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Developmental regulation of the heat shock response by nuclear transport factor karyopherin-3

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA

View larger version (27K): [in a new window]   Fig. 1. (A) Schematic diagram of the domain organization of karyopherin-3 and Drosophila HSF. Amino acid endpoints for each region, as well as their proposed functions, are indicated. Two Kap-3 deletion mutants used in the crosslinking experiments, 3-NLS and 3-ßBD, as well as three dHSF-NLS probes used in two hybrid screening or crosslinking experiments, are also shown. DBD, DNA-binding domain of dHSF; OLIGO, oligomerization domain of dHSF. (B) Two-hybrid cloning and amino acid sequence of dKap-3. Schematic diagram of the dKap-3 cDNAs isolated by two-hybrid screening. Two of the three distinctive positive clones, #158 and #161, were found to cover the entire coding region of dKap-3. (C) Amino acid sequence of Drosophila karyopherin-3. Amino acid sequence of full-length karyopherin-3 is shown. The functional domains of dKap-3 are illustrated as follows: boxed sequences include the internal NLS of dKap-3 and ARM repeats, respectively; dKap-3 binding domain is underlined and in italics.

View larger version (26K): [in a new window]   Fig. 2. Cross-linking of dKap-3 to mutant forms of dHSF-NLS polypeptides. The upper table summarizes the in vivo subcellular localization of the wild-type and mutant NLSs determined by immunostaining (Zandi et al., 1997b). Recombinant wild-type dKap-3 protein (1 µg) was cross linked to the following P32-labeled forms of mini-NLS: mini-NLS (lane 1); Q399L, K400R, L404R mini-NLS (lane 2); K405M mini-NLS (lane 3); L404P mini-NLS (lane 4); N408S, R411L mini-NLS (lane 5); K400E, R401L mini-NLS (lane 6); Q403L mini-NLS (lane 7); E406P mini-NLS (lane 8) and mini-NLS (lane 9). All probes were labeled with [32P]ATP and MAPK. 2 ng of probe (approximately 150,000 cpm) was used in each reaction. The NLS mutation Q399L/K400R/L404R (lane 2), and two constitutive nuclear entry mutations (Q403L, lane 7 and E406P, lane 8) formed complexes with dKap-3 of approximately the same size (approx. 90 kDa) as the wild-type mini-NLS probe (lane 1). The remaining mutant forms, which are not transported in vivo (lanes 3-6), did not form specific complexes in vitro. The lower molecular mass complexes seen are dimers of probe and are mini-NLS probe specific.

View larger version (29K): [in a new window]   Fig. 3. (A) The effect of dKap-ß on dKap-3 binding to Drosophila HSF’s NLS. 200 ng of recombinant dKap-3 protein was cross linked to either mini-NLS or mini-NLS probe. Increasing amounts of recombinant dKap-ß was added to the indicated reactions with mini-NLS probe (lanes 1-3). Lane 1, no dKap-ß; lane 2, 200 ng dKap-ß; lane 3, 1 µg dKap-ß. No enhancement of dKap-3 binding to the mini-NLS reaction was observed even with the addition of 1 µg of dKap-ß (lane 4). (B) Domains of dKap-3 required for Drosophila HSF NLS binding. Recombinant wild-type or mutant dKap-3 protein (1 µg) was cross linked to either mini-NLS or mini-NLS probes, which were labeled with [32P]ATP using MAP kinase. 2 ng of probe (150,000 cpm) was used in each reaction. Both wild-type dKap-3 and dKap-3-NLS cross-linked to mini-NLS probe very efficiently (lanes 1 and 3). Deletion of the dKap-ß binding domain, dKap-3-ßBD, eliminated binding to the mini-NLS probe (lane 5). The mini-NLS probe did not bind to any of the dKap-3 proteins, and no complex is observed (lanes 2,4 and 6).

View larger version (80K): [in a new window]   Fig. 4. Karyopherin-3 is required for nuclear docking in vitro. Digitonin-permeabilized SL2 cells were incubated with NLS-EGFP fusion proteins, recombinant dKap-3 and dKap-ß. The nuclear docking of EGFP fusion proteins was examined by fluorescent microscopy. (A) Wild-type NLS-EGFP fusion protein only. (B) Wild-type NLS-EGFP fusion protein, kap-3 and kap-ß. (C) K405M mutant NLS-EGFP fusion protein, dKap-3 and dKap-ß. (D) Q403L mutant NLS-EGFP fusion protein, dKap-3 and Kap-ß.

View larger version (56K): [in a new window]   Fig. 5. Effect of heat shock on the sub-cellular distribution of dKap-3 in SL2 cells. Schneider cells stained with DAPI are shown on the left side and immunofluorescent staining with anti-dKap-3 monoclonal antibodies at 1:1000 dilution is shown on the right side. NS, Non shocked cells; HS, cells heat shocked at 37°C.

View larger version (73K): [in a new window]   Fig. 6. Developmental western analysis of dKpa-3 expression in Drosophila embryos. 0- to 2-hour, 0- to 4-hour and 0- to 6-hour (after egg laying) embryos were collected and homogenized. Whole embryo extracts obtained from homogenization were then analyzed by SDS-PAGE, probed with anti-3 antibody for western blotting analysis (A) or stained with Coomassie Blue (B). M, molecular mass standards; NS, non-shock embryos; HS, heat-shocked embryos

View larger version (71K): [in a new window]   Fig. 7. Developmental timing of dKap-3 expression correlates with nuclear entry of dHSF. (A-L) Cycle-11 to cycle-13 embryos were stained with either DAPI, anti-dHSF monoclonal antibodies (HSF panels) or anti-dKap-3 monoclonal antibodies (Kap-3 panels). NS, non-shocked Drosophila embryos; HS, heat-shocked embryos. (A,E,I) Cycle 11 embryos; (C,G,K) cycle 12 embryos; (B,F,J,D,H,L) cycle 13 embryos. Clearly present but excluded from the nucleus is dHSF in both non-shocked cycle 11 embryos (E) and heat-shocked cycle 12 embryos (G). Similarly staged embryos display essentially no dKap-3 staining (I and K). At cycle 13 in non-shocked embryos the dHSF is constitutively nuclear and dKap-3 is expressed yet largely excluded from the nucleus (J). In heat-shocked cycle 12 embryos the dHSF remains excluded from the nucleus 9 (G) and no dKap-3 is observed as in non-shocked embryos (K). At cycle 13 in heat-shocked embryos the characteristic puntate pattern of dHSF nuclear staining is observed (panel H) and dKap-3 remains excluded from the nucleus (L). (M) Analysis of the Kap-3 mRNA distribution in the cycle 9-10 embryo demonstrates essentially no RNA except in the posterior region (indicated by the arrowhead, panel 1). Adjacent to this embryo is a cycle 13-14 embryo that shows significant levels of Kap-3 mRNA (panel 1). Analysis of dHSF protein distribution in the cycle 12 embryo shows that dHSF is clearly present within the nucleus of pole cells at this early stage (arrowhead, panel 2), corresponding to the presence of Kap-3 mRNA in this region of the pre-cycle 13 embryo (panel 1).