(A) Two-hybrid interactions between ODC, antizyme and AZI. The yeast two-hybrid strain EGY48 was transformed with plasmid pRB1840 (LacZ reporter), plasmid pEG202-AZ1, pEG202-AZ2, pEG202-AZ3 or pEG202-AZ4 (antizymes 1–4 respectively fused to the DNA-binding domain of LexA) and plasmid pJG4-5 (empty vector) or pJG-ODC or pJG-AZI (ODC or AZI respectively fused to the B42 AD). Transformants were grown on selective glucose medium and then analysed for reporter gene activation (LacZ) on selective galactose plates containing X-gal. Results are representative for multiple experiments with each interaction analysed in triplicate. (B) Expression of fusion proteins in two-hybrid interaction assay. The yeast two-hybrid strain EGY48 was transformed with antizyme–LexA fusion constructs (pEG202-AZ1, -AZ2, -AZ3, -AZ4 respectively) and co-transformed with either pJG-AZI or pJG-ODC AD vectors plus the LacZ reporter pRB1840. Protein expression of the AD-fusion proteins was induced by growing the transformants in galactose-containing medium. Equal amounts of protein were loaded in each lane, and expression levels of the LexA and AD-fusion proteins were monitored by immunoblot as described in the Materials and methods section using either anti-LexA or anti-HA antibodies. The immunoblot shown is representative for multiple experiments.

Wild-type yeast cells (strain Y10000) were transformed with the control plasmid p413ADH (+), isogenic spe1 deleted cells (strain Y15034) were transformed with either the control plasmid p413ADH (O) or plasmid p413ADH-ODC carrying human ODC behind the yeast ADH1 promoter (Δ). Yeast cell growth in polyamine-free, selective glucose medium was followed by determination of A₆₀₀. The data shown are representative of several experiments.

(A) Wild-type (WT) yeast cells (strain Y10000, lane 1) or cells deleted for spe1 (strain Y15034, lane 2) were transformed with the control plasmids p413GALL, p426GPD and p415GALL. Cells deleted for spe1 (strain Y15034) were transformed with plasmid p413GALL-ODC (carrying human ODC behind the GALL promoter) and the control plasmids p426GPD and p415GALL (lane 3) or transformed with p413GALL-ODC and plasmid p426GPD-AZ1 (carrying human antizyme 1 behind the GPD1 promoter) and the control plasmid p415GALL (lane 4) or transformed with plasmids p413GALL-ODC, p426GPD-AZ1 and p415GALL-AZI (carrying human AZI behind the GALL promoter, lane 5). (B) Cells deleted for spe1 (strain Y15034) were transformed with either p413GALL-ODC and the control plasmids p426GPD and p415GALL (lane 1) or p413GALL-ODC and plasmid p426GPD-AZ2 (carrying human antizyme 2 behind the yeast GPD1 promoter) and the control plasmid p415GALL (lane 2) or plasmids p413GALL-ODC, p426GPD-AZ2 and p415GALL-AZI (lane 3). (C) Cells deleted for spe1 (strain Y15034) were transformed with either p413GALL-ODC and the control plasmids p426GPD and p415GALL (lane 1) or p413GALL-ODC and plasmid p426GPD-AZ3 (carrying human antizyme 3 behind the yeast GPD1 promoter) and the control plasmid p415GALL (lane 2) or plasmids p413GALL-ODC, p426GPD-AZ3 and p415GALL-AZI (lane 3). (D) Cells deleted for spe1 (strain Y15034) were transformed with either p413GALL-ODC and the control plasmids p426GPD and p415GALL (lane 1) or p413GALL-ODC, plasmid p426GPD-AZ4 (carrying human antizyme 4 behind the GPD1 promoter) and the control plasmid p415GALL (lane 2) or plasmids p413GALL-ODC, p426GPD-AZ4 and p415GALL-AZI (lane 3). Liquid cultures were grown overnight in selective polyamine-free glucose medium. The overnight cultures were adjusted to A₆₀₀ 3.0/ml and 2 μl aliquots of the overnight culture were spotted at the indicated serial dilutions on to polyamine-free selective galactose plates. The plates were then incubated for 3 days at 30 °C. The results shown are representative for multiple experiments.

(A) Schematic representation of the three-hybrid assay system for ODC, antizyme and AZI. The two-hybrid interaction between ODC and antizyme leads to the activation of a reporter gene. Co-expression of AZI disrupts this interaction and thereby prevents the activation of the reporter gene. AD, activation domain; DBD, DNA-binding domain. (B) Inhibition of ODC–antizyme interaction by AZI. W303-1a cells were transformed with plasmid pRB1840 (LacZ reporter), plasmid pEG202-ODC (ODC fused to the DNA-binding domain of LexA), the plasmids pJG-AZ1, pJG-AZ2, pJG-AZ3 or pJG-AZ4 (antizymes 1–4 respectively fused to the B42 AD), and either plasmid p415GPD (white bars) or p415GPD-AZI carrying AZI behind the GPD1 promoter (black bars). Transformants were grown on selective plates, and then analysed for reporter gene activation (LacZ) using the CPRG assay as described in the Materials and methods section. Data represent means±S.D. values for four independent experiments.

(A) Functional expression of antizyme 1 in yeast. Yeast cells deleted for spe1 (strain Y15034) were transformed with plasmid p413GALL-ODC (carrying human ODC behind the yeast GALL promoter) and either the control plasmids p426ADH (–), p426GAL1 (–) and p426GPD (–) or plasmids p426ADH-AZ1 (+), p426GAL1-AZ1 (+) or p426GPD-AZ1 (+) (carrying human antizyme 1 behind the yeast ADH1, GAL1 and GPD1 promoters respectively). Liquid cultures were grown overnight in selective polyamine-free glucose medium. The overnight cultures were adjusted to A₆₀₀ 3.0/ml and serial dilutions starting with 3.0/ml were spotted (2 μl aliquots) as indicated on polyamine-free selective galactose plates (left panel) or selective galactose plates containing 7.5 μM of putrescine (right panel). The plates were then incubated for 3 days at 30 °C. The colony assays shown are representative for multiple experiments. (B) Functional expression of antizymes 2–4 in yeast. Yeast cells deleted for spe1 (strain Y15034) were transformed with plasmid p413GALL-ODC alone (lane 1, carrying human ODC behind the yeast GALL promoter) or co-transformed with plasmid p426GAL1-AZ2 (lane 2, carrying antizyme 2 behind the yeast GAL1 promoter), p426GAL1-AZ3 (lane 3, carrying antizyme 3 behind the yeast GAL1 promoter) or p426GPD-AZ4 (lane 4, carrying antizyme 4 behind the yeast GPD1 promoter). Liquid cultures were grown overnight in selective polyamine-free glucose medium. The overnight culture was adjusted to A₆₀₀ 3.0/ml and serial dilutions were spotted (2 μl of aliquots) as indicated on polyamine-free selective galactose plates. The plates were then incubated for 3 days at 30 °C. The results shown are representative for multiple experiments.

(A) Stabilizing effect of lactacystin on ODC, antizyme 2 and AZI expression. HEK-293 cells were transiently transfected with either pcDNA3.1-ODC, pcDNA3.1-HA-AZ2, pcDNA3.1-Myc-AZI or pcDNA3.1 alone (control). Lactacystin (8 μM) or an equal volume of DMSO was added. Antizyme 2 was detected with an anti-HA antibody (clone 3F10). AZI was detected with an anti-Myc antibody (clone 9E10). ODC was detected using a polyclonal anti-ODC antibody. Proteins were visualized with ECL®Plus enhanced chemiluminescence. The immunoblots shown are representative for multiple experiments. (B) In vivo association of ODC, antizyme 2 (AZ2) and AZI. HEK-293 cells were transiently transfected with either pcDNA3.1-ODC and pcDNA3.1-HA-AZ2 or with pcDNA3.1-ODC, pcDNA3.1-HA-AZ2 and pcDNA3.1-Myc-AZI. Left panel: expression of ODC, HA-tagged antizyme 2 and Myc-tagged AZI in the lysate. Immunoblot analysis with anti-HA, ODC or anti-Myc antibody. Right panel: association of ODC and antizyme 2 in the absence or presence of AZI. HA-tagged antizyme 2 was immunoprecipitated with anti-HA affinity matrix. Antizyme 2 interacting proteins were detected with antibodies against ODC or Myc. The immunoblots shown are representative for multiple experiments.