammalian CRY1 and CRY2 are integral components of the circadian oscillator. However, the function of their C terminus remains to be resolved. Here, we show that the C-terminal extension of mCRY1 harbors a nuclear localization signal and a putative coiled-coil domain that drive nuclear localization via two independent mechanisms and shift the equilibrium of shuttling mammalian CRY1 (mCRY1)/mammalian PER2 (mPER2) complexes towards the nucleus. Importantly, deletion of the complete C terminus prevents mCRY1 from repressing CLOCK/BMAL1-mediated transcription, whereas a plant photolyase gains this key clock function upon fusion to the last 100 amino acids of the mCRY1 core and its C terminus. Thus, the acquirement of different (species-specific) C termini during evolution not only functionally separated cryptochromes from photolyase but also caused diversity within the cryptochrome family.

scholarly journals Phosphorylation of Serine 114 on Atg32 mediates mitophagy

2011 ◽  
Vol 22 (17) ◽  
pp. 3206-3217 ◽  
Author(s):  
Yoshimasa Aoki ◽  
Tomotake Kanki ◽  
Yuko Hirota ◽  
Yusuke Kurihara ◽  
Tetsu Saigusa ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Quality Control ◽  
Significant Role ◽  
Coiled Coil ◽  
Adaptor Protein ◽  
Mitochondrial Quality Control ◽  
Signal Transduction Cascade ◽  
Coiled Coil Domain ◽  
C Terminus ◽  
N Terminus

Mitophagy, which selectively degrades mitochondria via autophagy, has a significant role in mitochondrial quality control. When mitophagy is induced in yeast, mitochondrial residential protein Atg32 binds Atg11, an adaptor protein for selective types of autophagy, and it is recruited into the vacuole along with mitochondria. The Atg11–Atg32 interaction is believed to be the initial molecular step in which the autophagic machinery recognizes mitochondria as a cargo, although how this interaction is mediated is poorly understood. Therefore, we studied the Atg11–Atg32 interaction in detail. We found that the C-terminus region of Atg11, which included the fourth coiled-coil domain, interacted with the N-terminus region of Atg32 (residues 100–120). When mitophagy was induced, Ser-114 and Ser-119 on Atg32 were phosphorylated, and then the phosphorylation of Atg32, especially phosphorylation of Ser-114 on Atg32, mediated the Atg11–Atg32 interaction and mitophagy. These findings suggest that cells can regulate the amount of mitochondria, or select specific mitochondria (damaged or aged) that are degraded by mitophagy, by controlling the activity and/or localization of the kinase that phosphorylates Atg32. We also found that Hog1 and Pbs2, which are involved in the osmoregulatory signal transduction cascade, are related to Atg32 phosphorylation and mitophagy.

scholarly journals The homo-oligomerisation of both Sas-6 and Ana2 is required for efficient centriole assembly in flies

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Matthew A Cottee ◽  
Nadine Muschalik ◽  
Steven Johnson ◽  
Joanna Leveson ◽  
Jordan W Raff ◽  
...  
Keyword(s):  
Point Mutations ◽  
Coiled Coil ◽  
Higher Order ◽  
Centriole Duplication ◽  
Terminal Domain ◽  
Coiled Coil Domain ◽  
Centriole Assembly ◽  
Tetramer Structure

Sas-6 and Ana2/STIL proteins are required for centriole duplication and the homo-oligomerisation properties of Sas-6 help establish the ninefold symmetry of the central cartwheel that initiates centriole assembly. Ana2/STIL proteins are poorly conserved, but they all contain a predicted Central Coiled-Coil Domain (CCCD). Here we show that the Drosophila Ana2 CCCD forms a tetramer, and we solve its structure to 0.8 Å, revealing that it adopts an unusual parallel-coil topology. We also solve the structure of the Drosophila Sas-6 N-terminal domain to 2.9 Å revealing that it forms higher-order oligomers through canonical interactions. Point mutations that perturb Sas-6 or Ana2 homo-oligomerisation in vitro strongly perturb centriole assembly in vivo. Thus, efficient centriole duplication in flies requires the homo-oligomerisation of both Sas-6 and Ana2, and the Ana2 CCCD tetramer structure provides important information on how these proteins might cooperate to form a cartwheel structure.

scholarly journals The C Terminus of Rotavirus VP4 Protein Contains an Actin Binding Domain Which Requires Cooperation with the Coiled-Coil Domain for Actin Remodeling

2018 ◽  
Vol 93 (1) ◽  
Author(s):  
Wilfried Condemine ◽  
Thibaut Eguether ◽  
Nathalie Couroussé ◽  
Catherine Etchebest ◽  
Agnes Gardet ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Coiled Coil ◽  
Binding Domain ◽  
Spike Protein ◽  
Actin Binding ◽  
Intestinal Cells ◽  
Actin Remodeling ◽  
Coiled Coil Domain ◽  
C Terminus ◽  
Actin Binding Domain

ABSTRACTThe interactions between viruses and actin cytoskeleton have been widely studied. We showed that rotaviruses remodel microfilaments in intestinal cells and demonstrated that this was due to the VP4 spike protein. Microfilaments mainly occur in the apical domain of infected polarized enterocytes and favor the polarized apical exit of viral progeny. The present work aims at the identification of molecular determinants of actin-VP4 interactions. We used various deletion mutants of VP4 that were transfected into Cos-7 cells and analyzed interactions by immunofluorescence confocal microscopy. It has been established that the C-terminal part of VP4 is embedded within viral particles when rotavirus assembles. The use of specific monoclonal antibodies demonstrated that VP4 is expressed in different forms in infected cells: classically as spike on the outer layer of virus particles, but also as free soluble protein in the cytosol. The C terminus of free VP4 was identified as interacting with actin microfilaments. The VP4 actin binding domain is unable to promote microfilament remodeling by itself; the coiled-coil domain is also required in this process. This actin-binding domain was shown to dominate a previously identified peroxisomal targeting signal, located in the three last amino acids of VP4. The newly identified actin-binding domain is highly conserved in rotavirus strains from species A, B, and C, suggesting that actin binding and remodeling is a general strategy for rotavirus exit. This provides a novel mechanism of protein-protein interactions, not involving cell signaling pathways, to facilitate rotavirus exit.IMPORTANCERotaviruses are causal agents of acute infantile viral diarrhea. In intestinal cells,in vitroas well asin vivo, virus assembly and exit do not imply cell lysis but rely on an active process in which the cytoskeleton plays a major role. We describe here a novel molecular mechanism by which the rotavirus spike protein VP4 drives actin remodeling. This relies on the fact that VP4 occurs in different forms. Besides its structural function within the virion, a large proportion of VP4 is expressed as free protein. Here, we show that free VP4 possesses a functional actin-binding domain. This domain, in coordination with a coiled-coil domain, promotes actin cytoskeleton remodeling, thereby providing the capacity to destabilize the cell membrane and allow efficient rotavirus exit.

scholarly journals The Conserved RIC-3 Coiled-Coil Domain Mediates Receptor-specific Interactions with Nicotinic Acetylcholine Receptors

2009 ◽  
Vol 20 (5) ◽  
pp. 1419-1427 ◽  
Author(s):  
Yoav Biala ◽  
Jana F. Liewald ◽  
Hagit Cohen Ben-Ami ◽  
Alexander Gottschalk ◽  
Millet Treinin
Keyword(s):  
Alternative Splicing ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Coiled Coil ◽  
Nicotinic Acetylcholine ◽  
Coiled Coil Domain ◽  
C Terminus ◽  
Pharyngeal Muscles ◽  
Specific Regulation

RIC-3 belongs to a conserved family of proteins influencing nicotinic acetylcholine receptor (nAChR) maturation. RIC-3 proteins are integral membrane proteins residing in the endoplasmic reticulum (ER), and containing a C-terminal coiled-coil domain (CC-I). Conservation of CC-I in all RIC-3 family members indicates its importance; however, previous studies could not show its function. To examine the role of CC-I, we studied effects of its deletion on Caenorhabditis elegans nAChRs in vivo. Presence of CC-I promoted maturation of particular nAChRs expressed in body-wall muscle, whereas it was not required for other nAChR subtypes expressed in neurons or pharyngeal muscles. This effect is receptor-specific, because it could be reproduced after heterologous expression. Consistently, coimmunoprecipitation analysis showed that CC-I enhances the interaction of RIC-3 with a nAChR that requires CC-I in vivo; thus CC-I appears to enhance affinity of RIC-3 to specific nAChRs. However, we found that this function of CC-I is redundant with functions of sequences downstream to CC-I, potentially a second coiled-coil. Alternative splicing in both vertebrates and invertebrates generates RIC-3 transcripts that lack the entire C-terminus, or only CC-I. Thus, our results suggest that RIC-3 alternative splicing enables subtype specific regulation of nAChR maturation.

scholarly journals JRAB/MICAL-L2 Is a Junctional Rab13-binding Protein Mediating the Endocytic Recycling of Occludin

2006 ◽  
Vol 17 (5) ◽  
pp. 2465-2475 ◽  
Author(s):  
Tomoya Terai ◽  
Noriyuki Nishimura ◽  
Ikuno Kanda ◽  
Natsuo Yasui ◽  
Takuya Sasaki
Keyword(s):  
Immunofluorescence Microscopy ◽  
Binding Protein ◽  
Coiled Coil ◽  
Epithelial Morphogenesis ◽  
Endocytic Recycling ◽  
Nih3t3 Cells ◽  
Coiled Coil Domain ◽  
C Terminus ◽  
Actin Cables ◽  
Paracellular Diffusion

The dynamic turnover of tight junctions (TJs) is essential for epithelial-mesenchymal transitions and/or mesenchymal-epithelial transitions during epithelial morphogenesis. We previously demonstrated that Rab13 specifically mediates the endocytic recycling of occludin. Here, we identified MICAL-L2 (molecule interacting with CasL-like 2) as a novel Rab13-binding protein. Immunoprecipitation and immunofluorescence microscopy showed that MICAL-L2 specifically bound to the GTP-bound form of Rab13 via its C terminus, which contained a coiled-coil domain, and localized at TJs in epithelial MTD-1A cells. Recycling assay demonstrated that a MICAL-L2 mutant lacking the Rab13-binding domain (MICAL-L2-N) specifically inhibited the endocytic recycling of occludin but not transferrin receptor. Ca2+ switch assay further revealed that MICAL-L2-N as well as Rab13 Q67L inhibited the recruitment of occludin to the plasma membrane, the development of transepithelial electrical resistance, and the formation of a paracellular diffusion barrier. MICAL-L2 was displaced from TJs upon actin depolymerization and was distributed along radiating actin cables and stress fibers in Ca2+-depleted MTD-1A and fibroblastic NIH3T3 cells, respectively. These results suggest that MICAL-L2 mediates the endocytic recycling of occludin and the formation of functional TJs by linking Rab13 to actin cytoskeleton. We rename MICAL-L2 as JRAB (junctional Rab13-binding protein).

scholarly journals N-terminus-mediated dimerization of ROCK-I is required for RhoE binding and actin reorganization

2008 ◽  
Vol 411 (2) ◽  
pp. 407-414 ◽  
Author(s):  
Ritu Garg ◽  
Kirsi Riento ◽  
Nicholas Keep ◽  
Jonathan D. H. Morris ◽  
Anne J. Ridley
Keyword(s):  
Kinase Activity ◽  
Coiled Coil ◽  
Kinase Domain ◽  
Serine Threonine Kinase ◽  
Actin Reorganization ◽  
Coiled Coil Domain ◽  
C Terminus ◽  
Coiled Coil Region ◽  
N Terminus ◽  
In Cells

ROCK-I (Rho-associated kinase 1) is a serine/threonine kinase that can be activated by RhoA and inhibited by RhoE. ROCK-I has an N-terminal kinase domain, a central coiled-coil region and a RhoA-binding domain near the C-terminus. We have previously shown that RhoE binds to the N-terminal 420 amino acids of ROCK-I, which includes the kinase domain as well as N-terminal and C-terminal extensions. In the present study, we show that N-terminus-mediated dimerization of ROCK-I is required for RhoE binding. The central coiled-coil domain can also dimerize ROCK-I in cells, but this is insufficient in the absence of the N-terminus to allow RhoE binding. The kinase activity of ROCK-I1–420 is required for dimerization and RhoE binding; however, inclusion of part of the coiled-coil domain compensates for lack of kinase activity, allowing RhoE to bind. N-terminus-mediated dimerization is also required for ROCK-I to induce the formation of stellate actin stress fibres in cells. These results indicate that dimerization via the N-terminus is critical for ROCK-I function in cells and for its regulation by RhoE.

scholarly journals Missense mutation in immunodeficient patients shows the multifunctional roles of coiled-coil domain 3 (CC3) in STIM1 activation

2015 ◽  
Vol 112 (19) ◽  
pp. 6206-6211 ◽  
Author(s):  
Mate Maus ◽  
Amit Jairaman ◽  
Peter B. Stathopulos ◽  
Martin Muik ◽  
Marc Fahrner ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Severe Combined Immunodeficiency ◽  
Coiled Coil ◽  
Cell Types ◽  
Combined Immunodeficiency ◽  
Loss Of Function ◽  
The Third ◽  
Coiled Coil Domain ◽  
C Terminus ◽  
Domain 3

Store-operated Ca2+ entry (SOCE) is a universal Ca2+ influx pathway that is important for the function of many cell types. SOCE occurs upon depletion of endoplasmic reticulum (ER) Ca2+ stores and relies on a complex molecular interplay between the plasma membrane (PM) Ca2+ channel ORAI1 and the ER Ca2+ sensor stromal interaction molecule (STIM) 1. Patients with null mutations in ORAI1 or STIM1 genes present with severe combined immunodeficiency (SCID)-like disease. Here, we describe the molecular mechanisms by which a loss-of-function STIM1 mutation (R429C) in human patients abolishes SOCE. R429 is located in the third coiled-coil (CC3) domain of the cytoplasmic C terminus of STIM1. Mutation of R429 destabilizes the CC3 structure and alters the conformation of the STIM1 C terminus, thereby releasing a polybasic domain that promotes STIM1 recruitment to ER–PM junctions. However, the mutation also impairs cytoplasmic STIM1 oligomerization and abolishes STIM1–ORAI1 interactions. Thus, despite its constitutive localization at ER–PM junctions, mutant STIM1 fails to activate SOCE. Our results demonstrate multifunctional roles of the CC3 domain in regulating intra- and intermolecular STIM1 interactions that control (i) transition of STIM1 from a quiescent to an active conformational state, (ii) cytoplasmic STIM1 oligomerization, and (iii) STIM1–ORAI1 binding required for ORAI1 activation.

scholarly journals Cep57, a multidomain protein with unique microtubule and centrosomal localization domains

2008 ◽  
Vol 412 (2) ◽  
pp. 265-273 ◽  
Author(s):  
Ko Momotani ◽  
Alexander S. Khromov ◽  
Tsuyoshi Miyake ◽  
P. Todd Stukenberg ◽  
Avril V. Somlyo
Keyword(s):  
Ectopic Expression ◽  
Coiled Coil ◽  
Full Length ◽  
Centrosomal Protein ◽  
Multidomain Protein ◽  
Coiled Coil Domain ◽  
C Terminus ◽  
N Terminus

The present study demonstrates different functional domains of a recently described centrosomal protein, Cep57 (centrosomal protein 57). Endogenous Cep57 protein and ectopic expression of full-length protein or the N-terminal coiled-coil domain localize to the centrosome internal to γ-tubulin, suggesting that it is either on both centrioles or on a centromatrix component. The N-terminus can also multimerize with the N-terminus of other Cep57 molecules. The C-terminus contains a second coiled-coil domain that directly binds to MTs (microtubules). This domain both nucleates and bundles MTs in vitro. This activity was also seen in vivo, as overexpression of full-length Cep57 or the C-terminus generates nocodazole-resistant MT cables in cells. Based on the present findings, we propose that Cep57 serves as a link with its N-terminus anchored to the centriole or centromatrix and its C-terminus to MTs.

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