Figure 3.

The export of Gbp2 is dependent on mRNA transport. (A) Gbp2 export is dependent on RNA polymerase II transcription and on the mRNA export factors Mex67 and Nup159/Rat7, but not on the arginine methyltransferase Hmt1. rpb1-1, mex67-5, rat7-1 or hmt1::HIS3 strains that express gbp2-S15A–GFP (green fluorescent protein) or nuclear localization signal–GFP were grown to log phase before they were shifted to 37 °C for 30 min. The intracellular localization of the GFP fusion proteins was analysed by fluorescence microscopy. The localization of npl3-27 was determined by immunofluorescence using anti-Npl3 antibodies. (B) Overexpression of gbp2 is toxic. Wild-type strains that express Gbp2–GFP under control of the strong GAL1 promoter or as a control NLS–GFP–GFP were spotted in 1:10 dilutions onto –uracil galactose plates. (C) Cells that overexpress Gbp2 accumulate poly(A)⁺ RNA in the nucleus. The same strains shown in (B) were grown to log phase in liquid –URA raffinose medium, shifted to galactose-containing medium for 4 h, followed by a 2-h repression in glucose-containing medium, before the intracellular mRNA localization was analysed by in situ hybridization using a digoxygenin-labelled oligo d(T)₅₀ probe and rhodamine-labelled anti-digoxygenin antibodies. The subcellular localization of the GFP-fusion proteins was determined by fluorescence microscopy.