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. 2002 Jun 10;157(6):1005–1016. doi: 10.1083/jcb.200201003

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Copyright © 2002, The Rockefeller University Press

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Figure 3. — Ypt32p overexpression partially restores the localization of Sec4p. (A) For these experiments, sec2-78 cells (NY2179) were transformed with a 2μ URA3 plasmid overproducing YPT32 from the GPD promoter (NY2431). Overexpression was confirmed by Western blot probed with αYpt32p antibody. (B) For Sec4p immunofluorescence, cells were grown overnight in YPD at 25°C, shifted to 37°C for 2 h, fixed, and labeled with αSec4p antibody. At 25°C, wild-type (NY10), sec2-78 (NY2179), and sec2-78/2μYPT32 cells (NY 2431) show Sec4p staining preferentially at the bud tip and necks (arrows). In sec2-78 cells, diffuse staining is observed after a temperature shift to 37°C, but upon Ypt32p overexpression, localization of Sec4p is partially restored (arrows). (C) Approximately 100 cells were counted for each experiment and the percentage of cells showing bud tip and mother–daughter neck localization of Sec4p was calculated. Wild-type yeast have 60% localized Sec4p, whereas sec2-78 and sec2-78/2μYPT32 yeast exhibit 21% and 25% localization before the temperature shift. After shifting to 37°C, no localized Sec4p is observed in sec2-78 cells. However, sec2-78/2μYPT32 exhibit 25% localization of Sec4p.

Figure 3. — Ypt32p overexpression partially restores the localization of Sec4p. (A) For these experiments, sec2-78 cells (NY2179) were transformed with a 2μ URA3 plasmid overproducing YPT32 from the GPD promoter (NY2431). Overexpression was confirmed by Western blot probed with αYpt32p antibody. (B) For Sec4p immunofluorescence, cells were grown overnight in YPD at 25°C, shifted to 37°C for 2 h, fixed, and labeled with αSec4p antibody. At 25°C, wild-type (NY10), sec2-78 (NY2179), and sec2-78/2μYPT32 cells (NY 2431) show Sec4p staining preferentially at the bud tip and necks (arrows). In sec2-78 cells, diffuse staining is observed after a temperature shift to 37°C, but upon Ypt32p overexpression, localization of Sec4p is partially restored (arrows). (C) Approximately 100 cells were counted for each experiment and the percentage of cells showing bud tip and mother–daughter neck localization of Sec4p was calculated. Wild-type yeast have 60% localized Sec4p, whereas sec2-78 and sec2-78/2μYPT32 yeast exhibit 21% and 25% localization before the temperature shift. After shifting to 37°C, no localized Sec4p is observed in sec2-78 cells. However, sec2-78/2μYPT32 exhibit 25% localization of Sec4p.