scholarly journals GMAP-210, A Cis-Golgi Network-associated Protein, Is a Minus End Microtubule-binding Protein

1999 ◽  
Vol 145 (1) ◽  
pp. 83-98 ◽  
Author(s):  
Carlos Infante ◽  
Francisco Ramos-Morales ◽  
Concepción Fedriani ◽  
Michel Bornens ◽  
Rosa M. Rios
Keyword(s):  
Golgi Apparatus ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Coiled Coil ◽  
Microtubule Network ◽  
Microtubule Binding ◽  
Terminal Domain ◽  
Green Fluorescent ◽  
Golgi Network

We report that a peripheral Golgi protein with a molecular mass of 210 kD localized at the cis-Golgi network (Rios, R.M., A.M. Tassin, C. Celati, C. Antony, M.C. Boissier, J.C. Homberg, and M. Bornens. 1994. J. Cell Biol. 125:997–1013) is a microtubule-binding protein that associates in situ with a subpopulation of stable microtubules. Interaction of this protein, now called GMAP-210, for Golgi microtubule-associated protein 210, with microtubules in vitro is direct, tight and nucleotide-independent. Biochemical analysis further suggests that GMAP-210 specifically binds to microtubule ends. The full-length cDNA encoding GMAP-210 predicts a protein of 1,979 amino acids with a very long central coiled-coil domain. Deletion analyses in vitro show that the COOH terminus of GMAP-210 binds to microtubules whereas the NH2 terminus binds to Golgi membranes. Overexpression of GMAP-210–encoding cDNA induced a dramatic enlargement of the Golgi apparatus and perturbations in the microtubule network. These effects did not occur when a mutant lacking the COOH-terminal domain was expressed. When transfected in fusion with the green fluorescent protein, the NH2-terminal domain associated with the cis-Golgi network whereas the COOH-terminal microtubule-binding domain localized at the centrosome. Altogether these data support the view that GMAP-210 serves to link the cis-Golgi network to the minus ends of centrosome-nucleated microtubules. In addition, this interaction appears essential for ensuring the proper morphology and size of the Golgi apparatus.

Two splice variants of Golgi-microtubule-associated protein of 210 kDa (GMAP-210) differ in their binding to the cis-Golgi network

2001 ◽  
Vol 357 (3) ◽  
pp. 699-708 ◽  
Author(s):  
Francisco RAMOS-MORALES ◽  
Carmen VIME ◽  
Michel BORNENS ◽  
Concepción FEDRIANI ◽  
Rosa M. RIOS
Keyword(s):  
Golgi Apparatus ◽  
Structural Integrity ◽  
Fluorescent Protein ◽  
Genomic Sequence ◽  
Splice Variants ◽  
Microtubule Network ◽  
Terminal Domain ◽  
Golgi Network

GMAP-210 (Golgi-microtubule-associated protein of 210kDa) is a peripheral Golgi protein that interacts with the minus end of microtubules through its C-terminus and with cis-Golgi network membranes through its N-terminus; it participates in the maintenance of the structural integrity of the Golgi apparatus [Infante, Ramos-Morales, Fedriani, Bornens and Rios (1999) J. Cell Biol. 145, 83–98]. We report here the cloning of a new isoform of GMAP-210 that lacks amino acid residues 105–196. On the basis of the analysis of the gmap-210 genomic sequence, we propose that the small isoform, GMAP-200, arises from alternative splicing of exon 4 of the primary transcript. Overexpression of GMAP-200 induces perturbations in both the Golgi apparatus and the microtubule network that are similar to those previously reported for GMAP-210 overexpression. We show that both isoforms are able to oligomerize under overexpression conditions. Analysis in vitro and in vivo, with the green fluorescent protein as a marker, reveals that the binding of the N-terminal domain of GMAP-200 to the cis-Golgi network membranes is lower than that of the N-terminal domain of GMAP-210. Implications for the regulation of interaction between the cis-Golgi network and microtubules are discussed.

scholarly journals Systematic survey of deubiquitinase localization identifies USP21 as a regulator of centrosome- and microtubule-associated functions

2012 ◽  
Vol 23 (6) ◽  
pp. 1095-1103 ◽  
Author(s):  
Sylvie Urbé ◽  
Han Liu ◽  
Sebastian D. Hayes ◽  
Claire Heride ◽  
Daniel J. Rigden ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
A549 Cells ◽  
Binding Motif ◽  
Vitro Assay ◽  
Microtubule Binding ◽  
Functional Studies ◽  
Physiological Processes ◽  
Associated Functions ◽  
Green Fluorescent

Ubiquitination is a reversible modification that influences a broad range of physiological processes. There are approximately 90 deubiquitinases (DUBs) encoded in the human genome, of which 79 are predicted to have catalytic activity. We tagged 66 DUBs with green fluorescent protein and systematically surveyed their subcellular distribution, identifying enzymes specific to the nucleus, plasma membrane, and secretory and endocytic pathways. USP21 is unique in showing clear association with both centrosomes and microtubules. Using an in vitro assay, we show that microtubule binding is direct and identify a novel microtubule-binding motif encompassed within amino acids 59–75 of the N-terminus of USP21. Our functional studies indicate a key role for USP21 in the governance of microtubule- and centrosome-associated physiological processes: Depletion of USP21 in A549 cells compromises the reestablishment of a radial array of microtubules during recovery from cold-induced depolymerization and also reduces the probability of primary cilium formation, whereas USP21 knockdown in PC12 cells inhibits nerve growth factor–induced neurite outgrowth.

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 Luv1p/Rki1p/Tcs3p/Vps54p, a Yeast Protein That Localizes to the Late Golgi and Early Endosome, Is Required for Normal Vacuolar Morphology.

2000 ◽  
Vol 11 (7) ◽  
pp. 2429-2443 ◽  
Author(s):  
Michael J. Conboy ◽  
Martha S. Cyert
Keyword(s):  
Fluorescent Protein ◽  
Protein A ◽  
Coiled Coil ◽  
Specific Inhibitor ◽  
Early Endosome ◽  
Carboxypeptidase Y ◽  
Similar Drug ◽  
Green Fluorescent ◽  
Vacuolar Protein ◽  
Golgi Network

We have characterized LUV1/RKI1/TCS3/VPS54, a novel yeast gene required to maintain normal vacuolar morphology. Theluv1 mutant was identified in a genetic screen for mutants requiring the phosphatase calcineurin for vegetative growth.luv1 mutants lack a morphologically intact vacuole and instead accumulate small vesicles that are acidified and contain the vacuolar proteins alkaline phosphatase and carboxypeptidase Y and the vacuolar membrane H+-ATPase. Endocytosis appears qualitatively normal in luv1 mutants, but some portion (28%) of carboxypeptidase Y is secreted. luv1 mutants are sensitive to several ions (Zn2+, Mn2+, and Cd2+) and to pH extremes. These mutants are also sensitive to hygromycin B, caffeine, and FK506, a specific inhibitor of calcineurin. Some vacuolar protein-sorting mutants display similar drug and ion sensitivities, including sensitivity to FK506. Luv1p sediments at 100,000 × g and can be solubilized by salt or carbonate, indicating that it is a peripheral membrane protein. A Green Fluorescent Protein–Luv1 fusion protein colocalizes with the dye FM 4-64 at the endosome, and hemagglutinin-tagged Luv1p colocalizes with the trans-Golgi network/endosomal protease Kex2p. Computer analysis predicts a short coiled-coil domain in Luv1p. We propose that this protein maintains traffic through or the integrity of the early endosome and that this function is required for proper vacuolar morphology.

scholarly journals BIM1Encodes a Microtubule-binding Protein in Yeast

1997 ◽  
Vol 8 (12) ◽  
pp. 2677-2691 ◽  
Author(s):  
Katja Schwartz ◽  
Kristy Richards ◽  
David Botstein
Keyword(s):  
Fluorescent Protein ◽  
Synthetic Lethality ◽  
Binding Protein ◽  
Human Colon ◽  
Wild Type ◽  
Human Colon Cancer ◽  
Microtubule Binding ◽  
Green Fluorescent ◽  
Two Hybrid

A previously uncharacterized yeast gene (YER016w) that we have named BIM1 (binding to microtubules) was obtained from a two-hybrid screen of a yeast cDNA library using as bait the entire coding sequence of TUB1 (encoding α-tubulin). Deletion of BIM1 results in a strong bilateral karyogamy defect, hypersensitivity to benomyl, and aberrant spindle behavior, all phenotypes associated with mutations affecting microtubules in yeast, and inviability at extreme temperatures (i.e., ≥37°C or ≤14°C). Overexpression of BIM1 in wild-type cells is lethal. A fusion of Bim1p with green fluorescent protein that complements the bim1Δ phenotypes allows visualization in vivo of both intranuclear spindles and extranuclear microtubules in otherwise wild-type cells. A bim1deletion displays synthetic lethality with deletion alleles ofbik1, num1, and bub3 as well as a limited subset of tub1 conditional-lethal alleles. A systematic study of 51 tub1 alleles suggests a correlation between specific failure to interact with Bim1p in the two-hybrid assay and synthetic lethality with thebim1Δ allele. The sequence of BIM1shows substantial similarity to sequences from organisms across the evolutionary spectrum. One of the human homologues, EB1, has been reported previously as binding APC, itself a microtubule-binding protein and the product of a gene implicated in the etiology of human colon cancer.

The non-catalytic domain of the Xenopus laevis auroraA kinase localises the protein to the centrosome

2001 ◽  
Vol 114 (11) ◽  
pp. 2095-2104
Author(s):  
Régis Giet ◽  
Claude Prigent
Keyword(s):  
Xenopus Laevis ◽  
Fluorescent Protein ◽  
Catalytic Domain ◽  
Dependent Manner ◽  
Bacterial Cells ◽  
Terminal Domain ◽  
Egg Extracts ◽  
Green Fluorescent ◽  
Xenopus Egg Extracts

Aurora kinases are involved in mitotic events that control chromosome segregation. All members of this kinase subfamily possess two distinct domains, a highly conserved catalytic domain and an N-terminal non-catalytic extension that varies in size and sequence. To investigate the role of this variable non-catalytic region we overexpressed and purified Xenopus laevis auroraA (pEg2) histidine-tagged N-terminal peptide from bacterial cells. The peptide has no effect on the in vitro auroraA kinase activity, but it inhibits both bipolar spindle assembly and stability in Xenopus egg extracts. Unlike the full-length protein, the N-terminal domain shows only low affinity for paclitaxel-stabilised microtubules in vitro, but localises to the centrosomes in a microtubule-dependent manner. When expressed in Xenopus XL2 cells, it is able to target the green fluorescent protein to centrosomes. Surprisingly, this is also true of the pEg2 catalytic domain, although to a lesser extent. The centrosome localisation of the N-terminal peptide was disrupted by nocodazole whereas localisation of the catalytic domain was not, suggesting that in order to efficiently localise to the centrosome, pEg2 kinase required the non-catalytic N-terminal domain and the presence of microtubules.

Functional analysis of CLIP-115 and its binding to microtubules

2000 ◽  
Vol 113 (12) ◽  
pp. 2285-2297 ◽  
Author(s):  
C.C. Hoogenraad ◽  
A. Akhmanova ◽  
F. Grosveld ◽  
C.I. De Zeeuw ◽  
N. Galjart
Keyword(s):  
Functional Analysis ◽  
Coiled Coil ◽  
Single Domain ◽  
Disulfide Bridges ◽  
Microtubule Network ◽  
Microtubule Binding ◽  
Neuronal Dendrites ◽  
Cytoskeletal Network ◽  
Coil Structure

Cytoplasmic linker proteins (CLIPs) bind to microtubules and are proposed to link this cytoskeletal network to other intracellular structures. We are interested in CLIP-115, since this protein is enriched in neuronal dendrites and may operate in the control of brain-specific organelle translocations. Each CLIP monomer is characterized by two microtubule-binding (MTB) motifs, surrounded by basic, serine-rich regions. This head domain is connected to the C-terminal tail through a long coiled-coil structure. The MTB domains are conserved as a single domain in other proteins involved in microtubule based transport and dynamics, such as p150(Glued). Here we provide evidence that efficient binding of CLIP-115 to microtubules is sensitive to phosphorylation and is not mediated by the conserved MTB domains alone, but requires the presence of the basic, serine rich regions in addition to the MTB motifs. In transfected COS-1 cells, CLIP-115 initially accumulates at the distal ends of microtubules and coincides with CLIP-170, indicating that both proteins mark growing microtubule ends. However, when expressed at higher levels, CLIP-115 and -170 affect the microtubule network differently. This might be partly due to the divergent C-termini of the two proteins. We demonstrate that, similar to CLIP-170, CLIP-115 forms homodimers, which, at least in vitro, are linked by disulfide bridges. Cysteine(391) of CLIP-115, however, is specific in that it controls the microtubule bundling capacity of certain mutant CLIP-115 molecules. Therefore, both similar and specific mechanisms appear to regulate the conformation of CLIPs as well as their binding to microtubules.

scholarly journals Btn2, a Hook1 Ortholog and Potential Batten Disease-Related Protein, Mediates Late Endosome-Golgi Protein SortinginYeast

2007 ◽  
Vol 27 (2) ◽  
pp. 605-621 ◽  
Author(s):  
Rachel Kama ◽  
Micah Robinson ◽  
Jeffrey E. Gerst
Keyword(s):  
Golgi Apparatus ◽  
Protein Trafficking ◽  
Fluorescent Protein ◽  
Batten Disease ◽  
Specific Protein ◽  
Late Endosome ◽  
Snare Complex ◽  
Golgi Protein ◽  
Green Fluorescent

ABSTRACT BTN2 gene expression in the yeast Saccharomyces cerevisiae is up-regulated in response to the deletion of BTN1, which encodes the ortholog of a human Batten disease protein. We isolated Btn2 as a Snc1 v-SNARE binding protein using the two-hybrid assay and examined its role in intracellular protein trafficking. We show that Btn2 is an ortholog of theDrosophila and mammalian Hook1 proteins that interact with SNAREs, cargo proteins, and coat components involved in endosome-Golgi protein sorting. By immunoprecipitation, it was found that Btn2 bound the yeast endocytic SNARE complex (e.g., Snc1 and Snc2 [Snc1/2], Tlg1, Tlg2, and Vti1), the Snx4 sorting nexin, and retromer (e.g., Vps26 and Vps35). In in vitro binding assays, recombinant His6-tagged Btn2 bound glutathione S-transferase (GST)-Snc1 and GST-Vps26. Btn2-green fluorescent protein and Btn2-red fluorescent protein colocalize with Tlg2, Snx4, and Vps27 to a compartment adjacent to the vacuole that corresponds to a late endosome. The deletion of BTN2 blocks Yif1 retrieval back to the Golgi apparatus, while the localization of Ste2, Fur4, Snc1, Vps10, carboxypeptidases Y (CPY) and S (CPS), Sed5, and Sec7 is unaltered in btn2Δ cells. Yif1 delivery to the vacuole was observed in other late endosome-Golgi trafficking mutants, including ypt6Δ, snx4Δ, and vps26Δ cells. Thus, Btn2 facilitates specific protein retrieval from a late endosome to the Golgi apparatus, a process which may be adversely affected in patients with Batten disease.

scholarly journals Rer1p, a Retrieval Receptor for Endoplasmic Reticulum Membrane Proteins, Is Dynamically Localized to the Golgi Apparatus by Coatomer

2001 ◽  
Vol 152 (5) ◽  
pp. 935-944 ◽  
Author(s):  
Ken Sato ◽  
Miyuki Sato ◽  
Akihiko Nakano
Keyword(s):  
Endoplasmic Reticulum ◽  
Green Fluorescent Protein ◽  
Membrane Proteins ◽  
Golgi Apparatus ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Endoplasmic Reticulum Membrane ◽  
Golgi Membrane ◽  
Green Fluorescent

Rer1p, a yeast Golgi membrane protein, is required for the retrieval of a set of endoplasmic reticulum (ER) membrane proteins. We present the first evidence that Rer1p directly interacts with the transmembrane domain (TMD) of Sec12p which contains a retrieval signal. A green fluorescent protein (GFP) fusion of Rer1p rapidly cycles between the Golgi and the ER. Either a lesion of coatomer or deletion of the COOH-terminal tail of Rer1p causes its mislocalization to the vacuole. The COOH-terminal Rer1p tail interacts in vitro with a coatomer complex containing α and γ subunits. These findings not only give the proof that Rer1p is a novel type of retrieval receptor recognizing the TMD in the Golgi but also indicate that coatomer actively regulates the function and localization of Rer1p.

scholarly journals A New Focal Adhesion Protein That Interacts with Integrin-Linked Kinase and Regulates Cell Adhesion and Spreading

2001 ◽  
Vol 153 (3) ◽  
pp. 585-598 ◽  
Author(s):  
Yizeng Tu ◽  
Yao Huang ◽  
Yongjun Zhang ◽  
Yun Hua ◽  
Chuanyue Wu
Keyword(s):  
Cell Adhesion ◽  
Focal Adhesion ◽  
Fluorescent Protein ◽  
Cytoskeleton Organization ◽  
Terminal Domain ◽  
Integrin Linked Kinase ◽  
Ch2 Domain ◽  
Green Fluorescent

Integrin-linked kinase (ILK) is a multidomain focal adhesion (FA) protein that functions as an important regulator of integrin-mediated processes. We report here the identification and characterization of a new calponin homology (CH) domain-containing ILK-binding protein (CH-ILKBP). CH-ILKBP is widely expressed and highly conserved among different organisms from nematodes to human. CH-ILKBP interacts with ILK in vitro and in vivo, and the ILK COOH-terminal domain and the CH-ILKBP CH2 domain mediate the interaction. CH-ILKBP, ILK, and PINCH, a FA protein that binds the NH2-terminal domain of ILK, form a complex in cells. Using multiple approaches (epitope-tagged CH-ILKBP, monoclonal anti–CH-ILKBP antibodies, and green fluorescent protein–CH-ILKBP), we demonstrate that CH-ILKBP localizes to FAs and associates with the cytoskeleton. Deletion of the ILK-binding CH2 domain abolished the ability of CH-ILKBP to localize to FAs. Furthermore, the CH2 domain alone is sufficient for FA targeting, and a point mutation that inhibits the ILK-binding impaired the FA localization of CH-ILKBP. Thus, the CH2 domain, through its interaction with ILK, mediates the FA localization of CH-ILKBP. Finally, we show that overexpression of the ILK-binding CH2 fragment or the ILK-binding defective point mutant inhibited cell adhesion and spreading. These findings reveal a novel CH-ILKBP–ILK–PINCH complex and provide important evidence for a crucial role of this complex in the regulation of cell adhesion and cytoskeleton organization.

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