A member of the Ran-binding protein family, Yrb2p, is involved in nuclear protein export

Abstract

Yeast cells mutated in YRB2, which encodes a nuclear protein with similarity to other Ran-binding proteins, fail to export nuclear export signal (NES)-containing proteins including HIV Rev out of the nucleus. Unlike Xpo1p/Crm1p/exportin, an NES receptor, Yrb2p does not shuttle between the nucleus and the cytoplasm but instead remains inside the nucleus. However, by both biochemical and genetic criteria, Yrb2p interacts with Xpo1p and not with other members of the importin/karyopherin beta superfamily. Moreover, the Yrb2p region containing nucleoporin-like FG repeats is important for NES-mediated protein export. Taken together, these data suggest that Yrb2p acts inside the nucleus to mediate the action of Xpo1p in at least one of several nuclear export pathways.

Figure 1

NES-mediated protein export in Δyrb2 cells. A NLS-NES-GFP- or Rev-GFP-encoding plasmid was introduced into wild-type, Δyrb2, or xpo1–1 cells. The localization of the GFP fusion proteins was examined at 30°C, after a 15-hr shift to 15°C or after a 1-hr shift to 37°C by fluorescence microscopy.

Figure 2

Yrb2p does not shuttle between the nucleus and the cytoplasm. The nup49–313 strain expressing GFP-Yrb2p or GFP-Npl3p was used for the protein-shuttling assay. After expression/repression at 25°C, cells were shifted to 37°C and incubated for 5.5 hr and the fusion proteins were localized by fluorescence microscopy.

Figure 3

Interactions between YRB2 and XPO1. (A) EGY42 × EGY48 diploid strains containing plexA-XPO1 (bait) and the indicated pJG4–5 derivatives (prey) were grown in selective media. After 2 hr of induction with galactose, β-galactosidase activity was measured as described in Materials and Methods. (B) EGY48 containing the indicated tester genes were streaked onto leucine drop-out media and grown at 30°C. (C) Cells expressing the GST-YRB2 fusion as well as XPO1-GFP or CSE1-GFP were lysed, and the resulting lysate was mixed with glutathione-Sepharose beads. After washing, proteins bound to the beads were detected by α-GFP immunoblotting. CBB, GST (lane 1) or GST-Yrb2 fusion protein (lanes 2–4) stained with Coomassie brilliant blue; B, bound; FT, flow through. (D) Cells containing a GAL1-YRB2 or a vector as well as a plasmid encoding XPO1-GFP were grown in raffinose media followed by 2 hr in galactose (induced) or glucose (uninduced) and were viewed by fluorescence microscopy. The nuclear rim is indicated with arrowheads.

Figure 4

Synthetic relationship of YRB2 and XPO1. Δyrb2 cells containing a YRB2 CEN URA3 plasmid were crossed to xpo1–1 cells. After sporulation and dissection of the resulting tetrads, haploid progeny with the indicated genotypes were spotted onto a uracil drop-out plate (ura do) or a plate with 1 mg/ml 5-fluoroorotic acid (5 FOA) and grown at 25°C.

Figure 5

The FG repeats are important for Yrb2p activity. Δyrb2 cells expressing NLS-NES-GFP and the indicated Yrb2p derivatives were grown at 30°C and shifted to 15°C for 13 hr, and the localization of the NLS-NES-GFP fusion protein was determined by fluorescence microscopy. All mutant Yrb2 proteins are expressed at levels similar to the intact protein. Parentheses indicate that a fraction of the protein still appears in either the nucleus or cytoplasm.