Article Figures & Data
Figures
- Fig. 1.
Purification of NPA-binding proteins with Tyr-AP activity from Arabidopsis PM vesicles. A, Silver stained SDS-PAGE gels of Tyr-AP peak fractions (1 μg of total protein per lane) from each purification stage. PM, Solubilized PM vesicle proteins; S300, Sephacryl S300 gel permeation chromatography peak fraction; Q, Q resin anion-exchange peak fraction; NPA, NPA affinity fraction (350 mm KCl). B, Tyr-aminofluoromethylcoumarin (AFC) enzyme overlay visualization of AP activity of NPA-binding proteins analyzed by native PAGE. C, Periodic acid-Schiff's reagent (PAS) staining of NPA-binding glycoproteins. D, Elution profiles (50–300 mmKCl) of proteins from NPA affinity beads. Dotted line,A 280; solid line, Tyr-AP activity assayed fluorometrically with Tyr-AFC substrate. Protein peak fractions identified with Roman numerals. E, Silver-stained 4% to 20% (w/v) SDS-PAGE gel of combined NPA affinity fractions II and III (100 ng of total protein).
- Fig. 2.
Flavonoid binding of NPA affinity chromatography peak fractions (Fig. 1D). Aglycone flavonoid binding of solubilized PM and NPA affinity fraction proteins was detected fluorometrically with DPBA. Values shown are means and sd of three independent experiments.
- Fig. 3.
Multiple sequence alignments of AtAPM1 and AtAPP1 with orthologous animal proteins. Consensus sequences are shadowed in black; conserved sequences are in white boxes. Putatative catalytic sites are indicated by solid lines. Glycosylation sites are indicated by triangles. A, AtAPM1. Mouse PSA, Mouse puromycin-sensitive microsomal AP (Q11011); DmAPM, Drosophila melanogasterpuromycin-sensitive AP (AAG48733). B, AtAPP1. HuAPP1, Human cytosolic APP1 (AAH05126). Human membrane-bound APP2 (AAG28480) used to determine consensus alignment is not shown.
- Fig. 4.
AtAPM1 and AtAPP1 expression in adult and seedling tissue. A, Northern blots of adult tissue. Total RNA (7.26 μg) was extracted from roots (R), rosette leaves (RL), cauline leaves (CL), lower inflorescence stems (LI), upper inflorescence stems (UI), young flowers (YF), mature flowers (MF), and siliques (S). Blots were probed and visualized with digoxygenin (DIG)-labeled AtAPM1and AtAPP1 probes. rRNA is shown as gel loading control. B, Northern-blot analysis of AtAPM1 and AtAPP1 mRNA in 2- to 10-d-old seedlings. Ten micrograms of total RNA was extracted and probed with DIG-labeled probes.
- Fig. 5.
Immunolocalization of AtAPM1 and AtAPP1 in Arabidopsis. Western blots with polyclonal anti-AtAPM1 and AtAPP1 antibodies. In western blots utilizing the anti-AtAPP1 polyclonal antibodies, only protein bands that did not cross-react with pre-immune antisera are shown. A, Cellular partitioning of the native AtAPM1 and AtAPP1 proteins. Total protein (10 μg) from 5-d-old light-grown seedlings was used in all cases except lane P5.5, which used protein from 5.5-d-old seedlings. D, Cellular debris; S, soluble; M, microsomal; P5, PM proteins from 5-d-old seedlings; P5.5, PM proteins from 5.5-d-old seedlings. B, Microsomal preparations (10 μg of total protein) from adult tissues. R, Roots; L, rosette leaves; UI, upper inflorescence; LI, lower inflorescence; F, flowers. C, Western-blot analysis of Tyr-AP peak fractions from each purification stage with anti-AtAPM1 and AtAPP1 polyclonal antibodies. All samples are from 5-d-old light-grown Arabidopsis seedlings. Ten micrograms of total protein was used in lanes M, S, and Q. Two hundred nanograms of total protein was used in lane NPA. M, Microsomal; S, Sephacryl S-300; Q, Q anion-exchange; NPA, NPA affinity.
Tables
- Table I.
Specific AP and NPA amidase activities of PM proteins at each step of purification
Substrate PM Solubilized S300 Q Resin NPA Peak II NPA Peak III NPA Peak II + III nmol min−1 mg−1 Tyr-AFC 31.6 16.6 78.1 226.8 59.3 7.7 392.4 Ala-Pro-AFC 17.6 13.5 15.7 22.4 89.5 1.9 66.3 Trp-AFC 20.2 17.1 3.3 4.7 2.2 1.7 4.7 Pro-AFC 16.7 15.6 27.9 31.5 27.1 5.9 46.1 Ala-AFC 2.6 2.4 16.1 62.8 27.2 7.5 84.6 Leu-AFC 10.3 9.4 18.2 19.7 2.5 3.1 2.1 Ala-NA 2.1 1.6 2.7 31.6 32.1 18.4 45.7 NPA 6.3 4.4 5.8 25.1 17.1 9.5 42.1 Fractions (100 ng) from each purification step were assayed for activity after dialysis against AP assay buffer for 1 h at 4°C. AFC conjugate concentrations were 20 μm. Ala-β-naphthylamide (NA) and NPA concentrations were 100 μm. PM, PM-enriched membranes; Solubilized, detergent-solubilized PM proteins; S300, Sephacryl S-300 PMSF-insensitive peak fraction; Q resin, Q anion-exchange PMSF-insensitive peak fraction; NPA peaks I through III, fractions (or combinations of fractions) corresponding to NPA affinityA 280 peaks (Fig. 1D). All assays were repeated at least three times with similar results.
Substrate Control SA NPA (50 μm) EGTA (2 mm) PMSF (100 μm) Bestatin (20 μm) Amastatin (20 μm) Apstatin (20 μm) Puromycin (20 μm) PAQ22 (20 μm) Dithiothreitol (DTT; 5 mm) Vanadate (5 mm) Thiorphan (50 μm) nmol min−1 mg−1 % Tyr-AFC 381 ± 17.5 65 58 78 41 72 53 61 48 108 95 101 Ala-Pro-AFC 71 ± 7.7 76 84 71 84 87 26 87 98 69 93 97 Ala-AFC 89 ± 5.3 54 20 87 36 37 94 53 34 96 97 98 Pro-AFC 44 ± 9.2 81 79 79 58 88 61 66 99 73 90 97 Proteins from NPA affinity fractions II and III (Fig. 1D) were reconstituted by dialysis in AP assay buffer, then assayed fluorometrically for AP activity with AFC conjugates (20 μm) in the presence or absence of inhibitors. Values shown for inhibitor assays are percent control-specific activities (SA). Values calculated are derived from means and sd of three independent experiments. Table entries marked with an asterisk were significantly different from their control (P < 0.05) when analyzed by Student-Newman-Keuls ANOVA.
- Table III.
Identification of PM AP-associated proteins purified by NPA affinity chromatography
Purified Protein Post-translational Modifications Internal Fragment Sequenced GenBank Protein Identification No. Amino Acids (Predicted) Characterized Ortholog Similarity % Aminopeptidases FII-103 (AtAPM1) GP (E,P) (K)TLEVPTDLVALSN CAB36783 879 PS aminopeptidase M (mouse)/IRAP 53 FII-70 (AtAPP1) GP (E,P) (K)VSDEANSYFNGLG CAB16823 634 Aminopeptidase P1 (human) 60 FIII-42 (AtAPM1Δ) GP (E,P) (R)VATVVAHELAHQWF CAB36783 Unknown Truncated form of AtAPM1 ∼68 AP-associated proteins FIII-68 (AtMyA1) GP (P) (R)SVFASAALGK AAB63637 619 Myrosinase-associated protein (Brassica napus) 62 FII-55 (AtHSP70p) GP (E,P) (K)DVLK-(R)LVEHFAADEFNK CAB10440 484 HSP70 (Arabidopsis) ∼68 FII-44/66 (AtFAGP2) GPI; GP (E,P) (K)AFSDILKSTGADK CAB40990 403 Fasciclin-like arabinogalactan protein 2 (Arabidopsis) 59 FIII-40 (ATAPL1) GP (E,P) (K)LFVFGDSYADTGNI CAB82926 359 Anther-specific Pro-rich protein (Arabidopsis) 46 FIII-24 (AtGSTF2) GP (P) (K)NISQYAIMAIQ P46422 212 Auxin-binding GST II (Arabidopsis) 100 Amino acid sequences of tryptic fragments from PM proteins contained in NPA affinity peaks II and III were obtained as described (see “Materials and Methods”). Protein identifications were assigned by NPA affinity fraction (FII or FIII) and SDS-PAGE apparent molecular mass. GenBank protein identification nos. were assigned if there was a unique database match with a similar predicted molecular mass. GP, Glycoprotein; GPI, glycosyl-phosphatidylinositol anchor; E, experimental evidence; P, predicted. K or R, Inferred lysine or arginine residues.
Substrate/Inhibitor AtAPM1 AtAPP1 nmol min−1 mg−1 Tyr-AFC 59 ± 5.8 36 ± 4.9 Tyr-AFC + kaempferol 32 ± 3.1∗ 30 ± 5.5 Tyr-AFC +PAQ22 17 ± 5.4∗ 47 ± 3.1∗ Tyr-AFC + apstatin 54 ± 6.9 15 ± 4.7∗ Tyr-AFC +EGTA 16 ± 6.4∗ 19 ± 2.2∗ Ala-Pro-AFC 6 ± 4.2 74 ± 3.1 Ala-AFC 35 ± 4.2 5 ± 4.8 Pro-AFC 18 ± 5.3 29 ± 6.1 Trp-AFC 4 ± 3.2 20 ± 1.7 Leu-AFC 5 ± 3.1 7 ± 1.9 NPA 8 ± 3.4 3 ± 1.4 AP and amidase assays with substrates and inhibitors indicated. Inhibitor concentrations used were as in Table II. Units are nmol AFC or β-NA released min−1 mg−1. Values reported are means and sd of three independent experiments. Student-Newman-Keuls ANOVA indicates that the Tyr-AFC-related values reported are different from a normal distribution (P < 0.001). Values marked with an asterisk differ from controls in pair-wise comparisons (P < 0.05).
- Table V.
Specific [3H]NPA binding of PM vesicles, affinity fractions, and in vitro translation products of AtAPM1 and AtAPP1
Sample [3H]NPA Bound pmol mg−1 PM 0.1 ± 0.03 Fraction I 0.0 ± 0.03 Fraction II 7.1 ± 1.67 Fraction III 8.2 ± 1.25 Fraction IV 17.2 ± 3.07 Fraction II+ III 17.8 ± 2.52 Fraction II + III +APAb 5.9 ± 3.55∗ Fraction IV +APAb 16.8 ± 3.25 In vitro AtAPM1 3.2 ± 1.07 In vitro AtAPP1 1.9 ± 1.6 Specific activity is calculated as [3H]NPA binding − [3H]NPA binding in the presence of 1,000× cold NPA. Fractions I through IV, NPA affinity fractions I–IV; APAb, mixed polyclonal anti-AtAPM1 and AtAPP1 antibodies (see “Materials and Methods”); in vitro, in vitro translation products. Values reported are means and sd of three independent experiments. Student-Newman-Keuls ANOVA indicates that the values reported are different from a normal distribution (P < 0.001). Values with an asterisk differ from their control in pairwise Student's t tests (P < 0.01).
