She2 but not Khd1 accumulates in nucleoli when messenger RNA export is inhibited. (A–L) She2 localization. Representative cells of (A–D) wild-type (Mex67⁺) and (E–L) mex67-5^ts strains that were grown for 30 min at 37°C and co-stained for She2 and the nucleolar marker protein Nop1 are shown. Nuclei were stained with DAPI. Wild-type cells show normal localization of She2 at the bud tip (A–D), whereas in mex67-5 cells the signals of She2 and Nop1 largely overlap (H,L, yellow signal). (M–P) Puf6 localization. Puf6-Myc9 in a wild-type strain is detected in a crescent–shaped region and colocalizes with nucleolar Nhp2. (Q–X) Khd1 localization. (Q–T) Staining of Khd1-HA6 shows cytoplasmic distribution of Khd1 in wild-type cells. In mex67-5 cells, Khd1-HA6 accumulates in nuclei when shifted to 37°C. Note that, in contrast to She2, nuclear Khd1-HA6 largely overlaps with the DAPI-stained region (W). DAPI, 4,6-diamidino-2-phenylindole; DIC, differential interference contrast.

RNA transport mediated by a She3N–She2 fusion protein results in inefficient sorting of Ash1. (A) Schematic diagram of the She3N–She2 (She3N–S2)fusion protein. Numbers indicate amino acids used of the corresponding protein part. (B) Immunoprecipitation of She2 and She3N–S2 using She2 antibody. She2 (white arrowhead) or the She3N–S2 fusion protein (black arrow) were detected at similar levels in total extracts (T) and immunopellets (P) of a wild-type strain or strain RJY2414 (She3N–S2). The asterisk indicates the heavy chain of the antibody that co-eluted with the antigen. S, supernatant. (C) Immunoprecipitates were subjected to reverse transcription–PCR and analysed for bound ASH1 RNA. SDS E, elution with 10% SDS; G E, elution with 100 mM glycine (pH 2.5), −RT, control lacking reverse transcriptase. ASH1 co-precipitated with She2 and She3N–S2, but not in the absence of She2 (Δshe2). (D) Cytoplasmic retention of She3N–S2 after inhibition of mRNA export. In strain RJY2422 (Mex67 She3N–S2), She3N–S2 localized to the bud tip (upper panel). Nucleolar accumulation of She2 (middle panels) but not She3N–S2 (lower panels) was observed in mutant mex67-5 cells. (E) Cytoplasmic retention of She2 did not interfere with mRNA localization. In situ hybridization in strains expressing She2 (wild type) or She3N–S2 indicated normal ASH1 localization (left panels). ASH1 mRNA localization efficiency was quantified from 150 binucleate cells (right panel). The black columns show category percentage for wild type and grey columns for She3N–S2. DAPI, 4,6-diamidino-2-phenylindole; DIC, differential interference contrast.

Accelerated Ash1 synthesis in cells expressing She3–She2 fusion protein. After induction of Ash1-Myc9 from a GAL1 promoter in wild-type (WT) or She3N–S2 cells, samples were taken at the indicated time points and Ash1/actin protein ratios were determined by western blot analysis. (A) She3N–S2 cells show an accelerated Ash1 synthesis (arrow) compared with wild-type cells; the white arrowheads indicate actin signal. (B) Increase in ASH1 mRNA levels was similar in wild-type and She3N–S2 cells. ASH1 levels (black arrows) were determined by northern blot analysis and normalized against intensities of an unspecific crossreacting RNA (asterisks). (C) Plot of average Ash1-Myc9/actin ratios from three independent experiments.

An RNA-binding mutant of She2 accumulates in the nucleolus. (A) Filter-binding analysis of the ASH1 E3 localization element (left) or unrelated human immunodeficiency virus type I (HIV-I) TAR RNA (right) to She2 (black) and mutant She2-N36S-R63K (red). Binding constants (K_D) of wild-type She2 to ASH1 E3 RNA and HIV-I TAR RNA are shown. Mutant She2-N36S-R63K showed no significant binding to either RNA. (B) Representative cells of strain RJY2838 expressing the She2-N36S-R63K mutant were stained for She2 and Nop1. She2-N36S-R63K mutant (left panel) accumulates in a region that overlaps with Nop1 signals (right panel; merge), indicating nucleolar accumulation of She2-N36S-R63K.

Loss of asymmetrical Ash1 distribution in She3N–S2 cells. (A) Cellular distribution of nuclear Myc-tagged Ash1 was categorized as asymmetrical, partly asymmetrical or symmetrical. (B) Ash1-Myc9 distribution in a wild-type strain (She2; black columns), an She3N–S2 strain (She3N–She2; grey columns) and a strain expressing an She3–She2 fusion protein containing a tandem nuclear localization signal at the N terminus (2 × NLS-She3N–She2; white columns) was quantified by counting 351 binucleate cells. DAPI, 4,6-diamidino-2-phenylindole; NLS, nuclear localization signal.