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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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Binding of Dam1p to Duo1p. GST alone and GST– Duo1p fusion proteins were purified from yeast using glutathione  beads (see Materials and Methods). Beads containing these proteins were then incubated with in vitro–translated three-repeat  Tau (a negative control, see text) and with Dam1p (full-length  Dam1p at 39 kD, with early termination product at ∼29 kD). After incubation, the beads were pelleted, and proteins present in  the supernatants (S) and pellets (P) were examined on SDS-PAGE gels. The top panel shows an autoradiograph of the negative control, three-repeat Tau, which did not pellet with the  beads. The bottom panel shows that Dam1p appears to bind specifically to GST–Duo1p beads but not to GST beads.
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Figure 4: Binding of Dam1p to Duo1p. GST alone and GST– Duo1p fusion proteins were purified from yeast using glutathione beads (see Materials and Methods). Beads containing these proteins were then incubated with in vitro–translated three-repeat Tau (a negative control, see text) and with Dam1p (full-length Dam1p at 39 kD, with early termination product at ∼29 kD). After incubation, the beads were pelleted, and proteins present in the supernatants (S) and pellets (P) were examined on SDS-PAGE gels. The top panel shows an autoradiograph of the negative control, three-repeat Tau, which did not pellet with the beads. The bottom panel shows that Dam1p appears to bind specifically to GST–Duo1p beads but not to GST beads.

Mentions: To confirm that the two-hybrid interaction between Duo1p and Dam1p was the result of direct physical binding, in vitro–translated Dam1p was incubated with glutathione beads coated with GST or GST–Duo1p. Please note that several translation products, some presumably resulting from internal translation initiation or premature translation arrest, were evident when Dam1p was translated. The beads were pelleted and washed with buffer containing 50 mM NaCl, and the presence of the in vitro– translated Dam1p in the supernatant and pellet fractions was monitored by analysis on protein gels (Fig. 4). Dam1p showed markedly enhanced interaction with GST–Duo1p compared with GST alone. An in vitro–translated fragment of mammalian tau protein (see below) served as a negative control and did not interact with either GST- or GST–Duo1p–coated beads (Fig. 4).


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)

Binding of Dam1p to Duo1p. GST alone and GST– Duo1p fusion proteins were purified from yeast using glutathione  beads (see Materials and Methods). Beads containing these proteins were then incubated with in vitro–translated three-repeat  Tau (a negative control, see text) and with Dam1p (full-length  Dam1p at 39 kD, with early termination product at ∼29 kD). After incubation, the beads were pelleted, and proteins present in  the supernatants (S) and pellets (P) were examined on SDS-PAGE gels. The top panel shows an autoradiograph of the negative control, three-repeat Tau, which did not pellet with the  beads. The bottom panel shows that Dam1p appears to bind specifically to GST–Duo1p beads but not to GST beads.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132964&req=5

Figure 4: Binding of Dam1p to Duo1p. GST alone and GST– Duo1p fusion proteins were purified from yeast using glutathione beads (see Materials and Methods). Beads containing these proteins were then incubated with in vitro–translated three-repeat Tau (a negative control, see text) and with Dam1p (full-length Dam1p at 39 kD, with early termination product at ∼29 kD). After incubation, the beads were pelleted, and proteins present in the supernatants (S) and pellets (P) were examined on SDS-PAGE gels. The top panel shows an autoradiograph of the negative control, three-repeat Tau, which did not pellet with the beads. The bottom panel shows that Dam1p appears to bind specifically to GST–Duo1p beads but not to GST beads.
Mentions: To confirm that the two-hybrid interaction between Duo1p and Dam1p was the result of direct physical binding, in vitro–translated Dam1p was incubated with glutathione beads coated with GST or GST–Duo1p. Please note that several translation products, some presumably resulting from internal translation initiation or premature translation arrest, were evident when Dam1p was translated. The beads were pelleted and washed with buffer containing 50 mM NaCl, and the presence of the in vitro– translated Dam1p in the supernatant and pellet fractions was monitored by analysis on protein gels (Fig. 4). Dam1p showed markedly enhanced interaction with GST–Duo1p compared with GST alone. An in vitro–translated fragment of mammalian tau protein (see below) served as a negative control and did not interact with either GST- or GST–Duo1p–coated beads (Fig. 4).

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