Chennan Ge1,2, †, Caiji Gao3,4, †, Qingguo Chen2, Liwen Jiang3 and Yunde Zhao 2*

1.	National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, 430070, China
2.	Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA 92093-0116, USA
3.	School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
4.	Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou 510631, China
ESCRT-dependent Vacuolar Sorting and Degradation of the Auxin Biosynthetic Enzyme YUC1 flavin monooxygenase

SUPPLEMENTARY METHODS

Materials
Organelle markers and vesicle trafficking dominant mutants for protoplasts transient expression: Aleurain-RFP, Man1-RFP, RFP-VSR2, RFP-ARA7Q69L, VPS4E232Q, VPS24.1-1-152-myc, SAR1 and the Arabidopsis transgenic marker lines: mCherry-SYP32 (Golgi), mCherry-Rha1 (PVC), VHAa1-mRFP (TGN), mCherry-VAMP711 (Tonoplast) were obtained from Prof. Liwen Jiang’s lab.
Plasmids construction
For GFP-YUC1, YUC1-RFP, GFP-TAA1, TAA1-RFP transient expression, the cDNAs coding the YUC1 and TAA1 were cloned into the pBI221 vector under the 35S promoter with GFP or RFP in the C-terminal or N-terminal. 
To generate the Arabidopsis YUC1 inducible line, GFP-YUC1 coding sequence was introduced after the XVE system in the pER8 plasmid. To test the biological function of GFP-YUC1, the GFP-YUC1 coding sequence from pBI221-GFP-YUC1 was cloned and inserted into the Kpn I site of pCHF3 under a 35S promoter. Transgenic plants were obtained after agrobacterium Agrobacterium-mediated floral dipping transformation. For induction of GFP-YUC1 in transgenic plants, 7 days old transgenic plants were treated with 10 μM ß-estradiol for 16 hours before confocal microscope imaging and Western blotting. For auxin overproduction phenotype capturing, XVE::GFP-YUC1 seeds were germinated on 0.01 μM ß-estradiol containing MS plates for 7 days.
Transient Expression in Protoplasts and Confocal Imaging
Protoplasts isolation from dark type Arabidopsis culture and protoplasts transient expression methods were described previously (Zeng et al. 2015). Confocal fluorescent images were acquired 16 to 18 h after transformation in most of the experiments or 6 to 8 h during RFP-ARA7(Q69L) co-expression. Pictures were taken using Leica SP8 confocal system. For each experiment or construct, more than 30 individual cells were observed for confocal imaging that represented more than 70% of the cells showing similar patterns. Images were processed using Adobe Photoshop.
Protein Preparation and Immunoprecipitation
For protein extraction from Arabidopsis protoplasts and total protein extraction from plants, detailed protocols have been mentioned before (Zeng et al. 2015). 
Immunoprecipitation
Total protoplasts cell lysates were prepared in IP buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5 mM EDTA, 0.2% Nonidet P-40, 5% glycerol, 1 mM dithiobis and 1×Complete Protease Inhibitor Cocktail) and were then incubated with GFP-Trap agarose beads (ChromoTek) for 6 h at 4°C. The beads were washed four times with ice-cold washing buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.5 mM EDTA) after incubation, and then eluted by boiling in reducing SDS sample buffer. Samples were separated by SDS-PAGE and analyzed by immunoblot using appropriate antibodies.
Antibodies
GFP antibody was purchased from Abmart (Product No. M20004). FBPase antibody was purchased from Agrisera (Cat No. AS04 043). Ubiquitin antibody was purchased from Santa Cruz Biotechnology (Ub P4D1: sc-8017).
Drug treatments
For Wortmannin (Sigma W1628-1MG) treatment, Wortmannin was prepared as 1.65mM stock, and diluted to 1:500 with 10μM ß-estradiol in PVC enlargement experiment. For IAA (Sigma CAS-No.6505-45-9) treatment, protoplasts were cultured in different concertations of IAA after transient expression for 16 hours.
Supplemental References
Chen Q, Dai X, De-Paoli H, Cheng Y, Takebayashi Y, Kasahara H, Kamiya Y, Zhao Y (2014) Auxin overproduction in shoots cannot rescue auxin deficiencies in Arabidopsis roots. Plant Cell Physiol 55: 1072-1079
Zeng Y, Chung KP, Li B, Lai CM, Lam SK, Wang X, Cui Y, Gao C, Luo M, Wong KB, Schekman R, Jiang L (2015) Unique COPII component AtSar1a/AtSec23a pair is required for the distinct function of protein ER export in Arabidopsis thaliana. Proc Natl Acad Sci U S A 112: 14360-14365

Figure S1. Predicted localization of Arabidopsis YUC1
(A) Predicted Signal peptide in Arabidopsis YUC1 amino acid sequence using SignalP-4.1. C-score is the predicted cleavage site value, S-score is the predicted signal peptide value and Y-score is the cleavage site value calculated by combining C-and S-scores. No signal peptide predicted in YUC1. 
(B) YUC1 topology predication using the TMHMM program. No typical N-terminal hydrophobic transmembrane domains (TMD) predicted in the YUC1 amino acid sequence using TMHMM.

Figure S2. Subcellular localization of the Arabidopsis auxin biosynthesis enzymes in protoplasts. 
(A) GFP-YUC1 and YUC1-RFP fusion are localized to vacuole and in a punctate organelle. Scale Bar = 10 μm. 
(B) GFP-TAA1 and TAA1-RFP are localized in cytosol. Scale Bar = 10 μm. 
(C) GFP alone is localized in both cytosol and nucleus in Arabidopsis protoplasts. Scale Bar = 10 μm. 
(D) Overexpression of YUC1-GFP fusion caused auxin overproduction phenotypes: elongated hypocotyls, epinastic cotyledons, and short roots, suggesting that YUC1-GFP is functional.

Figure S3. Organelle co-localization analysis of YUC1. 
(A) GFP-YUC1 is not co-localized with the Golgi marker in Arabidopsis protoplasts. Scale Bar = 10 μm. 
(B) Development of an inducible GFP-YUC1 expression system.  The GFP-YUC1 fusion was under the control of a synthetic promoter XVE (Chen et al. 2014).  Upon induction by adding estradiol, GFP-YUC1 was expressed. 7 days XVE::GFP-YUC1 seedlings germinated on 0.01 μM estradiol containing MS plates had long hypocotyls, short root, and epinatic cotyledons, which are characteristic auxin overproduction phenotypes. 
(C) Estradiol induced production of GFP-YUC1 was not co-localized with mCherry-SYP32 (Golgi), VHAa1-mRFP (TGN) or mCherry-VAMP711 (Tonoplast) markers in Arabidopsis plants. Scale Bar = 10 μm. 
(D) Estradiol induced GFP-YUC1 displayed cytosolic and punctate signals in the XVE::GFP-YUC1 transgenic plants. Wortmannin treatments led to an enlargement of the GFP-containing punctate structures. Scale Bar = 10 μm.

Figure S4. The Effects of VPS24.1-152 in regulating GFP-YUC1 localization.  
Arabidopsis protoplasts expressing GFP-YUC1 and GFP-YUC1 + VPS24.1-152 were analyzed with a confocal microscope after 12h and 16h incubation. The cell numbers of different GFP-YUC1 localization cell types (cytosol and vacuole) were counted. The numbers of cells were shown in the histograms and the graphs represent the percentage of different cell types in each transient expression.

Figure S5. Western Blot analysis of GFP-YUC1. 
(A) XVE::GFP-YUC1 plants were induced with 10µM estradiol for 0.5, 6, 7, 8, 12 hours and proteins were extracted. Protein extracts were analyzed by Western Blot using the commercial anti-GFP antibody. Blotting with the Actin antibody was used as the loading control. 
(B) Arabidopsis protoplasts expressing GFP-YUC1 were treated with a series of IAA concentrations for 16 hours. Protein extracts were analyzed by WB using anti-GFP antibody and Ponceau staining as loading control.