Figure 2.

Localization of Sec3 in the tropomyosin mutant and cells treated with latrunculin. (A) The affinity-purified anti-Sec3 antibody recognizes Sec3 in wild-type (SEC3) but not sec3 deletion (sec3Δ) cells by Western blotting (left; molecular masses indicated to the left) and immunofluorescence microscopy (right). (B) Sec3 remains polarized in tpm1-2 tpm2Δ mutant cells. The TPM1 tpm2Δ (left) and tpm1-2 tpm2Δ (right) cells were grown at 25°C and then shifted to 34.5°C for 10 and 60 min before immunostaining with anti-Sec3 or anti-Sec4 antibodies. Both Sec4 and Sec3 were polarized in TPM1 tpm2Δ cells (left). Sec4 was completely depolarized in the tpm1-2 tpm2Δ cells after the temperature shift, whereas Sec3 was mostly polarized, albeit less concentrated than the control cells. Sec3 in these mutants monitored by GFP tagging (Sec3-GFP) was also polarized. (C) Yeast cells were arrested at G₀ phase and then released into fresh medium at 25°C for 90 min in the presence of 100 μM Lat B (left) or DMSO (right). Cells were fixed and then processed for immunostaining with the anti-Sec3 antibody. In cells treated with Lat B, Sec3 still formed a patch in the presumptive budding site. Actin cables were disrupted by this treatment. As controls, both Sec3 and actin were polarized in cells treated with DMSO (right). Bars, 5 μm.