FYVE1/FREE1 Interacts with the PYL4 ABA Receptor and Mediates Its Delivery to the Vacuolar Degradation Pathway

Abstract

Recently, we described the ubiquitylation of PYL4 and PYR1 by the RING E3 ubiquitin ligase RSL1 at the plasma membrane of Arabidopsis thaliana This suggested that ubiquitylated abscisic acid (ABA) receptors might be targeted to the vacuolar degradation pathway because such ubiquitylation is usually an internalization signal for the endocytic route. Here, we show that FYVE1 (previously termed FREE1), a recently described component of the endosomal sorting complex required for transport (ESCRT) machinery, interacted with RSL1-receptor complexes and recruited PYL4 to endosomal compartments. Although the ESCRT pathway has been assumed to be reserved for integral membrane proteins, we show the involvement of this pathway in the degradation of ABA receptors, which can be associated with membranes but are not integral membrane proteins. Knockdown fyve1 alleles are hypersensitive to ABA, illustrating the biological relevance of the ESCRT pathway for the modulation of ABA signaling. In addition, fyve1 mutants are impaired in the targeting of ABA receptors for vacuolar degradation, leading to increased accumulation of PYL4 and an enhanced response to ABA Pharmacological and genetic approaches revealed a dynamic turnover of ABA receptors from the plasma membrane to the endosomal/vacuolar degradation pathway, which was mediated by FYVE1 and was dependent on RSL1. This process involves clathrin-mediated endocytosis and trafficking of PYL4 through the ESCRT pathway, which helps to regulate the turnover of ABA receptors and attenuate ABA signaling.

Figure 1.

TAP/Mass Spectrometry and BiFC Analyses Reveal That PYL4 Interacts in Vivo with FYVE1. (A) Expression of GS-PYL4 in Arabidopsis cell suspension cultures and recovery of PYL4-interacting proteins following TAP and mass spectrometry analysis. Immunoblot analysis using an anti-GS antibody detects the GS-PYL4 bait in Arabidopsis protein extracts prepared from transformed suspension cells (left panel). Coomassie blue staining reveals the presence of S-PYL4 eluted after the second purification step plus accompanying proteins (right panel). The different sizes of GS-PYL4 and S-PYL4 are due to TEV cleavage of the G domain after the first purification step. (B) The structure of FYVE1 showing the N-terminal location of the IDR and the FYVE domain. Interaction of FYVE1 and PYL ABA receptors in Y2H assays was determined by growth assay on media lacking histidine and adenine. The presence of 50 μM ABA does not affect significantly the observed interaction. (C) BiFC interaction of ABA receptors and FYVE1 decorates endosomes and MVB/PVC. Photographs show epifluorescence confocal images of transiently transformed tobacco epidermal cells coexpressing FYVE1-YFP^N and YFP^C-PYL4 or PYR1. The merging of the fluorescent (left panels) and bright-field (BF) images reveals the subcellular location of the interaction in endosomal compartments. FYVE1-YFP^N does not interact with YFP^C-TOL9 in a BiFC assay (far right panels). Bars = 20 μm.

Figure 2.

Coexpression of Receptor-RSL1 Complexes with the Vacuolar Marker RFP-TMD23-Ub Reveals Transit of PYR1/PYL4 ABA Receptors to the Vacuole. (A) Colocalization of Receptor-RSL1 complexes with FYVE1. Epifluorescence confocal images were obtained 48 h after Agroinfiltration of tobacco epidermal cells with constructs encoding SCYAN^N-RSL1 and SCYAN^C-PYR1 or PYL4 plus FYVE1-GFP. Levels of colocalization for red boxed regions (n > 20) are depicted in relative intensity (x-, y axes) scatterplots. Values of Rp and Rs coefficients were calculated and are given next to scatterplots. Rp and Rs coefficients were greater than 0.6 for the endocytic vesicles analyzed. Bars = 30 μm. (B) Coexpression of Receptor-RSL1 complexes with the vacuolar marker RFP-TMD23-Ub. CLSM 3D projection through a full z-series of confocal images obtained 72 h after Agroinfiltration of tobacco epidermal cells with constructs encoding YFP^N-RSL1 and YFP^C-PYR1 or PYL4 plus RFP-TMD23-Ub. Plants were incubated in darkness for 4 h in order to promote stabilization of the fluorescent protein-tagged vacuolar marker and RSL1-receptor complexes. Bars = 30 μm. (C) Immunoblot analysis to monitor vacuolar delivery of GFP-PYL4. Seedlings expressing GFP-PYL4 were either mock- or E64-treated for 4 h. Protein extracts were analyzed by immunoblot using an anti-GFP antibody (upper panel) and Ponceau staining (middle panel) and were then quantified using Image Guache V4.0 software (lower panel). Bars show mean protein levels normalized to Rubisco protein. Values are averages ± se of three independent experiments. (D) Interaction of PYL4 and CAR1 generates punctate/globular structures in plasma membrane and cytosol that colocalize with RFP-FYVE1. Photographs show epifluorescence confocal images of transiently transformed tobacco epidermal cells coexpressing CAR1-YFP^N/YFP^C-PYL4 interacting proteins and RFP-FYVE1. Asterisks indicate the presence of CAR1-PYL4 in membrane complexes that colocalize with FYVE1. Levels of colocalization for regions labeled with an asterisk are depicted in relative intensity (x-, y axes) scatterplots. Values of Rp and Rs coefficients were calculated and are given next to scatterplots. pm, plasma membrane. Bars = 30 μm.

Figure 3.

BiFC Interaction of FYVE1-YFP^N with YFP^C-SNF7A Labels Globular-Shaped Structures Representing LEs/MVBs. (A) Epifluorescence confocal images of transiently transformed tobacco epidermal cells coexpressing FYVE1-YFP^N and YFP^C-Snf7A. The right panel shows a detail of the BiFC fluorescent signal. FYVE1-YFP^N does not interact with YFP^C-TOL4 in a BiFC assay. Bars = 20 μm. (B) Immunoblot analyses of protein extracts (20 μg of total protein) obtained from tobacco leaves infiltrated with the indicated constructs and revealed using anti-myc or anti-GFP^C antibodies. (C) Colocalization of GFP-FYVE1 and RFP-Snf7A (r = 0.64). BY-2 cells were cotransformed with GFP-FYVE1 and RFP-Snf7A gene constructs and the cells were processed, imaged, and analyzed by CLSM as described in Methods. Bar = 10 μm. (D) Cotransformation of GFP-FYVE1 and mCherry-Vps23A reveals colocalization in LEs/MVBs (r = 0.74). By contrast, GFP-FYVE1 and myc-TOL proteins do not colocalize significantly (r = 0.3). Bar = 10 μm.

Figure 4.

Localization of PYL4 in Endosomal Compartments. (A) GFP-PYL4 is localized mostly in the nucleus (N) and cytosol of tobacco leaf cells after Agroinfiltration. A minor fraction of GFP-PYL4 decorates endosomal vesicles (asterisks) in 1-d dark-adapted tobacco plants. Bars = 30 μm (B) Epifluorescence confocal images of transiently transformed tobacco epidermal cells coexpressing FYVE1-GFP and RFP-PYL4 (upper panels). Time-course photographs show comigration of RFP-PYL4 and FYVE1-GFP in endosomal compartments (lower panels). Bars = 15 μm. (C) GFP-PYL4 accumulates in BFA compartments and shows colocalization with the endocytic marker FM4-64. Rp and Rs coefficients were greater than 0.7 for the BFA bodies analyzed (n > 20) by CLSM of root epidermal cells from Arabidopsis transgenic lines expressing GFP-PYL4. Four-day-old seedlings were labeled with 4 µM FM4-64 for 10 min, followed by 50 µM BFA treatment for 1 h. After washing in MS medium for 2 h, GFP-PYL4 redistributes and appears localized mostly in the nucleus and cytosol. Arrows mark BFA bodies. Bars = 10 μm. (D) Colocalization of GFP-PYL4 and mCherry-ARA7 in WM-induced compartments. CLSM of root epidermal cells from seedlings coexpressing GFP-PYL4 and the LE-marker mCherry-ARA7 were analyzed after either a mock or a 33 µM WM treatment for 1 h. The 5× enlarged images (dotted boxes) were used for statistical analysis of GFP-PYL4 and mCherry-ARA7 colocalization in WM compartments. The intensity profiles of GFP (green) and mCherry (magenta) fluorescence were measured along the indicated distance (microns). Bars = 10 μm.

Figure 5.

Clathrin-Mediated Endocytosis of PYL4. (A) Coimmunoprecipitation of HA-PYL4 with CHC protein is inhibited by TyrA23. Anti-CHC1,2 antibody (Agrisera 10690) was used to immunoprecipitate CHC from Arabidopsis protein extracts (1 mg of total protein each) prepared from plants expressing 3HA-PYL4. Protein extracts were prepared from plants that were treated with 50 μM MG132 or 50 μM MG132+50 μM TyrA23 for 6 h. Input levels of CHC and HA-PYL4 in crude protein extracts (20 μg of total protein) were analyzed by immunoblotting. Immunoprecipitated CHC protein was probed with anti HA-HRP antibody to detect coimmunoprecipitation of PYL4. (B) Colocalization of GFP-PYL4 with CLC2-mOrange in clathrin-coated vesicles at the plasma membrane. Epifluorescence confocal images were obtained 48 h after agroinfiltration of tobacco epidermal cells with constructs encoding GFP-PYL4 and CLC2-mOrange (Konopka et al., 2008). Pearson’s and Spearman’s correlation coefficients indicate colocalization of PYL4 and CLC2. Bars = 20 μm.

Figure 6.

Reduction-of-Function fyve1 Alleles Cause Enhanced Sensitivity to ABA. (A) Photographs of Col wild type, fyve1-3/fyve1-4 alleles, and the hab1-1abi1-2 ABA-hypersensitive mutant grown for 7 d on MS medium either lacking or supplemented with 0.5 μM ABA (left panel) and quantification of ABA-mediated inhibition of seedling establishment in the indicated backgrounds (right panel). Values are averages ± se of three independent experiments (n > 100). Asterisk indicates P < 0.05 (Student’s t test) compared with the wild type in the same assay conditions. Bar = 1 cm. (B) Heterozygous fyve1-1 seedlings show enhanced ABA-mediated inhibition of seedling establishment and growth compared with the wild type. Photographs of Col wild-type and fyve1-1 plants grown for 13 d on MS medium either lacking (left panel) or supplemented with 0.5 μM ABA (middle panel). Quantification of seedling establishment at 7 d (right panel). Values are averages ± se of three independent experiments (n > 100). Asterisk indicates P < 0.05 (Student’s t test) compared with the wild type in the same assay conditions. Bars = 1 cm (C) Enhanced ABA-mediated inhibition of root growth in (+/−) fyve1-1 and fyve1-3 mutants compared with Col wild type. Seedlings (5 d old) germinated on MS plates were transferred to new plates lacking or supplemented with 10 μM ABA. (+/−) fyve1-1 seedlings were genotyped a posteriori (left and middle panels). Quantification of root growth after 10 d (right panels). Data are averages ± se from three independent experiments (n = 20). Asterisk indicates P < 0.05 (Student’s t test) compared with the wild type in the same assay conditions. Bars = 1 cm. (D) Enhanced ABA-mediated inhibition of seed germination in (+/−) fyve1-1 seed progeny and the fyve1-3 mutant compared with Col wild type. Values are averages ± se of three independent experiments (n > 100). Asterisk indicates P < 0.05 (Student’s t test) compared with the wild type in the same assay conditions. (E) Enhanced expression of the ABA-responsive genes RAB18 and RD29B in (+/−) fyve1-1 seedlings and fyve1-3 mutant compared with the Col wild type. mRNAs were prepared from 10-d-old seedlings of Col-0 wild type, fyve1-1 heterozygous (+/−) individuals, and fyve1-4 homozygous mutant and expression of RAB18 and RD29B in response to endogenous ABA was quantified in real-time quantitative PCR experiments. Data are averages ± se from three independent experiments (n = 20). Asterisk indicates P < 0.05 (Student’s t test) compared with the wild-type in the same assay conditions. (F) Diminished water loss in the fyve1-3 mutant compared with Col wild type. Photographs show representative excised plants submitted for 2 h to the drying atmosphere of a flow laminar hood. Quantification of the loss of fresh weight in 15-d-old plants submitted to the drying atmosphere of a flow laminar hood. The graphic shows a polynomial fitting of the water loss kinetics. Data are averages ± se from three independent experiments (n = 5). Bar = 1 cm.

Figure 7.

fyve1 Mutants Show Enhanced Accumulation of Ubiquitylated PYL4. (A) GFP-PYL4 decorates endocytic vesicles in fyve1-3. Asterisks (far right panels) indicate endocytic vesicles labeled by FM4-64 that contain GFP-PYL4. Epifluorescence confocal images of Arabidopsis root cells expressing GFP-PYL4 in wild-type or fyve1-3 background stained with FM4-64. Bars = 15 μm. (B) GFP-PYL4 is found in WM-induced rings of wild-type seedlings. Upon WM treatment, in wild-type seedlings GFP-PYL4 is observed in late endocytic vesicles decorated by LysoTrackerRed (indicated by asterisks), whereas GFP-PYL4 is absent from late endocytic compartments in fyve1-3. Bars = 20 μm. (C) fyve1-3 accumulates more PYL4 than the wild type and contains more ubiquitylated PYL4. Immunoblots with anti-HA, anti-RBC, and anti-ubiquitin P4D1 antibody from total protein extracts (left panel). Immunoblot with anti-HA antibody from ubiquitylated proteins pulled down using p62-agarose (right panel). Ubiquitylated HA-PYL4 was pulled down using p62-agarose from Col wild-type or fyve1-3 protein extracts, and anti-HA immunoblotting was performed to detect HA-tagged ubiquitylated PYL4 (indicated by the open arrowhead). (D) FREE1 DEX-RNAi plants induced with DEX accumulate more GFP-PYL4 than mock-treated plants and contain more ubiquitylated GFP-PYL4. Immunoblots with anti-GFP, anti-FREE1, and anti-FBP antibody from total protein extracts (left panel). Immunoblot analysis with anti-GFP and anti-Ub antibody from proteins that were immunoprecipitated using GFP-Trap (right panel).

Figure 8.

A Proposed Model of the Endosomal Trafficking of ABA Receptors and the Role of FYVE1 in Recruiting Ubiquitylated PYL4. Ubiquitylation of PYL4 in the plasma membrane by RSL1 acts as a trigger for endocytosis. FYVE1 recognizes ubiquitylated PYL4 in the RSL1-PYL4 complex and recruits it to the ESCRT pathway, promoting degradative sorting of PYL4 at MVB/PVC. MVBs gain competence to fuse with the vacuole, where the RSL1-PYL4 complex is degraded. fyve1 mutants are impaired in the targeting of PYL4 to the MVB/PVC for vacuolar degradation, which increases the half-life of PYL4 and leads to an enhanced response to ABA. Our model predicts that the ESCRT machinery is required for the turnover of ABA receptors; hence, additional ESCRT components participate in degradative sorting of ABA receptors. The possible participation of deubiquitinating enzymes (DUBs) in this pathway as well as the pharmacological interference with TyrA23, BFA, WM, E64, and ConA at different steps of endocytosis, endosomal trafficking, and vacuolar degradation is indicated.