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Saccharomyces cerevisiae Duo1p and Dam1p, novel proteins involved in mitotic spindle function.

Hofmann C, Cheeseman IM, Goode BL, McDonald KL, Barnes G, Drubin DG - J. Cell Biol. (1998)

Bottom Line: By expressing a GFP-Dam1p fusion protein in yeast, Dam1p was also shown to be associated with intranuclear spindle microtubules and spindle pole bodies in vivo.Biochemical experiments demonstrated that Dam1p binds directly to microtubules with micromolar affinity.We suggest that Dam1p might localize Duo1p to intranuclear microtubules and spindle pole bodies to provide a previously unrecognized function (or functions) required for mitosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202, USA.

ABSTRACT
In this paper, we describe the identification and characterization of two novel and essential mitotic spindle proteins, Duo1p and Dam1p. Duo1p was isolated because its overexpression caused defects in mitosis and a mitotic arrest. Duo1p was localized by immunofluorescence, by immunoelectron microscopy, and by tagging with green fluorescent protein (GFP), to intranuclear spindle microtubules and spindle pole bodies. Temperature-sensitive duo1 mutants arrest with short spindles. This arrest is dependent on the mitotic checkpoint. Dam1p was identified by two-hybrid analysis as a protein that binds to Duo1p. By expressing a GFP-Dam1p fusion protein in yeast, Dam1p was also shown to be associated with intranuclear spindle microtubules and spindle pole bodies in vivo. As with Duo1p, overproduction of Dam1p caused mitotic defects. Biochemical experiments demonstrated that Dam1p binds directly to microtubules with micromolar affinity. We suggest that Dam1p might localize Duo1p to intranuclear microtubules and spindle pole bodies to provide a previously unrecognized function (or functions) required for mitosis.

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Dam1p cosediments with microtubules.  Different concentrations of  taxol-stabilized microtubules  were mixed with radio- labeled Dam1p or tau in  vitro–translation products.  The reactions were then centrifuged at high speeds to pellet the microtubules. The percentage of Dam1p and tau  that copelleted with the microtubules in each reaction  was determined by fractionating the pellets and supernatants on SDS-PAGE gels and  quantitating levels by autoradiography and densitometry.  (See text for discussion of  three bands resulting when  Dam1p is produced by translation in vitro.)
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Figure 7: Dam1p cosediments with microtubules. Different concentrations of taxol-stabilized microtubules were mixed with radio- labeled Dam1p or tau in vitro–translation products. The reactions were then centrifuged at high speeds to pellet the microtubules. The percentage of Dam1p and tau that copelleted with the microtubules in each reaction was determined by fractionating the pellets and supernatants on SDS-PAGE gels and quantitating levels by autoradiography and densitometry. (See text for discussion of three bands resulting when Dam1p is produced by translation in vitro.)

Mentions: Attempts to test whether Duo1p binds to microtubules directly were inconclusive. Therefore, the question of what interaction accounts for the colocalization of Duo1p with spindle microtubules remained open. We next tested whether Dam1p binds to microtubules. The experiment in Fig. 7 A shows that in vitro–translated Dam1p binds to microtubules. In vitro translation of Dam1p resulted in three major products, all specific to the Dam1p construct. As discussed earlier in the Results, it is likely that these represent full-length Dam1p and truncated Dam1p products resulting from in frame alternative translation start site usage and/or early translational termination due to RNA secondary structure. While all three Dam1p translation products copelleted with microtubules, the two truncated Dam1p products demonstrated solubility (i.e., lack of pelleting in the absence of microtubules) and concentration-dependent microtubule binding, while the largest product pelleted partially on its own. Therefore, for Kd calculations, we focused on the product marked by the arrow in Fig. 7 A. Binding of Dam1p was concentration dependent and saturable. Moreover, when binding reactions were diluted 10-fold after binding reached equilibrium, the percent binding dropped markedly, demonstrating that binding is reversible (not shown). These data show that Dam1p binding to microtubules is specific. The estimated Kd of the Dam1p–microtubule interaction is 1 μM, approximately threefold weaker than the interaction between tau and microtubules (Fig. 7 B).


Saccharomyces cerevisiae Duo1p and Dam1p, novel proteins involved in mitotic spindle function.

Hofmann C, Cheeseman IM, Goode BL, McDonald KL, Barnes G, Drubin DG - J. Cell Biol. (1998)

Dam1p cosediments with microtubules.  Different concentrations of  taxol-stabilized microtubules  were mixed with radio- labeled Dam1p or tau in  vitro–translation products.  The reactions were then centrifuged at high speeds to pellet the microtubules. The percentage of Dam1p and tau  that copelleted with the microtubules in each reaction  was determined by fractionating the pellets and supernatants on SDS-PAGE gels and  quantitating levels by autoradiography and densitometry.  (See text for discussion of  three bands resulting when  Dam1p is produced by translation in vitro.)
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Related In: Results  -  Collection

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Figure 7: Dam1p cosediments with microtubules. Different concentrations of taxol-stabilized microtubules were mixed with radio- labeled Dam1p or tau in vitro–translation products. The reactions were then centrifuged at high speeds to pellet the microtubules. The percentage of Dam1p and tau that copelleted with the microtubules in each reaction was determined by fractionating the pellets and supernatants on SDS-PAGE gels and quantitating levels by autoradiography and densitometry. (See text for discussion of three bands resulting when Dam1p is produced by translation in vitro.)
Mentions: Attempts to test whether Duo1p binds to microtubules directly were inconclusive. Therefore, the question of what interaction accounts for the colocalization of Duo1p with spindle microtubules remained open. We next tested whether Dam1p binds to microtubules. The experiment in Fig. 7 A shows that in vitro–translated Dam1p binds to microtubules. In vitro translation of Dam1p resulted in three major products, all specific to the Dam1p construct. As discussed earlier in the Results, it is likely that these represent full-length Dam1p and truncated Dam1p products resulting from in frame alternative translation start site usage and/or early translational termination due to RNA secondary structure. While all three Dam1p translation products copelleted with microtubules, the two truncated Dam1p products demonstrated solubility (i.e., lack of pelleting in the absence of microtubules) and concentration-dependent microtubule binding, while the largest product pelleted partially on its own. Therefore, for Kd calculations, we focused on the product marked by the arrow in Fig. 7 A. Binding of Dam1p was concentration dependent and saturable. Moreover, when binding reactions were diluted 10-fold after binding reached equilibrium, the percent binding dropped markedly, demonstrating that binding is reversible (not shown). These data show that Dam1p binding to microtubules is specific. The estimated Kd of the Dam1p–microtubule interaction is 1 μM, approximately threefold weaker than the interaction between tau and microtubules (Fig. 7 B).

Bottom Line: By expressing a GFP-Dam1p fusion protein in yeast, Dam1p was also shown to be associated with intranuclear spindle microtubules and spindle pole bodies in vivo.Biochemical experiments demonstrated that Dam1p binds directly to microtubules with micromolar affinity.We suggest that Dam1p might localize Duo1p to intranuclear microtubules and spindle pole bodies to provide a previously unrecognized function (or functions) required for mitosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202, USA.

ABSTRACT
In this paper, we describe the identification and characterization of two novel and essential mitotic spindle proteins, Duo1p and Dam1p. Duo1p was isolated because its overexpression caused defects in mitosis and a mitotic arrest. Duo1p was localized by immunofluorescence, by immunoelectron microscopy, and by tagging with green fluorescent protein (GFP), to intranuclear spindle microtubules and spindle pole bodies. Temperature-sensitive duo1 mutants arrest with short spindles. This arrest is dependent on the mitotic checkpoint. Dam1p was identified by two-hybrid analysis as a protein that binds to Duo1p. By expressing a GFP-Dam1p fusion protein in yeast, Dam1p was also shown to be associated with intranuclear spindle microtubules and spindle pole bodies in vivo. As with Duo1p, overproduction of Dam1p caused mitotic defects. Biochemical experiments demonstrated that Dam1p binds directly to microtubules with micromolar affinity. We suggest that Dam1p might localize Duo1p to intranuclear microtubules and spindle pole bodies to provide a previously unrecognized function (or functions) required for mitosis.

Show MeSH