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Cortical Num1p interacts with the dynein intermediate chain Pac11p and cytoplasmic microtubules in budding yeast.

Farkasovsky M, Küntzel H - J. Cell Biol. (2001)

Bottom Line: The bud tip location of Num1p depends on Bni1p but does not require Kar9p, Dyn1p, or cMTs, whereas cMT contacts with polar Num1p dots are reduced in cells lacking Dyn1p.Num1p also forms a complex with Bni1p and Kar9p, although Num1p is not required for Bni1p- and Kar9p-dependent nuclear migration to the bud neck in preanaphase cells.In addition, Num1p may transiently cooperate with an associated Bni1p-Kar9p complex at the bud tip of early anaphase cells.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck Institute for Experimental Medicine, D-37075 Göttingen, Germany.

ABSTRACT
Num1p, a cortical 313-kD protein, controls cytoplasmic microtubule (cMT) functions and nuclear migration through the bud neck in anaphase cells. A green fluorescent protein (GFP)-Num1p fusion protein localizes at the bud tip and the distal mother pole of living cells, apparently forming cMT capture sites at late anaphase. In addition, galactose-induced GFP-Num1p is seen at the bud neck and in lateral regions of the mother cortex. The bud tip location of Num1p depends on Bni1p but does not require Kar9p, Dyn1p, or cMTs, whereas cMT contacts with polar Num1p dots are reduced in cells lacking Dyn1p. Num1p associates with the dynein intermediate chain Pac11p in the presence of Dyn1p, and with the alpha-tubulin Tub3p, as shown by coimmune precipitation of tagged proteins. Num1p also forms a complex with Bni1p and Kar9p, although Num1p is not required for Bni1p- and Kar9p-dependent nuclear migration to the bud neck in preanaphase cells. Our data suggest that Num1p controls nuclear migration during late anaphase by forming dynein-interacting cortical cMT capture sites at both cellular poles. In addition, Num1p may transiently cooperate with an associated Bni1p-Kar9p complex at the bud tip of early anaphase cells.

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Coimmune precipitation of 3xMyc-Num1p with 3xHA-Bni1p and 3xHA-Kar9p. Anti-Myc immunoprecipitates obtained from strains FMY684 (lanes 1 and 6), FMY78 (lanes 2 and 7), FMY810 (lanes 3 and 8), FMY809 (lanes 4 and 9), and FMY805 (lanes 5 and 10) were probed with anti-HA (lanes 1–5) and anti-Myc (lanes 6–10) antibodies.
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Figure 10: Coimmune precipitation of 3xMyc-Num1p with 3xHA-Bni1p and 3xHA-Kar9p. Anti-Myc immunoprecipitates obtained from strains FMY684 (lanes 1 and 6), FMY78 (lanes 2 and 7), FMY810 (lanes 3 and 8), FMY809 (lanes 4 and 9), and FMY805 (lanes 5 and 10) were probed with anti-HA (lanes 1–5) and anti-Myc (lanes 6–10) antibodies.

Mentions: The close neighborhood of Num1p and Kar9p dots at the bud tip of early anaphase cells may indicate a transient physical association of Num1p with Kar9p-binding Bni1p at the bud tip cortex. Indeed, we observe a coimmunoprecipitation of Myc-Num1p with HA-Bni1p and HA-Kar9p, as shown in Fig. 10, lanes 4 and 5, respectively. Anti-Myc precipitates of cells expressing only HA-Bni1p (FMY783) or HA-Kar9p (FMY810) do not contain the 219-kD HA-Bni1p band (Fig. 10, lane 2) or the 74 kD HA-Kar9p band (Fig. 10, lane 3), supporting the specificity of the coprecipitation. The presence of Myc-Num1p in the anti-Myc precipitates was confirmed by anti-Myc probing (Fig. 10, lanes 6, 9, and 10).


Cortical Num1p interacts with the dynein intermediate chain Pac11p and cytoplasmic microtubules in budding yeast.

Farkasovsky M, Küntzel H - J. Cell Biol. (2001)

Coimmune precipitation of 3xMyc-Num1p with 3xHA-Bni1p and 3xHA-Kar9p. Anti-Myc immunoprecipitates obtained from strains FMY684 (lanes 1 and 6), FMY78 (lanes 2 and 7), FMY810 (lanes 3 and 8), FMY809 (lanes 4 and 9), and FMY805 (lanes 5 and 10) were probed with anti-HA (lanes 1–5) and anti-Myc (lanes 6–10) antibodies.
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Related In: Results  -  Collection

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Figure 10: Coimmune precipitation of 3xMyc-Num1p with 3xHA-Bni1p and 3xHA-Kar9p. Anti-Myc immunoprecipitates obtained from strains FMY684 (lanes 1 and 6), FMY78 (lanes 2 and 7), FMY810 (lanes 3 and 8), FMY809 (lanes 4 and 9), and FMY805 (lanes 5 and 10) were probed with anti-HA (lanes 1–5) and anti-Myc (lanes 6–10) antibodies.
Mentions: The close neighborhood of Num1p and Kar9p dots at the bud tip of early anaphase cells may indicate a transient physical association of Num1p with Kar9p-binding Bni1p at the bud tip cortex. Indeed, we observe a coimmunoprecipitation of Myc-Num1p with HA-Bni1p and HA-Kar9p, as shown in Fig. 10, lanes 4 and 5, respectively. Anti-Myc precipitates of cells expressing only HA-Bni1p (FMY783) or HA-Kar9p (FMY810) do not contain the 219-kD HA-Bni1p band (Fig. 10, lane 2) or the 74 kD HA-Kar9p band (Fig. 10, lane 3), supporting the specificity of the coprecipitation. The presence of Myc-Num1p in the anti-Myc precipitates was confirmed by anti-Myc probing (Fig. 10, lanes 6, 9, and 10).

Bottom Line: The bud tip location of Num1p depends on Bni1p but does not require Kar9p, Dyn1p, or cMTs, whereas cMT contacts with polar Num1p dots are reduced in cells lacking Dyn1p.Num1p also forms a complex with Bni1p and Kar9p, although Num1p is not required for Bni1p- and Kar9p-dependent nuclear migration to the bud neck in preanaphase cells.In addition, Num1p may transiently cooperate with an associated Bni1p-Kar9p complex at the bud tip of early anaphase cells.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck Institute for Experimental Medicine, D-37075 Göttingen, Germany.

ABSTRACT
Num1p, a cortical 313-kD protein, controls cytoplasmic microtubule (cMT) functions and nuclear migration through the bud neck in anaphase cells. A green fluorescent protein (GFP)-Num1p fusion protein localizes at the bud tip and the distal mother pole of living cells, apparently forming cMT capture sites at late anaphase. In addition, galactose-induced GFP-Num1p is seen at the bud neck and in lateral regions of the mother cortex. The bud tip location of Num1p depends on Bni1p but does not require Kar9p, Dyn1p, or cMTs, whereas cMT contacts with polar Num1p dots are reduced in cells lacking Dyn1p. Num1p associates with the dynein intermediate chain Pac11p in the presence of Dyn1p, and with the alpha-tubulin Tub3p, as shown by coimmune precipitation of tagged proteins. Num1p also forms a complex with Bni1p and Kar9p, although Num1p is not required for Bni1p- and Kar9p-dependent nuclear migration to the bud neck in preanaphase cells. Our data suggest that Num1p controls nuclear migration during late anaphase by forming dynein-interacting cortical cMT capture sites at both cellular poles. In addition, Num1p may transiently cooperate with an associated Bni1p-Kar9p complex at the bud tip of early anaphase cells.

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