The Saccharomyces cerevisiae homolog of p24 is essential for maintaining the association of p150Glued with the dynactin complex

Abstract

Stu1 is the Saccharomyces cerevisiae member of the CLASP family of microtubule plus-end tracking proteins and is essential for spindle formation. A genomewide screen for gene deletions that are lethal in combination with the temperature-sensitive stu1-5 allele identified ldb18Delta. ldb18Delta cells exhibit defects in spindle orientation similar to those caused by a block in the dynein pathway. Consistent with this observation, ldb18Delta is synthetic lethal with mutations affecting the Kar9 spindle orientation pathway, but not with those affecting the dynein pathway. We show that Ldb18 is a component of dynactin, a complex required for dynein activity in yeast and mammalian cells. Ldb18 shares modest sequence and structural homology with the mammalian dynactin component p24. It interacts with dynactin proteins in two-hybrid and co-immunoprecipitation assays, and comigrates with them as a 20 S complex during sucrose gradient sedimentation. In ldb18Delta cells, the interaction between Nip100 (p150(Glued)) and Jnm1 (dynamitin) is disrupted, while the interaction between Jnm1 and Arp1 is not affected. These results indicate that p24 is required for attachment of the p150(Glued) arm to dynamitin and the remainder of the dynactin complex. The genetic interaction of ldb18Delta with stu1-5 also supports the notion that dynein/dynactin helps to generate a spindle pole separating force.

Figure 1.—

Ldb18 plays a role in the dynein pathway of spindle orientation. (A) Wild-type (CUY1928), ldb18Δ (CUY1929), and dyn1Δ (CUY1930) strains expressing GFP-Tub1. Arrows designate cells with elongated spindles in the mother cell. (B) Wild-type (BY4741), ldb18Δ (CUY1816), and dyn1Δ (CUY1823) cells stained with DAPI. Arrows designate binucleate mother cells. (C) Quantification of binucleate mother cells by DAPI indicating spindle elongation within the mother cell. More than 250 budded cells were counted.

Figure 2.—

Ldb18 is a component of the dynactin complex. (A) Diagram of the mammalian dynactin complex. Yeast homologs are indicated in parentheses. (B) Ldb18 can co-immunoprecipitate with all components of the dynactin complex. Lysates from Ldb18-13Myc (CUY1932), Ldb18-13Myc Nip100-3HA (CUY1933), and Ldb18-13Myc Arp10-HA (CUY1934) strains were immunoprecipitated using a Myc antibody. Strains lacking Ldb18-13Myc (CUY26, MY8912, and MY8895) were used as controls. Immunoprecipitated proteins were run on SDS–PAGE and immunoblotted using Myc antibody and either a Jnm1, Arp1, or HA antibody. (C) Lysate from an Ldb18-13Myc Nip100-3HA strain (CUY1933) was run over a 5–20% sucrose gradient. Fractions were run on SDS–PAGE and immunoblotted using Myc, HA, Jnm1, and Arp1 antibodies. Arrows indicate migration of size standards: yeast alcohol dehydrogenase (7.4 S) and thyroglobulin (19.4 S).

Figure 3.—

Ldb18 shares sequential and structural properties with mammalian p24, a component of the dynactin complex. (A) Sequence alignment of yeast Ldb18 and human p24. Similar residues have a shaded background and identical residues have a solid background. (B) Ldb18 and p24 are both predicted to contain coiled-coil domains near the amino terminus using a 14-residue window in the COILS algorithm.

Figure 4.—

Localization of Ldb18. Z-series projections of cells (CUY1951) expressing Ldb18-3GFP (green) and mCherry-Tub1 (red) to visualize microtubules. Ldb18 localizes near the daughter-bound SPB (A) and at the plus ends of astral microtubules (B and C). MT, microtubule.

Figure 5.—

Ldb18 is involved in shoulder–sidearm interactions. LDB18 and ldb18Δ strains expressing Jnm1-3HA and Nip100-13Myc or Arp1-13Myc were constructed (CUY1935, CUY1936, CUY1937, and CUY1938). Lysates were immunoprecipitated using a Myc antibody. Samples were separated by SDS–PAGE and blotted using Myc and HA antibodies. Loss of Ldb18 does not affect the interaction of Jnm1 with Arp1 (A), but disrupts its interaction with Nip100 (B). (C) Extended exposure of the Nip100-Myc immunoprecipitation shown in the bottom right of B with serial dilutions of the Nip100-Myc immunoprecipitation from the LDB18 strain. The asterisk indicates the IgG heavy chain; WCE, whole cell extracts; IP, immunoprecipitation.