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. 2010 Feb;11(2):185-97.
doi: 10.1111/j.1600-0854.2009.01010.x. Epub 2009 Nov 19.

Nap1 and Chz1 have separate Htz1 nuclear import and assembly functions

Korinna Straube  1 , Jeffrey S Blackwell Jr, Lucy F Pemberton
Affiliations

Nap1 and Chz1 have separate Htz1 nuclear import and assembly functions

Korinna Straube et al. Traffic. 2010 Feb.

Abstract

We analyzed the nuclear import and regulation of the yeast histone variant Htz1 (H2A.Z), and the role of histone chaperones Nap1 and Chz1 in this process. Copurification suggested that Htz1 and H2B dimerized in the cytoplasm prior to import. Like H2B, Htz1 contained a nuclear localization signal (NLS) in its N-terminus that is recognized by multiple karyopherins (also called importins), indicating multiple transport pathways into the nucleus. However, Kap114 and Kap123 appeared to play the major role in Htz1 import. We also identified a role for Nap1 in the import of Htz1/H2B heterodimers, and Nap1 formed a RanGTP-insensitive import complex with Htz1/H2B and Kap114. Nap1 was necessary for maintaining a soluble pool of Htz1, indicating that its chaperone function may be important for the dynamic exchange of histones within nucleosomes. In contrast, Chz1 was imported by a distinct import pathway, and Chz1 did not appear to interact with Htz1 in the cytoplasm. Genetic analysis indicated that NAP1 has a function in the absence of HTZ1 that is not shared with CHZ1. This provides further evidence that the histone chaperones Nap1 and Chz1 have separate Htz1-dependent and -independent functions.

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Figures

Figure 1
Figure 1
Htz1 associates with Kap114, Kap123, and Nap1 in yeast cytosol. (A) Htz1-PrA and associated proteins were purified from cytosol prepared from a strain expressing Kap114-13myc, eluted with a MgCl2 step gradient, separated by SDS-PAGE and analyzed by Western blot (WB) using anti-Myc and anti-Nap1 antibodies. Htz1-PrA is recognized by IgG. (B) Nap1-PrA and associated proteins were purified from cytosol as above and probed with anti-Htz1 antibodies. (C) Htz1-PrA was purified from a strain expressing Kap123-13myc or (D) Kap120-13myc and analyzed as in (A). `Un' lane contains 0.02% of depleted post-bead starting cytosol. (E) Htz1-PrA was purified as in (A) and probed with anti-Chz1 and anti-Nap1 antibodies. W lane contains the last wash fraction. * indicates a proteolytic product of Htz1-PrA.
Figure 2
Figure 2
Htz1 contains an NLS in its amino terminus (A) Wild type (WT) yeast containing the indicated plasmid, were induced to express Htz1 residues 1–53, 1–24, 24–53, 32–134 or 52–134 fused to GFP2 and visualized by fluorescent microscopy. Coincident Hoechst staining and DIC is shown. Htz1 32–134 or 52–134 GFP2 expressing strains were induced for an additional 2 hours. (B) JHY200 strains deleted for HTZ1, and expressing wild type H2B or H2BΔ1–32, were induced to express the Htz1- GFP2 fusions indicated. Htz1 32–134 GFP2 and Htz1 52–134 GFP2 expressing strains were induced for an additional 5 hours, and only a small fraction of cells expressed Htz1 52–134 GFP2. Coincident Hoechst staining or DIC is shown. (C) Wild type yeast expressing mutant NLS reporters htz11–53K3,8,10,14Q or htz11–53K3,8,10,14R-GFP2 were visualized by fluorescent microscopy as above. (D) JHY200 strains deleted for HTZ1, and expressing wild type H2B or H2BΔ1–32, were induced to express the Htz1-GFP2 or htz1K3,8,10,14R-GFP2 fusions as above.
Figure 3
Figure 3
Htz1, Nap1, Kap114 form a co-complex. (A) The Htz1 NLS (residues 1–53) or Asf1 GFP2 reporter was expressed in yeast strains containing mutations of individual KAP genes as indicated and localized by fluorescent microscopy. Coincident Hoechst staining is shown. (B) Recombinant GST-Htz11–53 (250 nM) was immobilized on glutathione sepharose beads and incubated with the indicated recombinant Kap protein (MBP-Kap114 100 nM, MBP-Kap123 500 nM, Kap95 500 nM). MBP-LacZ (500 nM) was utilized as a negative control. After extensive washing, bound protein was analyzed by SDS-PAGE and Coomassie blue staining (CBB). A 50% input of MBP-LacZ is shown. * indicates a proteolytic product of MBP-Kap123. (C) Recombinant GST-Htz11–53 (400 nM) was immobilized on glutathione sepharose beads and incubated with either recombinant Nap1 (250 nM) or H6-PrA-Chz1 (500 nM). Similarly, GST (500 nM) was incubated with Nap1 (250 nM) as a control. After washing, protein was eluted from the beads and analyzed by SDS-PAGE and Coomassie stain. 50% input of Nap1 and H6-PrA-Chz1 are indicated. (D) Recombinant GST-Htz11–53 (400 nM) was immobilized on glutathione sepharose in the presence or absence of Nap1 (250 nM). MBP-Kap114 (100 nM) or MBP-Kap123 (300 nM) was pre-incubated with or without H6-Gsp1Q71L-GTP (50 μM) and/or MBP-LacZ (400nM) and then added to the binding reaction. After extensive washing, bound protein was analyzed by SDS-PAGE and Coomassie stain.
Figure 4
Figure 4
Chz1 contains an NLS and associates directly with Srp1/Kap95 in cytosol. (A) Fragments of Chz1, bearing the indicated mutations, fused to GFP2 were expressed in wild type yeast as indicated and visualized by fluorescence microscopy. Coincident Hoechst staining is shown. (B) Chz1-TAP was immunoprecipitated from cytosol prepared from strains expressing also Kap95-Myc or Kap114-Myc. Bound protein was eluted with MgCl2, precipitated, and analyzed by SDS-PAGE and Western blotting (WB) using anti-Myc antibodies. Chz1-TAP is recognized by IgG. `Un' lane contains 0.02% of depleted post-bead starting cytosol. (C) Recombinant H6-PrA-Chz1 (250 nM) was immobilized on IgG sepharose and incubated with H6-Srp1 (500 nM) and Kap95 (500 nM). zz protein (~250 nM) was used as a negative control. Bound protein was eluted and analyzed by SDS-PAGE and Coomassie blue staining (CBB).
Figure 5
Figure 5
Nap1 is important to maintain soluble pool of Htz1. (A) Htz1-FLAG was immunopurified from yeast cytosol from the indicated strain backgrounds. Bound protein was eluted with the indicated concentration of MgCl2 and analyzed by SDS-PAGE and Coomassie stain (CBB). The last wash fraction before elution is also shown (W). Indicated proteins are labeled according to molecular weight. Different cellular fractions were also analyzed for the presence of Htz1 as indicated. Cytosol: Proteins from the depleted, post-bead cytosol fractions (0.02% from above) were separated by SDS-PAGE and analyzed by Western blot (WB). Htz1 was detected via the Flag antibody. Pellet lysate: Nuclei enriched pellet fractions were analyzed by Western blot for the presence of Htz1-FLAG. WCE: Whole cell extracts of starting yeast cultures were analyzed by Western blot for the presence of Htz1-FLAG. Pgk1 levels were used as a loading control. (B) Htz11–53GFP2, Htz1-GFP2, or Asf1-GFP2 was expressed in wild type yeast containing pRS415GPD-NAP1 or empty vector (pRS415GPD). Localization of GFP was visualized by fluorescence and DIC microscopy.
Figure 6
Figure 6
NAP1, SWR1, and SWC2, but not CHZ1, exhibit genetic interactions with HTZ1. Strains of the indicated genotype were grown to logarithmic phase, equalized, and spotted as 10-fold serial dilutions onto CSM, CSM lacking inositol (CSM –INO), or CSM containing 2% galactose as the sole carbon source (CSM GAL) and grown at 30°C for 3 days.
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