Article Figures & Data
Figures
- Figure 1.
NLA Forms Homodimers via Its RING Finger Domain.
(A) Schematic diagrams of full-length NLA and its deletion derivatives used to test for NLA homodimerization. MBP or MBP-NLA was used as a bait protein and GST-NLA (amino acids 1 to 335), GST-SPX (amino acids 1 to 270), or GST-RING (amino acids 220 to 335) was used as a prey protein.
(B) In vitro pull-down assay of MBP-NLA and GST-NLA. Purified GST-NLA protein as prey was mixed with MBP or MBP-NLA as bait.
(C) In vitro pull-down assay of MBP-NLA and GST-SPX.
(D) In vitro pull-down assay of MBP-NLA and GST-RING. For (B) to (D), the reaction mix contained 1 μg each of prey and bait proteins. Pulled-down protein was analyzed by protein gel blot using anti-GST antibody.
- Figure 2.
UBC24 (PHO2) Is a Specific E2 Conjugase for NLA.
(A) In vitro pull-down assay of His-UBC8 with MBP-NLA full-length. His-UBC8 as prey was mixed with MBP or MBP-NLA as bait. His-UBC8 was analyzed by protein gel blot using anti-His antibody.
(B) In vitro pull-down assay of GST-UBC24 with MBP-NLA full-length. GST-UBC24 as prey was mixed with MBP or MBP-NLA as bait. GST-UBC24 was analyzed by protein gel blot using anti-GST antibody.
(C) UBC8 does not support NLA self-ubiquitination.
(D) UBC24 supports NLA autoubiquitination.
For (C) and (D), the reaction mix contained 25 ng UBE1 (rabbit E1), 100 ng UBC8 or UBC24 (E2), 500 ng MBP-NLA (E3), and 8 μg His-Ubiquitin. After the reaction, polyubiquitinated NLA was analyzed by protein gel blot using anti-MBP antibody.
- Figure 3.
UBC24 E2 Conjugase Is Required for PT2 Polyubiquitination by NLA E3 Ligase.
(A) and (C) In vitro pull-down assay of PT1 (C) and PT2 (A) with NLA. MBP or MBP-NLA as bait and GST-PT1 or GST-PT2 as prey were mixed. GST-PT1 or GST-PT2 was analyzed by protein gel blots using anti-GST antibody.
(B) and (D) In vitro ubiquitination of PT1 (D) and PT2 (B). The reaction mix contained 25 ng UBE1 (rabbit E1), 100 ng UBC8 or UBC24 (E2), 500 ng MBP-NLA (E3), 100 ng of substrate (GST-PT1-MYC or GST-PT2-MYC), and 8 μg His-Ubiquitin.
After the reaction, polyubiquitinated GST-PT1-MYC or GST-PT2-MYC was analyzed by protein gel blot using anti-MYC antibody.
- Figure 4.
NLA Interacts with PT2 and UBC24 in Arabidopsis.
NLA interaction with PT1 (C), PT2 (A), and UBC24 (B) in vivo was detected by coimmunoprecipitation with full-length NLA, PT1, and PT2 in double transgenic plants (35S:MYC-NLA/35S:PT1-HA, 35S:MYC-NLA/35S:PT2-HA, and 35S:MYC-NLA/35S:PHO2-HA). Pulled-down proteins were analyzed by protein gel blots using anti-HA antibody. PT1 was used as a negative control.
- Figure 5.
Pi Destabilizes PT2 Protein in Vivo.
(A) Wild-type transgenic plants expressing 35S:PT2 in 1 mM CHX were treated with (+) or without (−) 3.5 mM Pi.
(B) pho2 mutant plants expressing 35S:PT2 in 3.5 mM Pi and 1 mM CHX were treated with (+) or without (−) MG132.
(C) Wild-type transgenic plants expressing 35S:PT2 in 3.5 mM Pi and 1 mM CHX were treated with (+) or without (−) 50 μM MG132 (proteasome inhibitor). Plants were treated with 3.5 mM Pi and 1 mM CHX.
(D) Wild-type transgenic plants expressing 35S:PT2 in 3.5 mM Pi and 1 mM CHX were treated with or without 50 μM E64d (Cys protease inhibitor).
For (A) to (D), samples were taken at various time points, and PT2-HA protein levels were analyzed by protein gel blots using anti-HA antibody. Stained gel bands of the large subunit of Rubisco, rbcL, were used as a loading control.
- Figure 6.
NLA Targets PT2 but Not PT1 for Degradation in Vivo.
(A) Protein levels of PT1 were determined in wild-type transgenic plants carrying two transgenes, XVE:MYC-NLA (inducible) and 35S:PT1-HA. Plants were either treated or not with β-estradiol to induce NLA expression. Samples were taken at 0, 1, 3, and 6 h after induction, and the expression levels of PT1-HA and MYC-NLA were analyzed by protein gel blots. Stained gel bands of rbcL were used as a loading control. Two independent transgenic lines (no. 1 and 2) were analyzed.
(B) Wild-type transgenic plants carrying two transgenes, XVE:MYC-NLA (inducible) and 35S:PT2-HA, were used. Plants were treated with or without β-estradiol to induce the NLA E3 ubiquitin ligase. Proteins levels were analyzed by protein gel blots using anti-HA antibody. Stained gel bands of rbcL were used as a loading control. Two independent transgenic lines (no. 1 and 2) were analyzed.
- Figure 7.
NLA-Mediated Degradation of PT2 Is Dependent on PHO2 in Vivo.
pho2 mutant transgenic plants carrying two transgenes, XVE:MYC-NLA (inducible) and 35S:PT2-HA, were used. Plants were treated with or without β-estradiol to induce the NLA E3 ubiquitin ligase. Proteins levels were analyzed by protein gel blots using anti-HA antibody. Stained gel bands of rbcL were used as a loading control. Two independent transgenic lines (no. 1 and 2) were analyzed.
- Figure 8.
Interaction of NLA and PT2 on Plasma Membranes.
Fluorescent fusion genes (YFP-NLA, MYC2-YFP, nYFP, nYFP-NLA, cYFP, and cYFP-PT2) were transiently expressed in N. benthamiana leaves infiltrated with agrobacterial cultures. Confocal images were collected 3 days after infiltration. Experiments were repeated twice, and ∼25 to 30 cells were examined in each experiment. Bars = 20 μm.
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