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 ldb18D. ldb18D cells exhibit defects in spindle orientation similar to those caused by a block in the dynein pathway. Consistent with this observation, ldb18D 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 ldb18D 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 p150Glued arm to dynamitin and the remainder of the dynactin complex. The genetic interaction of ldb18D with stu1-5 also supports the notion that dynein/dynactin helps to generate a spindle pole separating force.

Parallel Genetic and Proteomic Screens Identify Msps as a CLASP–Abl Pathway Interactor in Drosophila

“…Double mutant analyses of the CLASP retinal gain-of-function (GOF) line combined with loss-offunction (LOF) or GOF mutants of these known interactors shows modification of the retinal phenotype. For example, when CLASP is overexpressed in combination with a transposon insertion in the Drosophila dynactin ortholog glued (a known interactor of CLASP and 1TIP complexes in other systems) (Amaro et al 2008;Manna et al 2008), suppression of the CLASP GOF phenotype occurs ( Figure 1D). Conversely, Abl GOF shows enhancement of the CLASP GOF retinal phenotype ( Figure 1E), demonstrating that the adult retinal system is a useful model for identifying genetic interactors of CLASP.…”

Stu1 inversely regulates kinetochore capture and spindle stability

“…Deprived of the restriction presented by the cohesion of bipolar-attached sister chromatids, spindle poles move apart (anaphase B). The forces that power the anaphase B movement are probably generated partly via astral MTs (Amaro et al 2008) and partly by elongating the ipMT array to form an (ultimately 9-mm-long) anaphase spindle. The latter requires that the spindle midzone (the overlap region of the interdigitizing ipMTs) is reinforced by a group of proteins that either facilitate MT bundling or stabilize MT plus ends (Khmelinskii and Schiebel 2008).…”

Dynactin and Num1 cooperate to establish the cortical anchoring of cytoplasmic dynein inS. pombe

“…Arp1 is a well-conserved homolog of the actin-related protein Arp1 in vertebrates, whereas Jnm1 is a homolog of p50/dynamitin in vertebrates and Jnm1 in S. cerevisiae (McMillan and Tatchell, 1994). Mug5 shows weak similarity to p24 in vertebrates and Ldb18 in S. cerevisiae (Amaro et al, 2008;Moore et al, 2008). The expression of arp1, mug5 and jnm1 was highly induced in diploid cells starved for nitrogen, indicating that these genes were expressed exclusively in meiotic cells (supplementary material Fig.…”