The yeast protein Stu2 belongs to the XMAP215 family of conserved

The yeast protein Stu2 belongs to the XMAP215 family of conserved microtubule-binding proteins which regulate microtubule plus end dynamics. plus ends. Permanently tethering Stu2 to SPBs by fusing it to a version Huperzine A of Spc72 that lacks the Stu2-binding site in part complements these defects in a manner which is dependent upon the microtubule-binding domain of Stu2. Thus the SPB-associated Spc72-Stu2 complex plays a key role in regulating microtubule properties. Huperzine A from pure tubulin due to the presence of regulatory factors. Huperzine A Members of the conserved family of MT-binding proteins named Stu2 in budding yeast XMAP215 in and the colonic-hepatic tumour-overexpressed gene (ch-TOG) in mammalian Ctsk cells represent proteins that regulate MT dynamics (Nabeshima et al. 1995 Wang and Huffaker 1997 Gr?f et al. 2000 XMAP215-like proteins regulate MT function by a number of mechanisms. One place of action is the MT plus end. Work in extract has shown that XMAP215 stabilizes MT plus ends by opposing the destabilizing activity of the kinesin XKCM1 (Tournebize et al. 2000 In contrast studies using stabilized MTs identified XMAP215 as an MT-destabilizing factor (Shirasu-Hiza et al. 2003 XMAP215-like proteins also bind to centrosomes and SPBs independently of MTs. In mutant that fails to bind Stu2 demonstrates that SPB-associated Stu2 is required for astral MTs anchorage and regulation of MT dynamics. Results The 35 most C-terminal amino acids of Stu2 confer binding to Spc72 and astral MT plus?ends In order to understand how Stu2 binds to SPBs we investigated which domain of Stu2 confers binding to the SPB component Spc72. In the yeast two-hybrid system the 35 most C-terminal amino acids of Stu2 (Stu2853-888) were sufficient to mediate interaction with Spc72 (Figure?1A lane?3). Consistently deletion of C-terminal amino acids of Stu2 (Stu21-855) completely abolished interaction with Spc72 (Figure?1A lane?2). The Stu21-855 two-hybrid construct was functional since it interacted strongly with approach. The C-terminal truncated Stu21-855 from insect cells had only a weak interaction with recombinant was terminated after codon 855. Although Online). Moreover localization studies showed that the Stu21-855 protein not only failed to bind to SPBs but also associated with strongly reduced efficiency (79.6% in wild-type and 16.7% in mutants with defects in only one of the two activities. Based on two-hybrid and co-immunoprecipitation experiments it was suggested that Spc72 interacts with the Tub4 complex and Stu2 (Chen et al. 1998 Knop and Schiebel 1998 Souès and Adams 1998 If Stu2 and the Huperzine A Tub4 complex bind to distinct regions of Spc72 it should be possible to construct mutants in which the association with Stu2 is defective yet the interaction with the Tub4 complex is unaffected. We therefore mapped the binding sites of Spc72 for the Tub4 complex subunit Spc98 (Geissler et al. 1996 and Stu2. As reported previously (Chen and with indicated fragments of binding experiments were performed to show direct binding of the Tub4 complex and Stu2 to Spc72. Reconstituted Tub4 complex from insect cells (Vinh et al. 2002 interacted strongly with recombinant N-Spc72 (Figure?2D lane?6). In contrast Spc72 association with individual subunits (Spc97 and Spc98) and heterodimers (Spc97-Tub4 and Spc98-Tub4) was much weaker (lanes 7-12). Thus only the fully assembled Tub4 complex binds with high efficiency to Spc72. When N-Spc72 subfragments were tested Stu2?but not the Tub4 complex interacted with N-Spc72ΔTub4 (Figure?2E lane?5). In Huperzine A contrast only the Tub4 complex but not Stu2 bound to N-Spc72ΔStu2 (lane?4). These binding data suggest that the N-terminal 100 amino acids of Spc72 interact directly with the Tub4 complex and amino acids 99-267 of Spc72 with Stu2. The Stu2-binding site is not required for Tub4 complex binding and vice versa. The Tub4 complex but not the Stu2-binding region of Spc72 is essential In our S288c strain background is an essential gene (Knop and Schiebel 1998 This allowed us to address whether the Stu2 or Tub4 complex binding domains of Spc72 were required for viability. Cells from which the chromosomal was erased and which were kept alive by on a derivatives. Cells were then tested for growth on 5-FOA. As the 5-FOA selects against the plasmid the construct was the only source of activity in the 5-FOA resistant colonies. Despite its manifestation appear (Number?3B lane?2). Similarly pRS316-cells were transformed with the indicated wild-type (Number?4A arrow head) and.