Fig. 6. alp4 mutants are defective in γ-tubulin localization to the SPB without affecting the capability of complex formation with γ-tubulin. (A) Gel filtration chromatography in alp4 mutants. Soluble cell extracts were prepared from wild type and alp4-1891 mutants that were incubated at 36°C for 6 h, separated through a Superose-6 column and immunoblotted as in Figure 2C. (B) Cellular localization of SPB (Cut12–GFP) and γ-tubulin. alp4 mutants in which cut12⁺ was tagged with GFP (LV25, Table II) were grown at 26°C in the presence of HU and shifted up to 36°C upon washout of HU. Immunofluorescence microscopy using affinity-purified anti-Gtb1 antibody was performed. At the same time, the location of Cut12 was determined by GFP. The bar indicates 10 µm.