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Mapmodulin: a Possible Modulator of the Interaction of Microtubule-Associated Proteins with Microtubules

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Mapmodulin: a Possible Modulator of the Interaction of Microtubule-Associated Proteins with Microtubules Proc. Natl. Acad. Sci. USA Vol. 94, pp. 5084–5089, May 1997 Cell Biology Mapmodulin: A possible modulator of the interaction of microtubule-associated proteins with microtubules NIRIT ULITZUR,MARTINE HUMBERT, AND SUZANNE R. PFEFFER* Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305-5307 Communicated by James A. Spudich, Stanford University, Stanford, CA, March 14, 1997 (received for review January 27, 1997) ABSTRACT We have purified and characterized a 31-kDa with motors for the same or adjacent binding sites on the protein named mapmodulin that binds to the microtubule- microtubules. Lo´pez and Sheetz (13) found that MAP2 inhib- associated proteins (MAPs) MAP2, MAP4, and tau. Map- ited both microtubule gliding and in vitro membrane-bound modulin binds free MAPs in strong preference to microtu- organelle motility. Specifically, MAP2-decorated microtu- bule-associated MAPs, and appears to do so via the MAP’s bules could not support the transport of vesicles in vitro in the tubulin-binding domain. Mapmodulin inhibits the initial rate presence of low concentrations of motor proteins, nor could of MAP2 binding to microtubules, a property that may allow they support formation of membranous networks (13). The mapmodulin to displace MAPs from the path of organelles large MAP mass was suggested to sterically inhibit motor translocating along microtubules. In support of this possibil- binding and movement along microtubules. While the results ity, mapmodulin stimulates the microtubule- and dynein- have not always been identical, it does seem that microtubules dependent localization of Golgi complexes in semi-intact CHO bind coverslip-attached dynein to a greater extent in the cells. To our knowledge, mapmodulin represents the first absence of MAP2. Similar results have been seen for kinesin example of a protein that can bind and potentially regulate at low (12, 13) but not high (14) motor concentrations. multiple MAP proteins. Factors are likely to exist that modulate MAP–microtubule interactions, thereby permitting motor-mediated transport. Microtubules play an important role in cytoplasmic organiza- Lo´pez and Sheetz (15) have suggested that MAP2 phosphor- tion, determination of cell polarity, cellular locomotion, and ylation could be used to modulate microtubule association. We chromosome segregation. The ability of microtubules to me- have taken the view that cells probably contain previously diate these processes is aided by their dynamic instability (1). undiscovered accessory proteins that regulate MAP– Microtubules undergo continual cycles of shrinkage and microtubule association directly. Indeed, we report here the growth and may be stabilized at specific locations within the purification and characterization of a protein that interacts cell andyor at different points in the cell cycle. Once stabilized, strongly with the microtubule-binding domains of MAP2, they act as tracks along which the molecular motors dynein and MAP4, and tau. We have named this protein mapmodulin kinesin move, carrying vesicular cargoes from one part of the because we show that it slows the rate of MAP binding to cell to another. microtubules. Mapmodulin stimulates an in vitro assay (16) Microtubule function is regulated by microtubule-associated that reconstitutes the overall process of dynein-mediated, proteins (MAPs). MAPs include the molecular motors, kine- microtubule-based Golgi localization in broken cells. We sin and dynein, as well as a group of proteins that can promote propose a speculative model in which mapmodulin clears microtubule assembly and contribute to polymer stability in microtubule tracks like a train’s cowcatcher to permit micro- vitro, and in some cases in vivo (2–4). This latter group of tubule-based motor-driven organelle motility. assembly-promoting proteins includes MAP2 and tau that are both specific to neuronal cells. In contrast, MAP4 is the major MAP in nonneuronal mammalian cells (5). MAP2, MAP4, MATERIALS AND METHODS and tau share a homologous, C-terminal microtubule-binding Mapmodulin Purification. CHO cytosol was prepared as domain that is comprised of three to five imperfect repeats of described by Balch et al. (17). Cytosol was desalted into cytosol an 18-amino acid motif (6, 7). An adjacent, proline-rich region buffer (25 mM Hepes, pH 7.5y50 mM KCly2 mM MgCl2y0.5 also contributes to microtubule binding. mM EDTAy1 mM DTT) using Bio-Gel P6DG (Bio-Rad) and Microtubule surfaces are essentially coated with MAPs in was immediately frozen in liquid nitrogen. Most preparations vivo, and the larger MAPs project a significant distance ('90 had protein concentrations of 4–6 mgyml, as determined using nm) away from the microtubule surface (8). It has been Bio-Rad reagent and BSA as standard. estimated that MAP4 alone occurs once per '40–50 tubulin Desalted CHO cytosol was loaded onto a 20 ml Fast Flow dimers, or '50 per mm of microtubule length (9, 10). Thus, Q (Pharmacia). The column was washed with 10 volumes, 0.35 motors moving organelles along microtubule tracks will en- M KCl (in 25 mM Tris, pH 7.6y1 mM DTT) and then eluted counter numerous bound MAPs as they move an organelle the with a 200 ml gradient of 0.35 M to 0.7 M KCl. The active equivalent of a single organelle diameter, because such or- fractions (corresponding to 0.5 M KCl) were collected, con- ganelles are usually larger than 0.1 mm. centrated ona1mlFast Flow Q column, and loaded onto a 120 Indications of MAP-mediated track blockage have come ml Superdex-200 fast protein liquid chromatography (FPLC) from in vitro motility assays. Paschal et al. (11) and Hagiwara column in cytosol buffer. Gel filtration and chemical crosslink- et al. (12) have shown that MAP2 and tau inhibit kinesin and ing revealed that mapmodulin is a dimer or trimer. Cytosol dynein-mediated microtubule gliding, possibly by competing levels of the protein assumed a dimeric mass and cytosol concentration of 50 mgyml in cells. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: MAP, microtubule-associated protein; PHAPI, puta- tive histocompatibility antigen-associated protein I; GlcNAc, N- Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA acetylglucosamine; GST, glutathione S-transferase. 0027-8424y97y945084-6$2.00y0 *To whom reprint requests should be addressed. e-mail: pfeffer@ PNAS is available online at http:yywww.pnas.org. cmgm.stanford.edu. 5084 Downloaded by guest on September 25, 2021 Cell Biology: Ulitzur et al. Proc. Natl. Acad. Sci. USA 94 (1997) 5085 Bacterial Expression. Mapmodulin is identical to a previ- type Golgi complexes at 378C. The ‘‘capture’’ of wild-type ously identified protein named PHAPI (putative histocom- Golgi complexes by mutant CHO cells is then measured after patibility antigen-associated protein I; see text and ref. 18). differential centrifugation: semi-intact mutant cells sediment The full-length cDNA of PHAPI (18), cloned into pBlueScript to the bottom of a sucrose cushion and carry with them bound, (KpnI–SalI) and fused to the enterokinase recognition site at wild-type Golgi complexes. The presence of cell-associated its N terminus, was subcloned into the pQE30 expression Golgi complexes is detected by subjecting cell pellets to a vector (Qiagen, Chatsworth, CA; KpnI–SalI). The His6-tagged GlcNAc transferase I enzyme assay. We have shown previously protein was purified by standard methods. C-terminally trun- that this process leads to the correct localization of Golgi cated His-tagged mapmodulinyPHAPI was constructed by complexes to the microtubule organizing center (MTOC) of blunt-end ligation of the BsaAI–PstI fragment from pBlue- recipient cells, and requires ATP hydrolysis, cytosolic and Script-PHAPI into pQE30, deleting residues 164–249 which peripheral membrane proteins, intact microtubules, and cyto- comprise the acidic tail. plasmic dynein (16). Antibodies. Polyclonal antibodies against CHO mapmodulin Treatment of Golgi complexes with 0.5–1 M KCl to remove and recombinant mapmodulinyPHAPI were affinity purified peripherally associated proteins destroys their ability to be using pure recombinant mapmodulinyPHAPI crosslinked to captured by semi-intact cells. However, if the released proteins AffiGel-15 (Bio-Rad). Monoclonal anti-MAP2 was from are dialyzed into a physiological buffer and added back to the Sigma; Texas red conjugated secondary antibodies were from salt-washed Golgi complexes, Golgi capture activity is re- The Jackson Laboratories. stored. CHO cytosol also restores activity to salt-treated Golgi Microtubule Protein Purification. Tubulin was purified complexes. These observations provided an assay for the from bovine brain using two cycles of polymerization and purification of proteins needed for Golgi complex localization. depolymerization followed by a phosphocellulose column (19). To succeed in the purification of a single protein that MAP2 and tau were purified from bovine brain by stripping a functions in a process requiring multiple proteins, reactions twice-cycled microtubule pellet with 0.75 M NaCl and boiling must contain limiting amounts of these other components. for 5 min after adding 2% 2-mercaptoethanol. The samples When cells are broken for the Golgi capture assay, endogenous were chilled on ice and then precipitates were sedimented at cytosolic proteins (including cytoplasmic dynein) are diluted 150,000 3 g for 20 min at 48C. The heat-stable MAP super- significantly, but are not removed. Thus, some Golgi capture natant was separated by FPLC on a Mono Q column (Phar- is seen in the absence of additional cytosolic proteins; the macia) using a 0–400 mM KCl gradient in 35 mM K-Pipes pH process is stimulated at least 3-fold when reactions are sup- 7.2, 5 mM MgSO4, 1 mM EGTA, and 0.5 mM EDTA (13). The plemented with crude CHO cytosol added to a final concen- expression and purification of MAP4-C and MAP4-N were tration of 0.5–1 mgyml.
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