Figure 3.
Localization and expression of GFP-IN, IN NLS and IN NLSmut in wildtype cells. (A) A schematic of the GFP-LacZ-Ty1 IN (32) and GFP2-IN NLS constructs is shown. Full-length IN is under control of the GAL1 promoter (DSM1); IN NLS and IN NLSmut are under control of the MET25 promoter (pAC1804 and pAC2431, respectively). Full-length IN contains three domains: an N-terminal zinc-finger domain, a central catalytic domain, and a C-terminal domain containing the IN NLS, consisting of basic regions 1 and 2 (BR1 and BR2). A GFP-fused truncated form of IN containing the last 54 amino acids (aa 582–636) (including BR1 and BR2 indicated in bold lettering—595SKKRSLEDNETEIKVSRDTWNTKNMRSLEPPRSKKRI631) was used in most experiments and is referred to as GFP2-IN NLS. Previously defined key lysine residues are highlighted in bold. (B) GFP-fused IN and GFP2-IN NLS are targeted to the nucleus while GFP2-IN NLSmut is not. Cells expressing either full-length IN, GFP2-IN NLS, or GFP2-IN NLSmut (596KKR598 → AAA and 628KKR630 → AAA) were analyzed using direct fluorescence microscopy (GFP). Cells were stained with Hoechst to indicate the location of the nucleus and corresponding DIC images are shown.