Fig. 3. yHst1p is enriched in the non-nucleolar nucleoplasm and yHst2p is cytoplasmic. The indicated proteins were localized by indirect immunofluorescence on fixed yeast cells as described in Materials and methods. In all cases, the nucleolar marker Nop1p was localized with anti-Nop1 (rabbit antiserum or mouse monoclonal antibody, as appropriate; Gotta et al., 1997) and a Cy5-coupled secondary antibody. This is shown in the first panel of each row and is red in the merged images. In the first row, localization of ectopically expressed ySir2p (α-Sir2, green in merged image) was examined in a diploid sir2::HIS3 strain (GA194) after transformation with pADH-ySir2. The inset shows the localization of ySir2p to the telomeric foci and the nucleolus, when the cells have been washed, after fixation, in 1% Triton–0.02% SDS to improve accessibility (see Gotta et al., 1997). yHst1-Myc is detected by the monoclonal 9E10 (α-Myc) in the haploid strain GA1154 (SIR2) and the isogenic sir2::HIS3 strain GA1155 (inset). yHst2-Myc was examined in GA1276 (SIR2) and the isogenic sir2::HIS3 strain GA1229 (inset). Both fusions are genomic and under their endogenous promoters. The NLS-containing HA-tagged yHst2p expressed from pJG45-yHst2 was examined in transformants of the diploid wild-type strain GA225 expressing either low or high levels of the protein after 4 h of galactose induction, as indicated. The localization of the HA-tagged yHst2 fusion protein, which is encoded by pGAL-yHst2 and lacks a detectable NLS, was examined in transformants of GA225 under conditions of low and high expression, as indicated. The merge is shown in colour, with Nop1p in red and ySir2p, c-Myc or HA epitopes in green. Coincidence of the two signals is yellow. Bar = 2 µm.