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. 2017 Dec 6;30(12):771–780. doi: 10.1093/protein/gzx059

Fig. 5.

Fig. 5

Labeling Pma1-SpyTag using SpyoIPD-EGFP: (A) Fluorescent images of yeast cells expressing Pma1-SpyTag, in which SpyoIPD-EGFP expression is induced by the indicated concentration of galactose. Note that due to the large difference in SpyoIPD-EGFP expression levels, it was necessary to image samples with different exposure times (1000 ms for [Gal] = 0%; 200 ms for [Gal] = 0.05%; 200 ms for [Gal] = 0.25% and 20 ms for [Gal] = 4.0%). (B) Yeast expressing Pma1-SpyTag and SpyoIPD-EGFP were induced for 8 h in 0.25% galactose, and then chased for an additional 2 h with glucose. Glucose inhibits the GAL1 promoter, turning off new synthesis of SpyoIPD-EGFP (100 ms exposure). Remaining cytosolic SpyoIPD-EGFP either reacts with Pma1-SpyTag, is degraded or is partitioned into the daughter cell. Cytosolic signal is significantly reduced, and the asymmetric segregation of Pma1 is visible, with no plasma membrane signal observed in the budding daughter cell.