Supplemental Data. Farmer et al. (2010) Plant Cell /tpc

Transcription

1 Supplemental Figure 1. Amino acid sequence comparison of RAD23 proteins. Identical and similar residues are shown in the black and gray boxes, respectively. Dots denote gaps. The sequence of plant Ub is included for comparison. The numbers at the beginning and end of each row refer to the amino acid position in Arabidopsis RAD23a. The residue length of each protein is listed at the end of each sequence. The locations of the UBL, UBA and STI1 sequences are indicated by the solid, dashed and dotted lines, respectively. The positions of the various T-DNA insertion mutants are located by the open arrowheads. The conserved lysine residues between Ub and the RAD23 UBL domains are denoted by solid arrowheads. The asterisks and diamonds demarcate the residues used to bind RPN10 (Leu8, Ile44, Val70) and Ub (MGF/G), respectively. Arabidopsis thaliana (At), Oryza sativa (Os), Saccharomyces cerevisiae (Sc). 1

2 Supplemental Figure 2. Phylogenetic comparison of plant RAD23 proteins. Phylogenetic tree was generated with RAD23 protein sequences from Arabidopsis thaliana (At), Daucus carota (Dc), Oryza sativa (Os), Physcomitrella patens (Pp), Populus trichocarpa (Pt), Saccharomyces cerevisiae (Sc), Selaginella moellendorffii (Sm), Vitis vinifera (Vv), and Zea mays (Zm). Bootstrap values are from no less than a thousand replicates, and the tree is rooted at the midpoint. The bar represents the branch length equivalent to 0.05 amino acid changes per residue. 2

3 Supplemental Figure 3. Specificity of the anti-rad23 antibodies. Panels show five-fold serial dilutions of extracts from E. coli induced to express recombinant 6His- RAD23b and c proteins. Uninduced (UI) lane contains lysates from bacterial cultures not treated with IPTG. (Top Panel) Immunoblot using anti-rad23b antibodies. (Middle Panel) Immunoblot using anti-rad23c antibodies. (Lower Panel) SDS-PAGE gel stained for total protein with silver nitrate. Arrowheads locate the positions of RAD23b and RAD23c. 3

4 Supplemental Figure 4. Nuclear enrichment of RAD23a-d proteins in Arabidopsis. (A) Subcellular localization of RAD23a-d transiently expressed by their native promoters as GFP fusions in protoplasts. Protoplasts were prepared from 2-week-old leaves and imaged by confocal fluorescence microscopy 24 hr after transfection with plasmids encoding GFP alone or fused inframe to the N-terminus of each RAD23 isoform. A protoplast transiently expressing GFP by the 35S promoter is included as a control. Green, GFP. Red, chloroplasts. Arrowheads identify nuclei. Bars represent 5 µm. (B) Co-localization of GFP-RAD23b with nuclei. Elongation zone of a rad23b-1 root stably expressing 35S-GFP-RAD23b was visualized by confocal fluorescence microscopy for GFP and for nuclei using the DNA stain Vybrant DyeCycle Orange. BF, bright field image merged with the fluorescent images. (C) Detection of stably expressed GFP-RAD23b in the nuclear (N)- and cytoplasm (C)-enriched fractions. The fractions were prepared from 1-week-old 35S-GFP-RAD23b rad23b-1 seedlings by Percoll gradient centrifugation and subjected to SDS-PAGE and immunoblot analyses with anti- GFP antibodies. Immunoblot analyses with anti-histone H3 (H3) and anti-rpn12a antibodies were used to verify enrichment of nuclei and the presence of the 26S proteasome, respectively. Cr, crude extract. 4

5 Supplemental Figure 5. Mature rad23a-1, rad23c-1, and rad23d-1 mutants resemble wildtype plants. Representative 8-week-old wild-type and single and double rad23 mutants grown under LD. 5

6 Supplemental Figure 6. Ub-conjugate profiles in the combinatorial rad23 mutants. Immunoblot analyses were performed with anti-ub antibodies on crude extracts from 4-week-old wild-type (WT) and rad23 single and combinatorial mutant seedlings. Immunoblot analyses with anti-pba1 antibodies were included to show equal protein loading. Arrowheads indicate mono-ub, and di- (Ub 2 ), tri- (Ub 3 ), tetra- (Ub 4 ), and penta-ub (Ub 5 ) chains. The bracket denotes the location of higher molecular mass Ub-protein conjugates. 6

7 Supplemental Figure 7. Molecular and biochemical descriptions of the Arabidopsis ddi1-1 mutant. (A) Diagram of the Arabidopsis DDI1 gene. Shaded boxes and broken lines denote protein coding regions and introns, respectively. Black boxes, Ub-like (UBL) and Ub-associated domains (UBA). Dark grey boxes, retroviral protease (RVP) domain. The arrowhead locates the predicted protease active-site aspartic acid (D). White box represents the 3 untranslated region. The location of the ddi1-1 T-DNA insertion is shown. Number to the right is the amino acid length of DDI1. (B) RT-PCR analyses of the ddi1-1 mutant. Total RNA isolated from wild-type (WT) and mutant seedlings was subjected to RT-PCR using the DDI1 primers located by the arrows in panel A. A primer pair specific to PAE2 was used as an internal control. (C) Immunoblot analyses of total protein from 1-week-old WT and the ddi1-1 and rad23a-d mutant seedlings with anti-ddi1 antibodies. Equal loading was confirmed by probing with anti-pba1 antibodies. (D) Immunoblot analyses of crude extracts from 1-week-old WT and ddi1-1 seedlings with anti-ub and anti-rad23b antibodies. The SDS-PAGE migration positions of the Ub monomer, dimer and trimer, and Ub-protein conjugates are indicated. Equal protein loads were confirmed by probing with anti-pba1 antibodies. (E) Pictures of homozygous ddi1-1 seedlings grown on solid GM medium under LD. 7

8 Supplemental Table 1. Arabidopsis proteins that interact with RAD23b by yeast two-hybrid assays a Name Chromosomal locus number of clones UBQ1 At3g UBQ10 At4g UBQ11 At4g UBQ7/RUB2 At2g UBQ5 At3g UBQ2 At2g IAA16 At3g a Arabidopsis cdna library was prepared from 14-d-old green seedlings. 8

9 Supplemental Table 2. Oligonucleotide primers used in this study. P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20 P21 P22 P23 P24 P25 P26 P27 P28 P29 P30 P31 P32 P33 P34 P35 P36 P37 P38 P39 P40 P41 P42 P43 P44 P45 P46 CATATGAAGCTCACCGTTAAA AAGCTTCAGTCTTCAAAGTCA CACCATGAAGCTCACTGTTAAGACTCTC TCAGTCTTCAAAATCTGCTGAGTGCGC CACCATGAAGCTCACCGTTAAAAC TCAGTCTTCAACTGCACCTG CACCATGAAGATATTTGTGAAAACTCTC TTATTCCTCGAATTCATGCATGTGATC CACCATGAAGATTTTCGTGAAGACTCTC TTATTGATCTTCAAACTCATGCATGTG CTGCAGCGGTTATGATCGGTGTG TCTAGAGTCGACGAATGAGACTAGAAG CTGCAGCGACCAAATATGAGAGGTT CCCGGGATCGTCGGAAAAATTGAAATCGA CTGCAGCCTACTCCATAGATACTCTGA TCTAGACTTCTCTCACCGTCGAAATTC CTGCAGGAGGTTCTACACCGTTTACC TCTAGACGTTTAGGGAGAAGGAAAAAAG TCCGAGCTCATGAAGCTCACCGTTAAAACTCTCAAGG GATAAGCTTTTGTGAACTCAGTCTTCAAAGTCACC TGAGAGCTCATGAAGATATTTGTGAAAACTCTCAAGG AATCTCGAGAAGCTTATTCCTCGAATTCATGCATGTG CCCGGATCCATGAAGATTTTCGTGAAGACTCTCAG TTGCTCGAGTTATTGATCTTCAAACTCATGCATGTG CACCTTTGGTGTGGACCCAAATATCGAT TCAAGCACCTGTAGCTTCACTTGACTC TGACTCCTCGCATATCCAGCTCATCTCCAA CGTGGTTGAAGATATGGTAGATGTCGTG AGAACATCGTGTGCTTAATCACGAATGGC GTCTTACCTGAAGAATTTGAGGGTTGG CCTGTTTCCGCTACCACATCTTCGACTAAG AACATATGGACTAGTCGAAAGATAGAGAGTAGGT CTCAACAACCTGCAGCAGCACCCGC TGGCTTCTTGTTTTCCCATCAACATTC GCAGCGGTTGCAGTTCCACTTGTAGG GTATTGCCCATGGTGTTG CGAGTCTCTTTAAAATCA TGGTTCACGTAGTGGGCCATCG TAGGTCTGTCTCTCTCTAGACACTCTCTCCGCCGT AAGAGCCAAAACTTTCTAGAAGAAGAGATAACGTT GGAGAGGTACGTATTTTTACAA AAGCTTCAGTCTTCAAAGTCA AAGCTTTATTCCTCGAATTCAT AAGCTTTATTGATCTTCAAACTC CTCAACTCGATAAAATCCATTATCTG TTAGCCGCCAAAGAGAAACCCAG 9

10 P47 P48 P49 P50 P51 P52 P53 P54 P55 P56 P57 P58 CCTGTTTCCGCTACCACATCTTCGACTAAG CATATGAAGATATTTGTGAAAAC CATATGAAGATTTTCGTGAAGA CTGACATTGAGGTTTATCTCAGATCG GTATTGCCCATGGTGTTG CACCATGAAGCTCACTGTTAAGACTCTCAAG GAGGACCTGCATATGAAGCTCACCGTTAAAACTC GATCCCCGG-GAATTCTCAGTCTTCAAAGTCACCTGAGTTC GAGCAGAAGCTGATCTCAGAGGAGGACCTGCATATG TGCGGCCGCTGCAGGTCGACGGATCCCCGGGAATTC CAAACCCAAAAAAAGAGATC GTTTTTCAGTATCTACGATT 10