Purification and Analysis of Authentic CLIP-170 and Recombinant Fragments

Scheel, Jochen; Pierre, Philippe; Rickard, Janet E.; Diamantopoulos, Georgios S.; Valetti, Caterina; Goot, F. Gisou van der; Häner, Markus; Aebi, Ueli; Kreis, Thomas E.

doi:10.1074/jbc.274.36.25883

Cited by 71 publications

(71 citation statements)

References 48 publications

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“…Recombinant constructs of CLIP-170 (an in-frame T7 tag was fused to the N-terminus of the previously characterized H2 construct [16] generously provided by Holly Goodson, Notre Dame University) and p150 Glued (residues 1-333) [17], which span the microtubule binding domains of these proteins, as well as full length EB1 [17], were expressed as His-tagged fusion proteins in E. coli and purified by Ni 2+ -chromatography.…”

Section: Generation Of Recombinant Proteinsmentioning

confidence: 99%

“…We added full-length recombinant EB1 or a wellcharacterized recombinant fragment of CLIP-170 (H2) [16] that includes the N-terminal tandem CAP-Gly microtubulebinding domains (Fig. 1A) to microtubules polymerized in vitro.…”

Section: Plus-end Proteins Do Not Preferentially Bind To Microtubule mentioning

confidence: 99%

“…2A) based upon a 1:1 stoichiometry of tubulin dimer to CLIP-170 H2 polypeptide. Alternatively, Scheel et al [16] have suggested that there is a 2:1 binding stoichiometry of tubulin dimers to CLIP-170-H2 polypeptide due to the presence of two tandem CAP-Gly domains. Fitting our data to a simple two site model, as shown in the inset to Fig.…”

Section: Clip-170 Binds To Tubulin Dimers and Co-polymerizesmentioning

confidence: 99%

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Microtubule binding proteins CLIP‐170, EB1, and p150^Glued form distinct plus‐end complexes

et al. 2006

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Microtubule plus-end proteins CLIP-170 and EB1 dynamically track the tips of growing microtubules in vivo. Here we examine the association of these proteins with microtubules in vitro. CLIP-170 binds tubulin dimers and co-assembles into growing microtubules. EB1 binds tubulin dimers more weakly, so no co-assembly is observed. However, EB1 binds to CLIP-170, and forms a co-complex with CLIP-170 and tubulin that is recruited to growing microtubule plus ends. The interaction between CLIP-170 and EB1 is competitively inhibited by the related CAP-Gly protein p150 Glued , which also localizes to microtubule plus ends in vivo. Based on these observations, we propose a model in which the formation of distinct plus-end complexes may differentially affect microtubule dynamics in vivo.

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Section: Generation Of Recombinant Proteinsmentioning

confidence: 99%

Section: Plus-end Proteins Do Not Preferentially Bind To Microtubule mentioning

confidence: 99%

Section: Clip-170 Binds To Tubulin Dimers and Co-polymerizesmentioning

confidence: 99%

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Microtubule binding proteins CLIP‐170, EB1, and p150^Glued form distinct plus‐end complexes

et al. 2006

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“…Briefly, varying lengths of coding sequence were PCR-amplified from a pET15b vector (Novagen) already containing the CLIP-170 H1-(1-350) sequence (24,25). The resulting PCR products were subcloned into the pENTR D-TOPO vector following the manufacturer's protocol.…”

Section: Methodsmentioning

confidence: 99%

Minimal Plus-end Tracking Unit of the Cytoplasmic Linker Protein CLIP-170

Gupta

Paulson

Folker

et al. 2009

Journal of Biological Chemistry

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Cytoplasmic linker protein 170 (CLIP-170) is the prototype microtubule (MT) plus-end tracking protein (؉TIP) and is involved in regulating MT dynamics. A comprehensive understanding of the process by which CLIP-170 tracks MT plus ends would provide insight into its function. However, the precise molecular mechanism of CLIP-170 ؉TIP behavior is unknown, and many potential models have been presented. Here, by separating the two CLIP-170 CAP-Gly domains and their adjacent serine-rich regions into fragments of varied size, we have characterized the minimal plus-end tracking unit of CLIP-170 in vivo. Each CLIP-170 fragment was also characterized for its tubulin polymerization activity in vitro. We found that the two CAP-Gly domains have different activities, whereas CAP-Gly-1 appears incompetent to mediate either ؉TIP behavior or MT nucleation, a CLIP-170 fragment consisting of the second CAPGly domain and its adjacent serine-rich region can both track MT plus ends in vivo and induce tubulin polymerization in vitro. These observations complement recent work on CLIP-170 fragments, demonstrate that CAP-Gly motifs do not require dimerization for ؉TIP and polymerization-promoting activities, and provide insight into CLIP-170 function and mechanism.

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“…5c; Kobe and Deisenhofer 1995;Scheel et al 1999). The X-ray-induced allele pfi-ML375 is associated with a 10-Mb inversion that disrupts the ORF, and two other alleles have early stop-codon mutations truncating the protein at amino acid residues 14 and 40, respectively (Fig.…”

Section: The Pilz Group and Kis Genes Encode Orthologs Of Mammalian Pmentioning

confidence: 99%

The Arabidopsis PILZ group genes encode tubulin-folding cofactor orthologs required for cell division but not cell growth

Steinborn¹,

Maulbetsch²,

Priester³

et al. 2002

Genes Dev.

163

130

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Plant microtubules are organized into specific cell cycle-dependent arrays that have been implicated in diverse cellular processes, including cell division and organized cell expansion. Mutations in four Arabidopsis genes collectively called the PILZ group result in lethal embryos that consist of one or a few grossly enlarged cells. The mutant embryos lack microtubules but not actin filaments. Whereas the cytokinesis-specific syntaxin KNOLLE is not localized properly, trafficking of the putative auxin efflux carrier PIN1 to the plasma membrane is normal. The four PILZ group genes were isolated by map-based cloning and are shown to encode orthologs of mammalian tubulin-folding cofactors (TFCs) C, D, and E, and associated small G-protein Arl2 that mediate the formation of ␣/␤-tubulin heterodimers in vitro. The TFC C ortholog, PORCINO, was detected in cytosolic protein complexes and did not colocalize with microtubules. Another gene with a related, although weaker, embryo-lethal phenotype, KIESEL, was shown to encode a TFC A ortholog. Our genetic ablation of microtubules shows their requirement in cell division and vesicle trafficking during cytokinesis, whereas cell growth is mediated by microtubule-independent vesicle trafficking to the plasma membrane during interphase. The microtubule cytoskeleton plays important roles in both nondividing and dividing cells of eukaryotes, assisting in vesicle trafficking and mediating proper segregation of daughter chromosomes to opposite poles during mitosis (for review, see Cole and Lippincott-Schwartz 1995;Straight and Field 2000). In fission yeast, microtubules have also been implicated in maintaining cell shape and polarity (Sawin and Nurse 1998). Higher-plant cells form their own specific microtubule arrays, such as cortical hoops of interphase microtubules that organize cell elongation (Whittington et al. 2001), and two arrays related to the plant-specific mode of cell division (for review, see Lloyd and Hussey 2001). The preprophase band forms transiently at the onset of mitosis and marks the cortical division site (for review, see Smith 2001). The phragmoplast forms at the center of the division plane in late anaphase and guides the delivery of Golgiderived vesicles. Their fusion results in the formation and lateral expansion of the cell plate that matures into a cell wall and flanking plasma membranes (for review, see Staehelin and Hepler 1996). Both preprophase band and phragmoplast also contain actin filaments; however, their specific role in plant cell division is not understood at present (Smith 2001).Microtubules are polymerized from ␣/␤-tubulin heterodimers (for review, see Nogales 2000). Newly synthesized ␣-and ␤-tubulin polypeptides undergo a sequence of folding steps catalyzed by chaperones. Initially, the tubulins are associated with the hexameric prefoldin complex that passes them on to the cytosolic chaperonin complex (Geissler et al. 1998;Vainberg et al. 1998;Hansen et al. 1999; for review, see Leroux and Hartl 2000;Llorca et al. 2000). Whereas fully func...

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Purification and Analysis of Authentic CLIP-170 and Recombinant Fragments

Cited by 71 publications

References 48 publications

Microtubule binding proteins CLIP‐170, EB1, and p150^Glued form distinct plus‐end complexes

Microtubule binding proteins CLIP‐170, EB1, and p150^Glued form distinct plus‐end complexes

Minimal Plus-end Tracking Unit of the Cytoplasmic Linker Protein CLIP-170

The Arabidopsis PILZ group genes encode tubulin-folding cofactor orthologs required for cell division but not cell growth

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Purification and Analysis of Authentic CLIP-170 and Recombinant Fragments

Cited by 71 publications

References 48 publications

Microtubule binding proteins CLIP‐170, EB1, and p150Glued form distinct plus‐end complexes

Microtubule binding proteins CLIP‐170, EB1, and p150Glued form distinct plus‐end complexes

Minimal Plus-end Tracking Unit of the Cytoplasmic Linker Protein CLIP-170

The Arabidopsis PILZ group genes encode tubulin-folding cofactor orthologs required for cell division but not cell growth

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Microtubule binding proteins CLIP‐170, EB1, and p150^Glued form distinct plus‐end complexes

Microtubule binding proteins CLIP‐170, EB1, and p150^Glued form distinct plus‐end complexes