scholarly journals MTCL2 promotes asymmetric microtubule organization by crosslinking microtubules on the Golgi membrane

2021 ◽  
Author(s):  
Risa Matsuoka ◽  
Masateru Miki ◽  
Sonoko Mizuno ◽  
Yurina Ito ◽  
Chihiro Yamada ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Active Role ◽  
Vesicle Transport ◽  
Golgi Membrane ◽  
Microtubule Organization ◽  
Directional Migration ◽  
Microtubule Stabilization ◽  
Coiled Coil Region ◽  
Assembly Structure

The Golgi complex plays an active role in organizing asymmetric microtubule arrays essential for polarized vesicle transport. The coiled-coil protein MTCL1 stabilizes microtubules nucleated from the Golgi membrane. Here, we report an MTCL1 paralog, MTCL2, which preferentially acts on the perinuclear microtubules accumulated around the Golgi. MTCL2 associates with the Golgi membrane through the N-terminal coiled-coil region and directly binds microtubules through the conserved C-terminal domain without promoting microtubule stabilization. Knockdown of MTCL2 significantly impaired microtubule accumulation around the Golgi as well as the compactness of the Golgi ribbon assembly structure. Given that MTCL2 forms parallel oligomers through homo-interaction of the central coiled-coil motifs, our results indicate that MTCL2 promotes asymmetric microtubule organization by crosslinking microtubules on the Golgi membrane. Results of in vitro wound healing assays further suggest that this function of MTCL2 enables integration of the centrosomal and Golgi-associated microtubules on the Golgi membrane, supporting directional migration. Additionally, the results demonstrated the involvement of CLASPs and giantin in mediating the Golgi association of MTCL2.

scholarly journals MTCL2 is a new Golgi-resident microtubule-regulating protein, essential for organizing asymmetric microtubule network

2020 ◽  
Author(s):  
Risa Matsuoka ◽  
Masateru Miki ◽  
Sonoko Mizuno ◽  
Yurina Ito ◽  
Atsushi Suzuki
Keyword(s):  
Coiled Coil ◽  
Golgi Membrane ◽  
Microtubule Network ◽  
Microtubule Binding ◽  
Microtubule Stabilization ◽  
Binding Domains ◽  
Microtubule Binding Domain ◽  
Ribbon Structure ◽  
Golgi Ribbon

AbstractThe Golgi apparatus plays important roles in organizing the asymmetric microtubule network essential for polarized vesicle transport. The Golgi-associated coiled-coil protein MTCL1 is crucially involved in Golgi functioning by interconnecting and stabilizing microtubules on the Golgi membrane through its N- and C-terminal microtubule-binding domains. Here, we report the presence of a mammalian paralog of MTCL1, named MTCL2, lacking the N-terminal microtubule-binding domain. MTCL2 localizes to the Golgi membrane through the N-terminal region and directly binds microtubules through the conserved C-terminal domain without promoting microtubule stabilization. Knockdown experiments demonstrated essential roles of MTCL2 in accumulating MTs around the Golgi and regulating the Golgi ribbon structure. In vitro wound healing assays further suggested a possible intriguing activity of MTCL2 in integrating the centrosomal and Golgi-associated microtubules around the Golgi ribbon, thus supporting directional migration. Altogether, the present results demonstrate that cells utilize two members of the MTCL protein family to differentially regulate the Golgi-associated microtubules for controlling cell polarity.

scholarly journals A Coiled-Coil Domain of Melanophilin Is Essential for Myosin Va Recruitment and Melanosome Transport in Melanocytes

2006 ◽  
Vol 17 (11) ◽  
pp. 4720-4735 ◽  
Author(s):  
Alistair N. Hume ◽  
Abul K. Tarafder ◽  
José S. Ramalho ◽  
Elena V. Sviderskaya ◽  
Miguel C. Seabra
Keyword(s):  
Coiled Coil ◽  
Binding Domain ◽  
Actin Binding ◽  
Binding Studies ◽  
Null Cell ◽  
Coiled Coil Region ◽  
Actin Binding Domain ◽  
Transport Assay ◽  
Myosin Va

Melanophilin (Mlph) regulates retention of melanosomes at the peripheral actin cytoskeleton of melanocytes, a process essential for normal mammalian pigmentation. Mlph is proposed to be a modular protein binding the melanosome-associated protein Rab27a, Myosin Va (MyoVa), actin, and microtubule end-binding protein (EB1), via distinct N-terminal Rab27a-binding domain (R27BD), medial MyoVa-binding domain (MBD), and C-terminal actin-binding domain (ABD), respectively. We developed a novel melanosome transport assay using a Mlph-null cell line to study formation of the active Rab27a:Mlph:MyoVa complex. Recruitment of MyoVa to melanosomes correlated with rescue of melanosome transport and required intact R27BD together with MBD exon F–binding region (EFBD) and unexpectedly a potential coiled-coil forming sequence within ABD. In vitro binding studies indicate that the coiled-coil region enhances binding of MyoVa by Mlph MBD. Other regions of Mlph reported to interact with MyoVa globular tail, actin, or EB1 are not essential for melanosome transport rescue. The strict correlation between melanosomal MyoVa recruitment and rescue of melanosome distribution suggests that stable interaction with Mlph and MyoVa activation are nondissociable events. Our results highlight the importance of the coiled-coil region together with R27BD and EFBD regions of Mlph in the formation of the active melanosomal Rab27a-Mlph-MyoVa complex.

scholarly journals 2066 Functional characterization of mutant BRCA1

2018 ◽  
Vol 2 (S1) ◽  
pp. 13-13
Author(s):  
John Barrows ◽  
David Long
Keyword(s):  
Functional Characterization ◽  
Coiled Coil ◽  
Wild Type ◽  
Coiled Coil Region ◽  
Egg Extracts ◽  
Study Population ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

OBJECTIVES/SPECIFIC AIMS: The objective of this work is to determine the mechanistic consequences of BRCA1 mutants in inter-strand crosslink (ICL) repair. METHODS/STUDY POPULATION: Our lab uses Xenopus egg extracts to study ICL repair. These extracts can be depleted of endogenous BRCA1 by immunoprecipitation. The goal of this work is to rescue endogenous depletion with in vitro translated, wild type BRCA1. Once achieved, we can supplement the depleted extract with BRCA1 mutants to access their function in ICL repair. RESULTS/ANTICIPATED RESULTS: We hypothesize that the BRCT and RING domain mutations will abrogate ICL repair, while mutations in the coiled coil region will not affect repair. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings will have an immense impact on the understanding of BRCA1 domains. Importantly these results will spur personalized therapy of BRCA1 mutants by showing which domains are sensitive to cross-linking agents.

scholarly journals Isolation and Characterization of EMILIN-2, a New Component of the Growing EMILINs Family and a Member of the EMI Domain-containing Superfamily

2001 ◽  
Vol 276 (15) ◽  
pp. 12003-12011 ◽  
Author(s):  
Roberto Doliana ◽  
Simonetta Bot ◽  
Gabriella Mungiguerra ◽  
Anna Canton ◽  
Stefano Paron Cilli ◽  
...  
Keyword(s):  
Elastic Fiber ◽  
Coiled Coil ◽  
Bac Clone ◽  
Protein Determination ◽  
Rich Domain ◽  
Isolation And Characterization ◽  
C Terminus ◽  
Coiled Coil Region ◽  
Novel Protein

EMILIN (elastinmicrofibrilinterfaselocated Protein) is an elastic fiber-associated glycoprotein consisting of a self-interacting globular C1q domain at the C terminus, a short collagenous stalk, an extended region of potential coiled-coil structure, and an N-terminal cysteine-rich domain (EMI domain). Using the globular C1q domain as a bait in the yeast two-hybrid system, we have isolated a cDNA encoding a novel protein. Determination of the entire primary structure demonstrated that this EMILIN-binding polypeptide is highly homologous to EMILIN. The domain organization is superimposable, one important difference being a proline-rich (41%) segment of 56 residues between the potential coiled-coil region and the collagenous domain absent in EMILIN. The entire gene (localized on chromosome 18p11.3) was isolated from a BAC clone, and it is structurally almost identical to that of EMILIN (8 exons, 7 introns with identical phases at the exon/intron boundaries) but much larger (about 40versus8 kilobases) than that of EMILIN. Given these findings we propose to name the novel protein EMILIN-2 and the prototype member of this family EMILIN-1 (formerly EMILIN). The mRNA expression of EMILIN-2 is more restricted compared with that of EMILIN-1; highest levels are present in fetal heart and adult lung, whereas, differently from EMILIN-1, adult aorta, small intestine, and appendix show very low expression, and adult uterus and fetal kidney are negative. Finally, the EMILIN-2 protein is secreted extracellularly byin vitro-grown cells, and in accordance with the partial coexpression in fetal and adult tissues, the two proteins shown extensive but not absolute immunocolocalizationin vitro.

scholarly journals Negative Regulation of Yeast Eps15-like Arp2/3 Complex Activator, Pan1p, by the Hip1R-related Protein, Sla2p, during Endocytosis

2007 ◽  
Vol 18 (2) ◽  
pp. 658-668 ◽  
Author(s):  
Jiro Toshima ◽  
Junko Y. Toshima ◽  
Mara C. Duncan ◽  
M. Jamie T.V. Cope ◽  
Yidi Sun ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Affinity Purification ◽  
Coiled Coil ◽  
Actin Assembly ◽  
Related Protein ◽  
Loss Of Function ◽  
Actin Organization ◽  
Coiled Coil Region ◽  
Kinase Phosphorylation

Control of actin assembly nucleated by the Arp2/3 complex plays a crucial role during budding yeast endocytosis. The yeast Eps15-related Arp2/3 complex activator, Pan1p, is essential for endocytic internalization and proper actin organization. Pan1p activity is negatively regulated by Prk1 kinase phosphorylation after endocytic internalization. Phosphorylated Pan1p is probably then dephosphorylated in the cytosol. Pan1p is recruited to endocytic sites ∼25 s before initiation of actin polymerization, suggesting that its Arp2/3 complex activation activity is kept inactive during early stages of endocytosis by a yet-to-be-identified mechanism. However, how Pan1p is maintained in an inactive state is not clear. Using tandem affinity purification–tagged Pan1p, we identified End3p as a stoichiometric component of the Pan1p complex, and Sla2p, a yeast Hip1R-related protein, as a novel binding partner of Pan1p. Interestingly, Sla2p specifically inhibited Pan1p Arp2/3 complex activation activity in vitro. The coiled-coil region of Sla2p was important for Pan1p inhibition, and a pan1 partial loss-of-function mutant suppressed the temperature sensitivity, endocytic phenotypes, and actin phenotypes observed in sla2ΔCC mutant cells that lack the coiled-coil region. Overall, our results establish that Sla2p's regulation of Pan1p plays an important role in controlling Pan1p-stimulated actin polymerization during endocytosis.

scholarly journals MICAL-L1 Links EHD1 to Tubular Recycling Endosomes and Regulates Receptor Recycling

2009 ◽  
Vol 20 (24) ◽  
pp. 5181-5194 ◽  
Author(s):  
Mahak Sharma ◽  
Sai Srinivas Panapakkam Giridharan ◽  
Juliati Rahajeng ◽  
Naava Naslavsky ◽  
Steve Caplan
Keyword(s):  
Coiled Coil ◽  
Live Cells ◽  
Endocytic Recycling ◽  
Interacting Protein ◽  
Recycling Endosomes ◽  
Coiled Coil Region ◽  
Membrane Tubules ◽  
Tubular Membranes

Endocytic recycling of receptors and lipids occurs via a complex network of tubular and vesicular membranes. EHD1 is a key regulator of endocytosis and associates with tubular membranes to facilitate recycling. Although EHD proteins tubulate membranes in vitro, EHD1 primarily associates with preexisting tubules in vivo. How EHD1 is recruited to these tubular endosomes remains unclear. We have determined that the Rab8-interacting protein, MICAL-L1, associates with EHD1, with both proteins colocalizing to long tubular membranes, in vitro and in live cells. MICAL-L1 is a largely uncharacterized member of the MICAL-family of proteins that uniquely contains two asparagine-proline-phenylalanine motifs, sequences that typically interact with EH-domains. Our data show that the MICAL-L1 C-terminal coiled-coil region is necessary and sufficient for its localization to tubular membranes. Moreover, we provide unexpected evidence that endogenous MICAL-L1 can link both EHD1 and Rab8a to these structures, as its depletion leads to loss of the EHD1-Rab8a interaction and the absence of both of these proteins from the membrane tubules. Finally, we demonstrate that MICAL-L1 is essential for efficient endocytic recycling. These data implicate MICAL-L1 as an unusual type of Rab effector that regulates endocytic recycling by recruiting and linking EHD1 and Rab8a on membrane tubules.

scholarly journals Dual function of the NDR-kinase Dbf2 in the regulation of the F-BAR protein Hof1 during cytokinesis

2013 ◽  
Vol 24 (9) ◽  
pp. 1290-1304 ◽  
Author(s):  
Franz Meitinger ◽  
Saravanan Palani ◽  
Birgit Hub ◽  
Gislene Pereira
Keyword(s):  
Critical Role ◽  
Coiled Coil ◽  
Dual Function ◽  
Serine Residue ◽  
Bar Domain ◽  
Division Site ◽  
Coiled Coil Region ◽  
Ndr Kinase

The conserved NDR-kinase Dbf2 plays a critical role in cytokinesis in budding yeast. Among its cytokinesis-related substrates is the F-BAR protein Hof1. Hof1 colocalizes at the cell division site with the septin complex and, as mitotic exit progresses, moves to the actomyosin ring (AMR). Neither the function of Hof1 at the septin complex nor the mechanism by which Hof1 supports AMR constriction is understood. Here we establish that Dbf2 has a dual function in Hof1 regulation. First, we show that the coiled-coil region, which is adjacent to the conserved F-BAR domain, is required for the binding of Hof1 to septins. The Dbf2-dependent phosphorylation of Hof1 at a single serine residue (serine 313) in this region diminishes the recruitment of Hof1 to septins both in vitro and in vivo. Genetic and functional analysis indicates that the binding of Hof1 to septins is important for septin rearrangement and integrity during cytokinesis. Furthermore, Dbf2 phosphorylation of Hof1 at serines 533 and 563 promotes AMR constriction most likely by inhibiting the SH3-domain–dependent interactions of Hof1. Thus our data show that Dbf2 coordinates septin and AMR functions during cytokinesis through the regulation/control of Hof1.

scholarly journals Functional Domains Present in the Mycobacterial Hemagglutinin, HBHA

1999 ◽  
Vol 181 (24) ◽  
pp. 7464-7469 ◽  
Author(s):  
Giovanni Delogu ◽  
Michael J. Brennan
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Coiled Coil ◽  
Surface Protein ◽  
Functional Domains ◽  
Heparin Binding ◽  
Coiled Coil Region ◽  
Host Tissues

ABSTRACT Identification and characterization of mycobacterial adhesins and complementary host receptors required for colonization and dissemination of mycobacteria in host tissues are needed for a more complete understanding of the pathogenesis of diseases caused by these bacteria and for the development of effective vaccines. Previous investigations have demonstrated that a 28-kDa heparin-binding mycobacterial surface protein, HBHA, can agglutinate erythrocytes and promote mycobacterial aggregation in vitro. In this study, further molecular and biochemical analysis of HBHA demonstrates that it has three functional domains: a transmembrane domain of 18 amino acids residing near the N terminus, a large domain of 81 amino acids consistent with an α-helical coiled-coil region, and a Lys-Pro-Ala-rich C-terminal domain that mediates binding to proteoglycans. Using His-tagged recombinant HBHA proteins and nickel chromatography we demonstrate that HBHA polypeptides which contain the coiled-coil region form multimers. This tendency to oligomerize may be responsible for the induction of mycobacterial aggregation since a truncated N-terminal HBHA fragment containing the coiled-coil domain promotes mycobacterial aggregation. Conversely, a truncated C-terminal HBHA fragment which contains Lys-Pro-Ala-rich repeats binds to the proteoglycan decorin. These results indicate that HBHA contains at least three distinct domains which facilitate intercalation into surface membranes, promote bacterium-bacterium interactions, and mediate the attachment to sulfated glycoconjugates found in host tissues.

scholarly journals Phasins, Multifaceted Polyhydroxyalkanoate Granule-Associated Proteins

2016 ◽  
Vol 82 (17) ◽  
pp. 5060-5067 ◽  
Author(s):  
Mariela P. Mezzina ◽  
M. Julia Pettinari
Keyword(s):  
Binding Capacity ◽  
Coiled Coil ◽  
Active Role ◽  
Structural Features ◽  
Structural Function ◽  
Associated Proteins ◽  
Α Helix ◽  
Pha Granule

ABSTRACTPhasins are the major polyhydroxyalkanoate (PHA) granule-associated proteins. They promote bacterial growth and PHA synthesis and affect the number, size, and distribution of the granules. These proteins can be classified in 4 families with distinctive characteristics. Low-resolution structural studies andin silicopredictions were performed in order to elucidate the structure of different phasins. Most of these proteins share some common structural features, such as a preponderant α-helix composition, the presence of disordered regions that provide flexibility to the protein, and coiled-coil interacting regions that form oligomerization domains. Due to their amphiphilic nature, these proteins play an important structural function, forming an interphase between the hydrophobic content of PHA granules and the hydrophilic cytoplasm content. Phasins have been observed to affect both PHA accumulation and utilization. Apart from their role as granule structural proteins, phasins have a remarkable variety of additional functions. Different phasins have been determined to (i) activate PHA depolymerization, (ii) increase the expression and activity of PHA synthases, (iii) participate in PHA granule segregation, and (iv) have bothin vivoandin vitrochaperone activities. These properties suggest that phasins might play an active role in PHA-related stress protection and fitness enhancement. Due to their granule binding capacity and structural flexibility, several biotechnological applications have been developed using different phasins, increasing the interest in the study of these remarkable proteins.

scholarly journals Bet1p activates the v-SNARE Bos1p.

1997 ◽  
Vol 8 (7) ◽  
pp. 1175-1181 ◽  
Author(s):  
S Stone ◽  
M Sacher ◽  
Y Mao ◽  
C Carr ◽  
P Lyons ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Coiled Coil ◽  
Direct Interaction ◽  
Binding Studies ◽  
Yeast Saccharomyces Cerevisiae ◽  
In Vitro Binding ◽  
Protein Receptor ◽  
Coiled Coil Region ◽  
Vitro Binding

Bet1p is a type II membrane protein that is required for vesicular transport between the endoplasmic reticulum and Golgi complex in the yeast Saccharomyces cerevisiae. A domain of Bet1p, that shows potential to be involved in a coiled-coil interaction, is homologous to a region of the neuronal protein SNAP-25. Here, we used in vitro binding studies to demonstrate that Bet1p plays a role in potentiating soluble NSF attachment protein receptor (SNARE) interactions. Mutational analysis points to the coiled-coil region as necessary for Bet1p function, and circular dichroism experiments support this theory. In vitro binding studies were also used to demonstrate that a direct interaction between Bet1p and Bos1p is required for the efficient interaction of the vesicle SNARE with its SNARE target. Genetic studies suggest that the interactions of Bet1p with Bos1p are regulated by the small GTP-binding protein Ypt1p.

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