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. 2005 Jan 20;24(3):567–579. doi: 10.1038/sj.emboj.7600547

Figure 5.

Figure 5

Ectopic expression of wild-type NMD3 and introduction of suppressor mutations allows Nmd3-GFP to recycle in the presence of lsg1 and rpl10 mutants. (A) Cultures of strain AJY1705 (NMD3-GFP CRM1[T539C]) containing pAJ363 (NMD3, 2μ) and either empty vector (pRS425), pAJ879 (GAL10∷LSG1) or pAJ1109 (GAL10∷LSG1[K349T]) were maintained in raffinose or galactose was added to induce the LSG1 alleles. Cells were then treated with LMB, fixed, DAPI stained and visualized as described in Materials and methods. For (B), AJY1657 (rpl10[G161D]) containing either pAJ1069 (NMD3[L291F]AAA-GFP) or pAJ1288 (NMD3[I112T, I362T]AAA-GFP) was grown constitutively at 25°C and treated as in (A). DEH221+ containing pAJ1069 (NMD3[L291F]AAA-GFP) was cultured and prepared for microscopy as described for Figure 3C. (C) Overnight cultures of strain AJY1896 (nmd3∷TRP1 CRM1[T539C]) containing pAJ582 (NMD3-GFP) or pAJ1287 (NMD3[I112T, I362T]-GFP) as the sole copies of NMD3 and either empty vector (pRS426), pAJ1312 (GAL10∷LSG1) or pAJ1278 (GAL10∷LSG1[K349T]) in medium containing raffinose were diluted two-fold in fresh medium in the presence of either raffinose or galactose to induce LSG1 expression. After 3 h, cells were fixed and treated for visualization as in (A).