Nab2 is phosphorylated upon heat shock. (A) Immunoblotting for Nab2 (lanes 1 to 4) or Nab2-TAP (lanes 5 to 8) was conducted with cell lysates isolated after shifting cells from growth in YPD at 30°C to 42°C (lanes 1 and 2 and lanes 5 and 6) or shifting to growth in YPD with 10% ethanol (EtOH) (lanes 3 and 7) or YPD with 0.4 M NaCl (lanes 4 and 8) for 1 h. P-Nab2-TAP, phosphorylated Nab2-TAP; P-Nab2, phosphorylated Nab2. (B) Nab2-TAP was immunoprecipitated from cells in early log phase grown in YPD at 30°C and shifted to 42°C for 1 h. Immunoprecipitates were treated with λ phosphatase (λ PPase) (lane 2), λ phosphatase plus phosphatase inhibitors (lane 3), or mock treatment (−) (lane 1) for 30 min at 30°C.

Nab2 phosphorylation occurs via the MAP kinase, Slt2, on residues 178 and 180. (A) S. cerevisiae kinase null mutants were screened by immunoblotting with anti-Nab2 antibodies for the Nab2 mobility shift. Cells were grown at 23°C (−) (lanes 1, 3, 5, 7, and 9), and shifted to 42°C for 1 h (+) (lanes 2, 4, 6, 8, and 10). WT, wild type; pS, phosphorylated serine; p^T, phosphorylated threonine. (B) Diagram of Nab2 domain structure (41), with Nab2 LC-MS data for a peptide from residues 170 to 185 shown. Mass spectrometry was used to determine the phosphorylation sites on Nab2-TAP isolated from heat-shocked cells. In the graph, mass spectrum intensity for the product b and y ions from the peptide are all shown. Overall, the mass spectrum indicates that two sites are phosphorylated. T178 and S180 were identified because ions b4 (Q-T-D-A) and b6 (Q-T-D-A-P-A) have masses indicative of the absence of phosphorylation on T171, and y3 (F-A-S) is indicative of the absence of phosphorylation on S183, leaving T178 and S180 as the only possible phosphorylation targets. (C) In vitro kinase reactions were conducted with Slt2-TAP (lanes 1 and 2), Bck1-TAP (lane 3), Mkk1-TAP (lane 4), and Ypk1-TAP (lane 5) purified from S. cerevisiae on IgG-coated magnetic beads and with wild-type recombinant Nab2 (lane 1 and lanes 3 to 5) or nab2-T178A/S180A (lane 2), with [γ-³²P]ATP for 30 min at 30°C. Reaction products were separated by SDS-PAGE and exposed to autoradiography film. The positions (in kilodaltons) of molecular mass markers (MM) are indicated to the left of the gel. (D) Coomassie blue-stained SDS-polyacrylamide gel of Nab2 protein used in vitro kinase reactions in panel C. (E) Immunoblotting for Nab2 was performed on lysates made from cells grown at either 23°C (−) (lanes 1, 3, and 5) or shifted to 42°C for 1 h (+) (lanes 2, 4, and 6) in nab2Δ strains containing plasmids expressing either wild-type NAB2 (lanes 1 and 2), nab2-T178A/S180A (lanes 3 and 4), or nab2-T178E/S180E (lanes 5 and 6).

Poly(A⁺) mRNA does not accumulate in the nucleus of heat-shocked slt2Δ cells. (A and B) In situ hybridization using an oligo[d(T)] probe was performed on WT and slt2Δ cells (A) and wild-type and nab2-T178A/S180A cells (B) grown to early log phase in YPD at 23°C or after shift to 42°C for 45 min. Bars = 3 μm.

Production of heat shock proteins is not affected in slt2Δ or bck1Δ cells. Wild-type (WT) (lanes 1 and 2), nup42Δ (lanes 3 and 4), bck1Δ (lanes 5 and 6), and slt2Δ (lanes 7 and 8) cells were grown at 23°C, shifted to 42°C for 15 min, and labeled with [³⁵S]methionine for an additional 15 min. Cell lysates were separated by SDS-PAGE, and proteins were visualized by autoradiography. The positions of proteins induced upon heat shock (Hsp104, Hsp82, and Hsp70 proteins, respectively) are indicated by asterisks to the right of the gel. The positions (in kilodaltons) of molecular mass markers (MM) are indicated to the left of the gel.

Nab2 phosphorylation is required for efficient recovery from heat shock. The cells were grown at 23°C prior to shifting the cells to 52°C for the times indicated (0, 5, 10, and 20 min). Samples with the same number of cells were then serially diluted and plated for growth at 23°C on YPD for 2.5 days.

Nab2 forms MLP1-dependent foci during heat shock. Cells were grown to early log phase at 23°C and shifted to 30°C for 1 h. In wild-type cells (left panels), Nab2-GFP shifts from total nuclear localization at 30°C to nuclear foci at 42°C. (A) Nab2-mCherry (Nab2-mCh) forms intranuclear foci at 42°C that overlap with the foci formed by Yra1-GFP. (B) In mlp1Δ cells (right panels), Nab2-GFP foci do not form at 42°C. Bars = 3 μm.

During heat shock, Nab2 phosphorylation and intranuclear focus formation are independent events. (A) Nab2-mCherry forms intranuclear foci in wild-type (WT) and slt2Δ cells shifted to growth at 42°C for 1 h. Bar = 3 μm. (B) Immunoblotting for Nab2 in lysates from yeast strains shows the mobility shift induced by heat shock (42°C for 1 h) in wild-type (lanes 1 and 2), nup42Δ (lanes 3 and 4), mlp1Δ (lanes 5 and 6), and gfd1Δ (lanes 7 and 8) cells.

Nab2-Mex67 directly interact and are not colocalized during heat shock. (A) Cells coexpressing Nab2-mCherry and Yra1-GFP were tested for formation of heat shock-induced intranuclear foci. Nab2-mCherry and Yra1-GFP colocalize in foci at 42°C. Bar = 3 μm. (B) Mex67-GFP localization does not change during heat shock and remains localized in the nuclear envelope. (C) Nab2 and Mex67-Mtr2 directly interact in vitro. Soluble binding assays with glutathione resin were conducted with purified recombinant Mex67-Mtr2 (lanes 2 and 3) or with Dbp5 (lanes 4 and 5) with lysate (Lys) from bacterial cells expressing GST-Nab2 (lane 1). Samples were separated by SDS-PAGE, and proteins were visualized by Coomassie blue staining. Input (In) and bound (Bd) fractions are shown. GST alone as a proteolytic product from GST-Nab2 was observed in lanes 3 and 5.