The ubiquitin E3 ligase LOSS OF GDU2 is required for GLUTAMINE DUMPER1-induced amino acid secretion in Arabidopsis

Abstract

Amino acids serve as transport forms for organic nitrogen in the plant, and multiple transport steps are involved in cellular import and export. While the nature of the export mechanism is unknown, overexpression of GLUTAMINE DUMPER1 (GDU1) in Arabidopsis (Arabidopsis thaliana) led to increased amino acid export. To gain insight into GDU1's role, we searched for ethyl-methanesulfonate suppressor mutants and performed yeast-two-hybrid screens. Both methods uncovered the same gene, LOSS OF GDU2 (LOG2), which encodes a RING-type E3 ubiquitin ligase. The interaction between LOG2 and GDU1 was confirmed by glutathione S-transferase pull-down, in vitro ubiquitination, and in planta coimmunoprecipitation experiments. Confocal microscopy and subcellular fractionation indicated that LOG2 and GDU1 both localized to membranes and were enriched at the plasma membrane. LOG2 expression overlapped with GDU1 in the xylem and phloem tissues of Arabidopsis. The GDU1 protein encoded by the previously characterized intragenic suppressor mutant log1-1, with an arginine in place of a conserved glycine, failed to interact in the multiple assays, suggesting that the Gdu1D phenotype requires the interaction of GDU1 with LOG2. This hypothesis was supported by suppression of the Gdu1D phenotype after reduction of LOG2 expression using either artificial microRNAs or a LOG2 T-DNA insertion. Altogether, in accordance with the emerging bulk of data showing membrane protein regulation via ubiquitination, these data suggest that the interaction of GDU1 and the ubiquitin ligase LOG2 plays a significant role in the regulation of amino acid export from plant cells.

Figure 1.

Interaction assays between GDU family members and the E3 ubiquitin ligases of the LOG2 family. A, yeast two-hybrid interaction of the cytosolic domain of the GDU proteins (cGDU) with LOG2 and LULs. The panels show swapping of inserts between the bait (pGBT9) and prey (pACT2) plasmids. Yeast coexpressing the protein pairs were grown for 4 d on medium selecting for protein interaction, lacking Leu, Trp, adenine, and His. All yeast grew on a medium lacking Leu and Trp, selecting for the plasmids only (data not shown). B, GST pull-down (PD) assay of flag-cGDU1 or flag-cGDU1^G100R with GST-LOG2, GST-LUL1, or GST alone. Top, pulled-down samples; middle, cGDU input; bottom, GST protein input. Arrowheads indicate GST (approximately 27 kD) or full-length GST-tagged LOG2 and LUL1. C, Coimmunoprecipitation (IP) assay after the expression of GDU1 or GDU1^G100R and mLOG2 or mLOG2^R12K in transiently infiltrated N. benthamiana leaves. Top, Myc coimmunoprecipitation samples probed with α-HA (H); middle, Myc coimmunoprecipitation samples probed with α-Myc (M); bottom, HA-protein input. Asterisks and diamonds indicate LOG2 and GDU1 proteins, respectively. Numbers on the right in B and C indicate molecular mass in kD. ORF, Open reading frame; WB, western blot.

Figure 2.

Ubiquitin ligase activity tests of LOG2 and LULs. A, In vitro ubiquitination assay of GST-LOG2 and GST-LUL1 to -4 visualized with an anti-ubiquitin antibody. −E2 and −E3, Omission of ubiquitin-conjugating enzyme (E2) and the indicated ubiquitin ligase (E3) from the assay, respectively; T, all ubiquitin pathway components were present in the assay. B, In vitro ubiquitination assay of flag-cGDU1 by GST-LOG2, GST-LUL1, or no added E3, visualized with anti-flag antibody. C, In vitro ubiquitination assay of flag-cGU1 by truncated and mutant forms of GST-LOG2. ΔN, LOG2 truncation lacking amino acids N terminal to DAR2; M, LOG2^CC354/347AA (RING-dead); I-A, LOG2^I321A (RING-E2 interaction impaired). D, In vitro ubiquitination assay of either flag-cGDU1 or flag-cGDU1^G100R by LOG2-V5. The arrowhead indicates nonubiquitinated flag-cGDU1; the pound symbol indicates ubiquitinated flag-cGDU1. Numbers on the left indicate molecular mass in kD. WT, Wild type.

Figure 3.

LOG2 promoter-GUS analysis. A, Rosette. B, Leaf. C, Stem cross-section. D and E, Root. F, Flower. G, Stamen. H, Pollen grain. Con, Connective; Ph, phloem; St, style; Xy, xylem.

Figure 4.

Analysis of membrane localization and the association of GDU1 and LOG2. A and C, Subcellular fractionation of transgenic Arabidopsis expressing GDU1-Myc (A) or LOG2-HA (C). The fractionation of plasma membrane-localized PMA2 in respective preparations is shown at the bottom. CYT, Cytosolic fraction; TM, total microsomes. B, Sensitivity of microsomal GDU1-Myc expressed in Arabidopsis to 1 m NaCl, 0.1 m Na₂CO₃, pH 11.5, or 1% (v/v) Triton X-100. P, Pellet; S, supernatant. D, Subcellular fractionation of LOG2-HA and LOG2^G2A-HA after transient expression in N. benthamiana. E, Sensitivity of microsomal LOG2 and LOG2^G2A to 1 m NaCl, 0.1 m Na₂CO₃, pH 11.5, or 1% (v/v) Triton X-100 after transient expression in N. benthamiana. F, In vitro transcription/translation assay reactions incubated with either [³H]myristic acid (left) or [³H]Leu (right) using plasmids expressing either wild-type LOG2 or LOG^G2A. G, Coimmunoprecipitation (IP) after transient expression of GDU1-HA (H) and mLOG2-Myc or mLOG2^G2A-Myc (M) in N. benthamiana leaves using an anti-Myc antibody. Top, Myc coimmunoprecipitation samples probed with α-HA; middle, Myc coimmunoprecipitation samples probed with α-Myc; bottom, HA-protein input. Asterisks and arrowheads indicate LOG2 and GDU1 proteins, respectively. Numbers on the right indicate molecular mass in kD.

Figure 5.

Subcellular localization of GDU1, LOG2, and LOG2 variants. All proteins were transiently expressed in N. benthamiana epidermis cells. Images are maximum projections of optical sections of the abaxial side of cells, obtained by confocal microscopy. A, Localization of GDU1-GFP protein. B, Colocalization of GDU1-GFP and FYVE-RFP, an endosome marker. C, Localization of mLOG2-GFP, mLOG2^G2A-GFP, and mLOG2^R12K-GFP. D, Colocalization of mLOG^G2A with cytosolic mCherry. E, Change of localization of GDU1-GFP when coexpressed with mLOG2-mCherry. The middle cell (arrow) expressed mLOG2-mCherry at a low level, while the two lateral cells expressed mLOG2-mCherry at higher levels. All three cells expressed GDU1-GFP. The central fluorescence of the middle cell is potentially due to internal reflection. Bars = 20 μm (A), 5 μm (B) or 10 μm (C to E).

Figure 6.

Suppression of the Gdu1D phenotype by T-DNA insertion in the LOG2 gene. A, gdu1-1D was crossed to plants harboring a T-DNA in the first intron of LOG2 (log2-2). Five-week old F3 plants were recovered from F2 parents with the indicated genotypes. B, gdu1-1D, log2-2, and log2-2 gdu1-1D seeds were sown on medium containing 10 mm of the indicated amino acid or no added amino acid (top left plate). Each plate is oriented with quadrants as shown in the model above. Clockwise from top left: the wild type (WT), gdu1-1D, log2-2, and log2-2 gdu1-1D double homozygote. Experiments were repeated three or more times with 25 seeds from each line. Representative images are shown.

Figure 7.

Suppression of the Gdu1D phenotype by LOG2-directed amiRNAs in two different Gdu1D overexpression backgrounds. A, Phenotypic segregation on soil of T2 plants expressing LOG2-amiRNA in the gdu1-1D background. Bar = 5 cm. B, Growth of seedlings expressing one of two LOG2-amiRNAs in the gdu1-1D background was assessed for the presence of green, expanded cotyledons after 14 d of growth on 10 mm Met, Leu, and Phe. Error bars represent sd. Asterisks indicate significant differences from gdu1-1D alone (−) on the same medium (t test with Bonferroni correction for multiple comparisons; P < 0.01). Experiments were repeated three or more times with 25 seeds from each line. C, Phenotype of plants overexpressing LOG2-miRNAb in the 35S-GDU1-Myc background. D, For each line overexpressing LOG2-miRNAb in the 35S-GDU1-Myc background, the phenotype of about 10 progeny was recorded (WT, wild type; Inter., intermediate between Gdu1D and the wild type), GDU1 protein levels were estimated by western blotting using an anti-Myc antibody (top), and GDU1 and LOG2 mRNA contents were measured by quantitative RT-PCR. Accumulation is expressed relative to the levels in the wild type (Col-7), and error bars represent the results of two independent experiments. CTRL, Control (35S-GDU1-Myc background only, no amiRNA present). [See online article for color version of this figure.]

Figure 8.

Analysis of the effect of the log2-1 mutation on LOG2 protein properties. A, Complementation of the log2-1 mutation. A wild-type (WT) genomic fragment (8,511-bp XhoI-PstI from bacterial artificial chromosome F11F8) was cloned into a hygromycin resistance-conferring binary vector and inserted into the genome of the log2-1 gdu1-5D double mutant. GDU1 mRNA content was estimated by quantitative RT-PCR and is given as the double difference between the qPCR cycle threshold (Ct) of GDU1 and Actin2 mRNAs obtained in the wild type and the mutants (ΔΔCt). Errors bars are from two biological replicates. B, GST-LOG2 and GST-LOG2^R12K ubiquitination assays without substrate. C, GST pull-down assay using flag-cGDU1 and GST-LOG2 or GST-LOG2^R12K. D, Yeast two-hybrid interaction assay of LOG2^R12K with cGDU1, cGDU1^G100R, or ΔcGDU1. E, In vitro ubiquitination assay with LOG2-V5 or LOG2^R12K-V5 and flag-cGDU1. F, GDU1-Myc accumulation in 35S-GDU1-Myc and the 35S-GDU1-Myc log2-1 double mutant. Lane 1, Wild type; lane 2, 35S-GDU1-Myc; lane 3, 35S-GDU1-Myc log2-1. Numbers on the side of western blots indicate molecular mass in kD. [See online article for color version of this figure.]