In vitro and in vivo interaction of Apg-2 with ZO-1. (A) Apg-2 binds to the SH3 domain of ZO-1 in vitro. Glutathione beads carrying equal amounts of GST fusion proteins of PDZ3SH3, PDZ3, and SH3 domains of ZO-1 or GST were incubated with His₆-Apg-2. Pull-downs were probed with anti-Apg-2 (C terminus) and anti-GST antibodies. (B) In vivo association of Apg-2 and ZO-1. Wild-type MDCK cell extracts were immunoprecipitated with a control antibody (neg. control) or anti-ZO-1 antibody (anti-ZO-1/wt-MDCK). As an additional control, ZO-1 was also precipitated from extracts of tetracycline-treated Apg-2 Ri z2 cells (anti-ZO-1/Apg-2 Ri). The precipitates and total cell extracts (inputs) were analyzed by immunoblotting using antibodies against Apg-2, ZO-1, and Hsc70.

Localization of Apg-2 in normal and heat-shocked MDCK cells. (A) MDCK cells were cultured on coverslips and then either fixed directly (control) or first incubated at 43°C for 2 h (heat shock). Cells were preextracted with Triton X-100 before fixation as indicated. The samples were then processed for indirect immunofluorescence using antibodies against Apg-2 and ZO-1. (B) Apg-2-depleted MDCK cells (Apg-2 Ri z2) were processed as described for the samples in A and were then stained for Apg-2. Panels A and B show confocal sections that were taken with identical microscope settings. (C) MDCK cells were incubated either at 37°C for 3 h (0 min) or at 43°C for the indicated times and were then lysed and expression of Apg-2, α-tubulin, Hsp70, and ZONAB was analyzed by immunoblotting.

Regulation of G₁/S phase transition by Apg-2. Control and Apg-2 RNAi cell lines were cultured on coverslips without (white bars) or with tetracycline (gray bars), and synchronized in G₀/G₁ phase by serum starvation. Entry into S phase was then stimulated by adding serum and monitored by bromodeoxyuridine labeling. Replicating cells were quantified by counting (10 different fields per sample, 3 samples per condition; * denotes statistically significant [p < 0.05] differences using t tests).

Regulation of transcriptional activation of ZONAB. (A) MDCK cells at low (A1) or high (A2) density were cotransfected with a plasmid containing a minimal promoter with a ZONAB binding site driving firefly luciferase expression, a plasmid containing an identical promoter sequence but with an inactivated ZONAB binding site driving Renilla luciferase expression together with plasmids resulting in either Apg-2- or ZONAB-specific RNAi, and/or Apg-2 overexpression. Apg-2 H z2 marks an Apg-2 construct that is resistant to RNAi induced by the z2 plasmid. The ratios between the two luciferase activities were calculated and results expressed as percentage of change from control plasmid transfections (shown are means ± 1 SD). (B) Wild-type MDCK cells and Apg-2 RNAi cell lines z2 and z5 were incubated with tetracycline and transfected with a plasmid driving the expression of VSV-tagged Apg-2 resistant to the z2 RNAi plasmid. Total cell extracts were blotted with antibodies against the VSV epitope and α-tubulin. (C) Cells grown to high density were transfected and ZONAB activity was assayed as in A. Plasmids coding for VSV-tagged constructs representing either full-length Apg-2, the ATPase domain, or the C-terminal (CTD-VSV) domain were cotransfected (* denotes statistically significant [p < 0.05] differences using t tests).

Domain structure and expression of Apg-2. (A) Domain structure of Apg-2. The ATPase, peptide binding (PBD), and C-terminal (CTD) domains are indicated (canine Apg-2: GenBank/EMBL/DDBJ accession no. AY911512). (B) Expression of Apg-2. For detection of endogenous Apg-2, total MDCK cell extracts were immunoblotted with a rabbit antibody against the C terminus of Apg-2. Regulated expression of full-length Apg-2-VSV was analyzed by immunoblotting with an anti-VSV antibody. (C) Regulated depletion of Apg-2. MDCK cells stably transfected with the Apg-2-directed regulated RNAi plasmids (z2 and z5) and a control RNAi clone were plated without or with tetracycline. After 3 d, the cells were harvested in SDS gel sample buffer, and expression of Apg-2 and α-tubulin was analyzed by immunoblotting.

Binding of the SH3 domain of ZO-1 to the ATPase domain of Apg-2. (A and B) Glutathione beads carrying either GST-SH3 or GST were incubated with His₆-ATPase, His₆-PBD, or His₆-CTD (A); or with His₆-ATPase domains derived from Apg-2 or Hsc70 (B). (C) Apg-2 His₆-ATPase domain was incubated with GST fusion proteins containing the indicated SH3 domains. In all panels, pull-down was assayed by immunoblotting with an antibody against His₆.

Stimulation of the Apg-2–ZO-1 interaction by heat shock. Wild-type (wt-MDCK) or Apg-2-depleted MDCK cells (Apg-2 Ri z2) were either kept at 37°C or heat shocked for 2 h at 43°C. The cells were then extracted, and ZO-1 was immunoprecipitated as described in Figure 2B. Precipitates and total extracts were then immunoblotted for Apg-2, ZO-1, and Hsc70.

Apg-2 and ZONAB compete for binding to the SH3 domain. (A) GST-SH3 samples conjugated to glutathione beads were incubated with or without His₆-ZONAB for 30 min. Increasing amounts of His₆-ATPase were then added and the reactions were incubated for 2 h (final concentrations: His₆-ZONAB, 1 μg/ml; His₆-ATPase, 0, 1, 2, and 4 μg/ml). As negative control, GST was incubated with His₆-ZONAB, and the highest concentration of His₆-ATPase. Pull-downs were assayed using antibodies against ZONAB and the N terminus of Apg-2. The quantification was obtained by densitometric scanning of two independent experiments with different batches of recombinant proteins. (B) Wild-type and Apg-2 RNAi cells were grown for 4 d on coverslips. Apg-2 was depleted by adding tetracycline during the last 2 d of culture. The cells were either left at 37°C (control) or heat shocked for 2 h before processing for immunofluorescence. The cells were then preextracted with Triton X-100, fixed in methanol, and the distribution of ZONAB was monitored by indirect immunofluorescence and epifluorescence microscopy. The distribution of ZONAB was not affected in tetracycline-treated control RNAi cells (our unpublished data). Quantification of fluorescence intensities showed that heat shock induced an increase in the ratio of nuclear to cytoplasmic fluorescence from 1.1 ± 0.2 to 2.4 ± 0.4 in control cells, whereas in heat-shocked Apg-2-depleted cells, the ratio remained at 1.2 ± 0.3 (10 images were quantified for each condition averaging 5 cells per image).

Heat shock induces ZONAB activity. High-density cells were transfected with the same reporter plasmids as in Figure 8A together with a control RNAi plasmid or RNAi constructs against ZONAB or Apg-2. Cells were either incubated continuously at 37°C (A, control) or incubated at 43°C for 2 h and then allowed to recover for 2 h at 37°C (B, heat shock) before measuring the luciferases. Values are given as percentage change from control transfections performed at the same conditions. Shown are means ± 1 SD (* marks statistically significant [p < 0.05] differences using t tests).