A band mobility shift assay identifies proteins that specifically bind the E3 element. Radiolabeled E3 transcripts were incubated with yeast protein extracts, and then with RNase T1 and heparin at room temperature. RNA–protein complexes were resolved in 4% native gel and visualized by autoradiography. Lane 1, E3 transcripts incubated with K699 extract; lanes 2 and 3, E3 transcripts incubated with K699 extracts in the presence of 200× molar excess of pGEM RNA and unlabeled E3 transcripts, respectively.

The formation of the specific RNA–protein complex is dependent on Loc1p. Band mobility shift assays were performed as mentioned in the legend to Fig. 1. Lane 1, E3 RNA probe only, no protein extract. Lanes 2 and 3, E3 transcripts were incubated with yeast extracts of YLM090 (loc1) and K699 (wild-type) strains. Lane 4, E3 transcripts incubated with extracts of YLM090 (loc1) complemented with a LOC1-myc plasmid.

Amino acid sequence of Loc1p as predicted from the Yeast Genome Sequencing project. One remarkable feature of Loc1p is the abundance of arginine and lysine in this protein. Loc1p contains 20.1% lysine and 9.8% arginine. The pI value of 10.79 for Loc1p is a reflection of the abundance of positive charge in this protein.

Affinity purification of Loc1p-myc from an E3 RNA column. RNA affinity-purified fractions were analyzed by Western blot with anti-myc antibody. Lane 1, starting extracts; lane 2, flowthrough fraction; lane 3, wash fraction; lane 4, elution fraction with high salt; lanes 5 and 6, elution fractions from RNA affinity columns using M9 and pGEM 3Z RNA as ligands, respectively; and lane 7, protein extract from strain K699 (no myc-tagged protein). Arrow, Loc1p-myc protein.

Recombinant Loc1p-GST fusion binds to E3. Lanes 1 and 2, E3 transcripts incubated with bacterial extracts containing recombinant GST-Loc1p but immunodepleted by GST antibody or glutathione beads, respectively. Lane 3, E3 transcripts incubated with the extract before immunodepletion. Lanes 4, 5, and 6, E3 transcripts incubated with bacterial extract containing recombinant GST-Loc1p in the presence of 100× excess of unlabeled E3, IRE, and pGEM 3Z RNAs, respectively. Lane 7, E3 transcripts incubated with SDS-PAGE purified recombinant Loc1p-GST fusion. The arrow indicates the E3/Loc1p–GST complex.

The ASH1 mRNA is delocalized in a loc1 yeast strain. (A) Fluorescent in situ hybridization for ASH1 mRNA in wild-type (K699) and loc1 (YLM090) yeast cells. ASH1 mRNA is expressed from a multicopy plasmid (YEPlac181). DAPI, DNA staining; NOM., Nomarski. The ASH1 mRNA is tightly localized at the bud tip of a wild-type cell, whereas it is delocalized in the cytoplasm of a late anaphase loc1 yeast cell. Bar, 10 μm. (B) Immunofluorescence detection of the Ash1p-myc in postanaphase YML094 (cla4) and YML093 (cla4 loc1) yeast cells. DAPI, DNA staining; NOM., Nomarski. Note the elongated bud neck caused by the deletion of the CLA4 gene. In the majority of cells, Ash1p-myc is symmetrically distributed in the loc1 strain. Bar, 10 μm.

ASH1 mRNA coimmunoprecipitates specifically with Loc1p-myc. (A) RT-PCR amplification of ASH1 mRNA extracted from pellet (top) or supernatant (bottom) after immunoprecipitation. Lane 1, RT-PCR reaction without RNA extract. Lanes 2 and 4, immunoprecipitation from strain expressing Loc1p-myc with (lane 2) or without (lane 4) anti-myc antibody. Lanes 3 and 5, immunoprecipitation from strain expressing Loc1p with (lane 3) or without (lane 5) anti-myc antibody. CTL, control PCR amplification of the ASH1 gene. MW, molecular weight marker. Arrows indicate the ASH1 PCR fragment of 375 bp. (B) RT-PCR amplifications of various mRNAs after immunoprecipitation of Loc1p-myc. P, RNA extracted from the pellet; S, RNA extracted from the supernatant; MW, molecular weight marker. (C) Competition of the immunoprecipitation of ASH1 mRNA by the localization elements. Yeast extract was incubated with either 10, 1, and 0.1 μg of an equimolar mix of the four ASH1 localization elements (ASH1) or the IRE RNA (IRE) before immunoprecipitation of the Loc1p-myc protein and RT-PCR of the ASH1 mRNA. MW, molecular weight marker; −, negative control (no yeast extract); +, positive control (IP of ASH1 mRNA and RT-PCR without competition).

Intracellular distribution of the Loc1p-myc protein. (A) Immunofluorescence detection of the Loc1p-myc. Strain YLM090 (loc1) was transformed with the plasmid pRL094 (LOC1-MYC). Loc1p-myc (in red) is expressed throughout the cell cycle and appears to be strictly nuclear (see DAPI, in blue). The two panels show cells before (left) and during (right) anaphase. Bar, 10 μm. (B) Shuttling assay for Loc1p-myc protein. (a–f) Npl3p-GFP at 24°C (a–c) and 36°C (d–f). (g–l) Loc1p-myc at 24°C (g–i) and 36°C (j–l). Whereas the Npl3p-GFP (in green) accumulates in the cytoplasm after a temperature shift from 24°C (a) to 36°C (d), Loc1p-myc (in red) remains in the nucleus at 24°C (g) and 36°C (j). g and j are an overlap of images h and i, and k and l, respectively. b, e, i, and l are DAPI staining (in blue) and c and f are Nomarski. Bar, 10 μm. (C) Heterokaryon shuttling assay for Loc1p-myc. (a–c) Loc1p-myc. (d–f) Npl3p-GFP. Binuclear yeasts show no accumulation of Loc1p-myc (in red) in the second nucleus (a), whereas Npl3p-GFP (in green) appears in both nuclei after cell fusion (d). b and e are DAPI staining (in blue) and c and f are Nomarski. Bar, 10 μm.