LAP2β interacts with HA95 via two distinct domains. (A) GST–LAP2β deletion peptides. (B) Indicated peptides were incubated in nuclear extracts of Bjab cells expressing HA95–Myc and sedimented by GST precipitation. Binding of HA95–Myc to peptides was analyzed by immunoblotting using anti-Myc antibodies. Control incubations contained GST or glutathione beads alone. (C) GST–LAP2β(137–373) was incubated in a nuclear extract of Myc-tagged p70S6 kinase-expressing cells, and binding (or lack thereof) of p70S6 kinase to the peptide was detected by GST precipitation and immunoblotting. (D) GST–LAP2β overlays of immunoprecipitated HA95 or B-type lamins. Peptide binding was detected using anti-GST antibodies. (E) Peptide-mediated dissociation of LAP2β from HA95. HA95-IPs were incubated with 100 μM GST–LAP2β peptides, HA95-IPs were sedimented, and pellets and supernatants were immunoblotted using the indicated antibodies. Arrows indicate incubation with LAP2β(137–298) followed by addition of LAP2β(299–373) (lanes 7 and 8) or vice versa (lanes 9 and 10).

Characterization of nuclei used in the in vitro DNA replication assay. (A) Nuclei purified from HeLa cells in G1 phase were loaded with GST–LAP2β(1–452). Peptide uptake was analyzed by immunofluorescence using anti-GST antibodies. (B) GST–LAP2β(1–452)-loaded G1 nuclei were exposed to an S-phase extract, and import of Cdc6 was visualized by immunofluorescence. Note that the G1 nuclei contain low detectable levels of Cdc6. (C) Cdc6 import was also examined in nuclei containing the indicated peptides. G1 nuclei were also analyzed by (D) TUNEL and (E) electrophoresis to monitor DNA degradation after exposure to replication extract at 4°C or 37°C. Bars, 10 μm.

Dissociation of HA95 from HA95-NBD in G1 elicits degradation of Cdc6. (A) HA95 (top three panels) or Cdc6 (bottom three panels) was immunoprecipitated from G1- or S-phase HeLa cells, and immune precipitates (P) and supernatants (S) were immunoblotted using either precipitating antibody. (B) Nuclei from G1-phase cells (lane 1) or G1 nuclei loaded with the indicated GST–LAP2β peptides (lanes 2–5) and incubated in nuclear isolation buffer for 1 h were immunoblotted using the indicated antibodies. In lanes 6–8, G1 nuclei loaded with LAP2β(137–298) were incubated in buffer for 1 h together with 25 μM β-lactone, LLnL, or LLM. In lane 9, whole samples (WS; nuclei + buffer) were immunoblotted. (C) G1 nuclei were loaded with GST–LAP2β(137–298) or GST–LAP2β(299–373) in the presence of no inhibitor (−), β-lactone, LLnL, or LLM. Nuclei were allowed to replicate in S-phase extract containing [α³²P]dCTP, and synthesized DNA was analyzed by autoradiography.

Nuclear injection of GST–LAP2β(137–298) in G1 inhibits replication. Nuclei of G1 HeLa cells were injected with 5 ng of the indicated GST–LAP2β peptide or GST alone together with a 150-kD FITC–dextran to visualize injections, and cells were cultured for 9–10 h with BrdU. Mock (buffer)-injected cells were also cultured with BrdU and 50 μM aphidicolin. BrdU incorporation was detected using TRITC-conjugated anti-BrdU antibodies. DNA was labeled with Hoechst 33342. (B) Percentages of cells labeled with BrdU 9–10 h after peptide injection (n = 45–50/treatment in two replicates). (C) Immunofluorescence analysis of Cdc6 and Orc2p in G1 cells injected as in A. Cells were fixed 2 h after peptide injection. Arrows point to noninjected cells. Bars, 10 μm.

HA95 coprecipitates with LAP2β in interphase. (A) HA95 or LAP2β was immunoprecipitated (IP) from interphase and mitotic Bjab cells. Precipitates (P) and supernatants (S) were immunoblotted using anti-LAP2β or anti-HA95 antibodies. (B) Myc-tagged HA95 was immunoprecipitated from Bjab cells stably expressing HA95–Myc using anti-Myc antibodies, and precipitates were immunoblotted using indicated antibodies. Control immunoprecipitations were done with preimmune rabbit IgGs. (C) HA95-IPs were extracted with 0, 0.25, 0.5, 0.75, 1.0, or 1.25 M NaCl before sedimentation and immunoblotting of pellets and supernatants.

Disruption of HA95 interaction with LAP2β does not interfere with nuclear assembly in vitro. (A) Purified HeLa nuclei (Nuc) were disassembled in mitotic extract, and chromosomes (Chr) were immunoblotted using the indicated antibodies. (B) Indicated GST–LAP2β peptides were allowed to bind to chromosomes, and binding was analyzed by immunofluorescence using anti-GST antibodies. Insets, DNA labeled with Hoescht 33342. (C) Chromosomes harboring the indicated peptides were incubated in interphase extract under conditions promoting nuclear formation. Membrane assembly was examined by DiOC₆ labeling and phase contrast microscopy. (D) At the end of incubation, the nuclear distribution of GST–LAP2β peptides, LAP2β, A- and B-type lamins, and BAF was examined by immunofluorescence. (E) Nuclei or chromatin masses were also sedimented through sucrose, and proteins were immunoblotted using the indicated antibodies. Bars, 10 μm.

LAP2β(137–298) inhibits initiation of DNA replication in G1 nuclei. (A) Peptide-loaded G1 nuclei were incubated in S-phase extract containing [α³²P]dCTP, dNTPs, GTP, and an ATP-regenerating system. [α³²P]dCTP incorporation was analyzed by phosphorImager (cpm/10⁵ nuclei). (B) Nuclei isolated from HeLa cells at the indicated cell cycle stages were incubated in extracts from S or G0 cells under conditions promoting replication. S-phase extracts also contained 1 mM olomoucine (+Olo) or H₂O (−Olo) (right panel). Synthesized DNA was analyzed by autoradiography. (C) Relative BAF content of G1 nuclei loaded with the indicated peptides and exposed to S-phase extract was examined by immunoblotting. HA95 was also detected as a loading control in the gel. (D) Summary of GST–LAP2β peptides harboring HA95-NBD supporting (+) or inhibiting (−) replication in G1 nuclei. (E) BrdU density substitution experiment. G1 nuclei containing either no peptide (circles), GST alone (triangles), or GST–LAP2β(137–298) (squares) were allowed to replicate in S-phase extract containing BrdU and [α³²P]dCTP. Substituted DNA was separated by centrifugation in CsCl gradients. cpm of each fraction was plotted against fractions of equal densities. HL indicates density of heavy-light replicated DNA. (F) Nuclei from S-phase HeLa cells were loaded with GST–LAP2β peptides and peptide uptake was analyzed as in Fig. 4 A (GST–LAP2β[1–452] is shown). Peptide-loaded nuclei were incubated in S-phase extract and incorporation of [α³²P]dCTP was measured by phosphorImager (cpm/10⁵ nuclei). Bar, 10 μM.

Nuclear injection of GST–LAP2β(137–298) in G1 does not alter distribution of NE proteins or BAF. Nuclei of G1 HeLa cells were injected with 5 ng of the indicated GST–LAP2β peptide or GST alone, cultured for 2–3 h, and analyzed by immunofluorescence using anti-GST, LAP2β, lamin B (goat polyclonal), or BAF antibodies. Arrows point to noninjected cells. Bar, 10 μm.

Functional domains of LAP2β. Identified functional regions of LAP2β (LEM and transmembrane [TM]) and domains binding to indicated ligands are shown. Numbers refer to amino acid positions. See text for references to binding domains.