Fig. 1.

The C terminus is required for some Lhp1p functions. (A) Constructs encoding Lhp1 proteins. Lhp1p contains a Kap108-dependent NLS that maps to the RRM (19). In some constructs, an SV40 NLS was added to a loop in the RRM (Fig. S2C). (B–D) Plasmids containing the indicated lhp1 alleles, or the empty vectors, were introduced into sup61-10 lhp1Δ (B), lsm8-1 lhp1Δ (C), or trr4-1 lhp1Δ (D) strains that also carried LHP1 on a URA3-containing vector (pSLL28). LHP1 was in the plasmid pAD12, which contains TRP1 and ADE2, while LHP1-NLS, lhp1(1-227), and lhp1(1-227)-NLS were in the TRP1 plasmid pRS314. After growing cells in media lacking tryptophan, fivefold serial dilutions were spotted on agar lacking tryptophan (-Trp) or containing 5-fluoroorotic acid (FOA) and grown at 25 °C (B, C, and D, left) or 16 °C (D, right). (E) Wild-type, lhp1Δ, and sup61-10 lhp1Δ cells carrying the indicated plasmids were subjected to immunofluorescence with anti-Lhp1p (green) and anti-Nop1p (red). DNA was stained with DAPI (blue). As described (21), staining of lhp1Δ cells with anti-Lhp1p reveals a background cytoplasmic fluorescence. (Scale bar, 10 μm).