PHT1;2-CFP YFP-PHF + PHT1;2-CFP YFP-PHF

Transcription

1 YFP-PHF1 CFP-PHT1;2 PHT1;2-CFP YFP-PHF + PHT1;2-CFP YFP-PHF + CFP-PHT1;2 Negative control!-gfp Supplemental Figure 1: PHT1;2 accumulation is PHF1 dependent. Immunoblot analysis on total protein extract corresponding to transient expression in N. benthamiana leaf epidermis 48h postinfiltration presented in figure 2. Protein detection is done using anti-gfp antibodies recognizing YFP-PHF1 (75kDa dark arrow) or CFP-PHT1;2 fusion proteins (90kDa white arrow). Coomassie blue acrylamide gel staining is shown as protein loading control. 1

2 PHF1-GFP ST-mRFP Merging D E F BFA 50!M 120min PHF1-GFP ST-mRFP Merging Supplemental Figure 2: Strict ER localization of PHF1. (A-F) A. thaliana epidermal root cells expressing PHF1-GFP under control of its own promoter (A) and Golgi marker ST-mRFP (B). (C-D) same line with BFA treatment (50!M, 2 hours). Scale bar:10!m. 2

3 YFP-channel Supplemental Figure 3: PHT1;2-CFP export from ER is COPII-dependent (A-L) Transient protein expression in N. benthamiana epidermal cells analyzed by confocal microscopy 48h post-infiltration. Transient expression of PHT1;2-CFP alone (B) showing weak plasma membrane fluorescence, together with Sec12-YFP (D-I) blue fluorescence is retained in ER structure with Sec12-YFP. Co-expression of PHT1;2-CFP together with YFP-PHF1 (J-L) shows an increase of blue fluorescence, colocalization at the ER and correct targeting of PHT1;2-CFP to the plasma membrane. Scale bars are 10µm. 3

4 Supplemental Figure 4: PHF1 is not associated with recruitment of COPII components to ERES. (A-L) Transient protein expression in N. benthamiana epidermal cells analyzed by confocal microscopy 48h post-infiltration. Transient expression ERES marker YFP-Sec24 expressed alone (B), or together with ST-CFP (G-I), PHT1;2-CFP (J- L) or CFP-PHF1 (J-L). YFP-Sec24 mainly localizes to cytoplasm and is recruited to ERES (white arrows). Scale bars are 10µm. 4

5 CFP-channel Sar1-YFP Merging CFP-PHF1 Sar1-YFP Merging G H I PHT1;2-CFP YFP-channel Merging J K L PHT1;2-CFP Sar1-YFP Merging Supplemental Figure 5: Co-expression of PHF1 and PHT1;2-CFP fusion protein together with ERES marker SAR1. (A-L) Transient protein expression in N. benthamiana epidermal cells analyzed by confocal microscopy 48h post-infiltrations. Transient expression of SAR1-YFP alone (B) or together with CFP-PHF1 (D-F) or PHT1;2-CFP (J-L). Transient expression PHT1;2-CFP alone is indicated as control (G-I) Scale bars = 10!M. 5

6 Supplemental Figure 6: MS/MS fragmentation spectrum of the diphosphopeptide SLEEL(pS)GEAEV(pS)HDEK (PHT1.1). Attributed y and b ions are indicated on the spectrum. Double Neutral Loss (NL) was present. S*: identified phosphorylated serine after neutral loss. 6

7 Control treatment M Control treatment PHT1;1-GFP 0!M Pi 500!M Pi N r =0,51±0,09 r = 0,53 SNX1-mRFP PHT1;1-GFP r = 0,55 SNX1-mRFP 0!M Pi 500!M Pi r r =0,0±0,034 r =0,53±0,09 r r =0,0±0,05 Supplemental Figure 7: Sorting endosomes localization of PHT1;1 independently of Pi external content. A. thaliana root tip cells co-expressing PHT1;1- GFP and the sorting endosome marker SNX1- mrfp, 60 minutes after 33!M Wm treatment. Plants were cultivated on Pi depleted medium (A-F) or Pi (500!M) containing medium (G-L) prior to treatment. (M) Quantitative analysis of intracellular colocalization between PHT1;1-GFP and SNXmRFP, cytofluorogram obtain for two images, (N) average Pearson s coefficient (r) and after Coste randomization based colocalization (r r ) are given. Scale bars = 5!M. The Pearson s coefficient was calculated based on the analysis of 350 root tip cells from 20 plants per condition. 7

8 A CHX 50!M T0 B T2 C T7 Control treatment D T7 -P E T0 F T2 G T7 H T7 +P Supplemental Figure 8: Kinetic analysis of Pi-induced PHT1 degradation. A. thaliana root tip cells co-expressing PHT1;1-GFP cultivated on medium containing 0!M (A-D) Pi or 500!M (E-H). (A and E) prior to drug treatment; (B and F) 2 hours and (C and G) 7 hours after CHX (50!M) treatment. (H and L) control untreated after 7 hours. Scale bars are 10!m 8

9 A relative fluorescence (AU) Inhibition 26S proteasome test P500 B relative fluorescence T0 2h CHX 6h MG132 2h CHX Effect of pho2-1 mutation on PHT1;1:GFP degradation P500 T0 2h CHX 0 PHT1;1 pho2 N 1 pho2 N 2 Supplemental Figure 9: Pi induced PHT1 degradation is not affected by MG-132 treatment or pho2-1 mutation. Relative fluorescence intensity of PHT1;1-GFP observed in root of plant grown on +Pi (500!M). (A) Effect of MG132 treatment (6h, 50!M), CHX was added during the two last hours (50!M). (B) Effect of pho2-1 mutation, PHT1;1-GFP marker was introgressed into pho2-1 mutant and treated with CHX drugs as previously described. For each experiment, 12 lateral roots were observed. The fluorescence from 20 (A) or 25 (B) regions of interests for each sample was quantified. The mean and standard deviation from the measurements are indicated here for one of the experiments performed. 9

10 A T0 B T15min C T60min 0!M Pi 500!M Pi D T0 E T15min F T60min Supplemental Figure 10 : Phosphate starvation does not affect internalization of endocytic tracer FM4-64 in root cells. Seedlings cultivatedeither in 500!M Pi (A to C) or Pi deprived (D to F) medium were subjected to kinetic analysis of FM4-64 internalization. Short time (A, B, D and E) shows endocytic tracer at the plasma membrane and endosomes, longer time shows tonoplast staining (C and F). Scale bar=5!m 10

11 Table 1 : Primers used for amplification and site-directed mutagenesis of PHT1;1 forward primer reverse primer PHT1;1 cdna 5'-CACCATGGCCGAACAACAACTAGG-3' 5'-TTTCTCGTCATGGCTAACCTCA-3' PHT1;1-S148A 5'-GTGACTACCCACTTGCTGCCACCATCATGTC-3' 5'-GACATGATGGTGGCAGCAAGTGGGTAGTCAC-3' PHT1;1-S148D 5'-GTGACTACCCACTTGATGCCACCATCATGTC-3' 5'-GACATGATGGTGGCATCAAGTGGGTAGTCAC-3' PHT1;1-S153A 5'-TGCCACCATCATGGCTGAATACGCAAACA-3' 5'-TGTTTGCGTATTCAGCCATGATGGTGGCA-3' PHT1;1-S153D 5'-TGCCACCATCATGGATGAATACGCAAACA-3' 5'-TGTTTGCGTATTCATCCATGATGGTGGCA-3' PHT1;1-T160A 5'-CGCAAACAAGAAGGCCCGTGGGGCTTTCA-3' 5'-TGAAAGCCCCACGGGCCTTCTTGTTTGCG-3' PHT1;1-T160D 5'-CGCAAACAAGAAGGACCGTGGGGCTTTCA-3' 5'-TGAAAGCCCCACGGTCCTTCTTGTTTGCG-3' PHT1;1-T239A 5'-GAAGATGCCTGAAGCTGCCCGTTACACCG-3' 5'-CGGTGTAACGGGCAGCTTCAGGCATCTTC-3' PHT1;1-T239D 5'-GAAGATGCCTGAAGATGCCCGTTACACCG-3' 5'-CGGTGTAACGGGCATCTTCAGGCATCTTC-3' PHT1;1-T368A 5'-GCGTTTATTGATGCCATTGGAAGGTTT-3' 5'-AAACCTTCCAATGGCATCAATAAACGC-3' PHT1;1-T368D 5'-GCGTTTATTGATGACATTGGAAGGTTT-3' 5'-AAACCTTCCAATGTCATCAATAAACGC-3' PHT1;1-S438A 5'-GGCCAGGCTAAGGGCTACATGTCATGGAA-3' 5'-TTCCATGACATGTAGCCCTTAGCCTGGCC-3' PHT1;1-S438D 5'-GGCCAGGCTAAGGGATACATGTCATGGAA-3' 5'-TTCCATGACATGTATCCCTTAGCCTGGCC-3' PHT1;1-S509A 5'-GCCCAAAGGCAAGGCCCTTGAAGAACTCT-3' 5'-AGAGTTCTTCAAGGGCCTTGCCTTTGGGC-3' PHT1;1-S509D 5'-GCCCAAAGGCAAGGACCTTGAAGAACTCT-3' 5'-AGAGTTCTTCAAGGTCCTTGCCTTTGGGC-3' PHT1;1-S514A 5'-CCTTGAAGAACTCGCTGGTGAGGCTGAGG-3' 5'-CCTCAGCCTCACCAGCGAGTTCTTCAAGG-3' PHT1;1-S514D 5'-CCTTGAAGAACTCGATGGTGAGGCTGAGG-3' 5'-CCTCAGCCTCACCATCGAGTTCTTCAAGG-3' PHT1;1-S520A 5'-TGAGGCTGAGGTTGCCCATGACGAGAAA-3' 5'-TTTCTCGTCATGGGCAACCTCAGCCTCA-3' PHT1;1-S520D 5'-TGAGGCTGAGGTTGACCATGACGAGAAA-3' 5'-TTTCTCGTCATGGTCAACCTCAGCCTCA-3' The CACC sequence allowing a directional cloning of PHT1;1 cdna into pentr is shown in bold. For site-directed mutagenesis, the subsitution sites for Ser and Thr in the primers to generate the mutations are underlined. 11