Article Figures & Data
Figures
- Figure 1.
Interaction of MFP1 with MAF1.
(A) Schematic representation of MFP1 indicating the two N-terminal hydrophobic domains as filled bars and the C-terminal DNA binding domain as a hatched bar. The MFP1 subfragments HHMFP1, Δ125HMFP1, and Δ125MFP1 are shown below. Numbers indicate the first and last amino acids of the protein subfragments.
(B) Test for specificity of the pBD–HHMFP1/pAD21-1 interaction. Yeast cells were contransformed with p53 and pSV40 (sector 1), pLaminC and pAD21-1 (sector 2), p53 and pAD21-1 (sector 3), pBD–HHMFP1 and pAD21-1 (sector 4), pLaminC and pSV40 (sector 5), and pBD–GAL4 and pAD21-1 (sector 6); transformants were streaked on Leu-, Trp-, and His- plates.
(C) Quantification of β-galactosidase activity by ONPG assays of yeast strains cotransformed with p53 and pSV40 (bar 1), pLaminC and pSV40 (bar 2), pBD–HHMFP1 and pAD21-1 (bar 3), pBD–GAL4 and pAD21-1 (bar 4), and pBD–HHMFP1 and pAD–GAL4 (bar 5). Mean values and standard deviations of three assays are shown. mU, milliunits.
(D) Confirmation of the MFP1–MAF1 interaction in yeast. Yeast cells were cotransformed with p53 and pSV40 (sector 1), pBD–GAL4 and pAD–HHMFP1 (sector 2), pBD–MAF1 and pAD–GAL4 (sector 3), pBD–MAF1 and pAD–HHMFP1 (sector 4), pBD–Δ125HMFP1 and pAD–GAL4 (sector 5), and pBD–Δ125HMFP1 and pAD6-3 (sector 6); transformants were streaked on Leu-, Trp-, and His- plates.
(E) Confirmation of the MFP1–MAF1 interaction by protein overlay analysis. SDS–polyacrylamide gels (10%) were loaded with 4 μL (lanes 1, 5, and 10) or 4 μL of a 1:10 dilution (lanes 2, 6, and 11) of extract from Bac-Δ125MFP1–infected Trichoplusia ni cells. The extract contained ∼0.5 μg/mL of Δ125MFP1 protein. As a negative control, adjoining lanes were loaded with 4 μL (lanes 3, 7, and 12) or 4 μL of a 1:10 dilution (lanes 4, 8, and 13) of wild-type baculovirus-infected Trichoplusia ni cell extract spiked with 0.5 μg/mL of BSA. After electrophoresis, the gels were stained with Bluprint Fast–PAGE Coomassie stain (lanes 10 to 14) or transferred to a nitrocellulose membrane (lanes 1 to 9). The membrane was divided in half and probed with purified MAF1 protein (lanes 1 to 4) or binding buffer alone (lanes 5 to 9), and immunoblot analysis was subsequently performed with anti-MAF1 antiserum. Lane 9, 15 ng MAF1 protein; lane 14, Benchmark protein ladder (Life Technologies), with molecular masses given in kilodaltons at right. The arrowhead indicates the position of Δ125MFP1.
- Figure 2.
Sequence and Structural Organization of MAF1.
(A) The deduced amino acid sequence of MAF1 is in single-letter code. The GenBank accession number is AF118113.
(B) Structural organization of MFP1. At top is the distribution of acidic and basic domains. Acidic domains are indicated by open bars, and basic domains are indicated by filled bars. Numbers indicate the calculated pI values of the respective domains. At bottom is the distribution of serine and threonine residues. Lines above the horizontal line indicate serine residues; lines below the horizontal line indicate threonine residues.
- Figure 3.
Genomic Organization and Expression of MAF1.
(A) Genomic DNA gel blot analysis. Tomato genomic DNA was digested with EcoRI (lane 1), HindIII (lane 2), EcoRV (lane 3), BamHI (lane 4), EcoRI and HindIII (lane 5), EcoRI and EcoRV (lane 6), and EcoRV and HindIII (lane 7). DNA length markers are indicated at left in kilobases. The 12-kb BamHI signal has reduced intensity due to partial transfer of high molecular weight DNA. The faint bands in the EcoRI digest (lane 1) are most likely due to incomplete digestion, because no additional bands were detected in the EcoRI and HindII and the EcoRI and EcoRV double digests (lanes 5 and 6).
(B) RNA gel blot analysis with total RNA isolated from different tomato tissues. RNA length markers are indicated at left in kilobases. Equal amounts of total RNA were loaded in each lane, as determined by ethidium bromide staining of the rRNAs. yl, young leaf; yf, young fruit; fl, flower.
(C) Immunoblot analysis with total protein extracts from different tomato tissues. Protein mass markers are indicated at left in kilodaltons. Equal amounts of protein were loaded in each lane as determined by staining with Bluprint Fast–PAGE stain of a replica gel. yl, young leaf; yf, young fruit; fl, flower; st, stem.
(D) Immunoblot analysis of leaf extracts from tomato (tom) and tobacco (tob). Equal amounts of protein were loaded in both lanes as determined by staining with Bluprint Fast–PAGE stain of a replica gel. Molecular mass markers are indicated at left in kilodaltons.
- Figure 4.
Localization of MAF1–mGFP–MAF1 Fusion Protein in Tobacco NT-1 Cells.
(A) Schematic representation of the proteins transiently expressed in tobacco NT-1 cells.
(B) Differential interference contrast image of the cell shown in (C).
(C) GFP fluorescence of a cell transiently expressing MAF1–mGFP–MAF1 after particle bombardment–mediated transformation.
(D) Differential interference contrast image of the cell shown in (E).
(E) GFP fluorescence of a cell transiently expressing mGFP after particle bombardment–mediated transformation.
Bars in (B) and (D) = 10 μm for (B) to (E).
- Figure 5.
Immunolocalization of MAF1 in NT-1 Cells, NT-1 Nuclei, and the NT-1 Nuclear Matrix.
(A), (D), and (F) Differential interference contrast images of fixed NT-1 cells (A), NT-1 nuclei (D), and an NT-1 nuclear matrix fraction (F).
(B), (E), and (G) Detection of MAF1 with the anti-MAF1 antibody R37 and a Cy5-conjugated goat anti–rabbit secondary antibody in NT-1 cells (B), NT-1 nuclei (E), and an NT-1 nuclear matrix fraction (G).
(C) 4′,6-Diamidino-2-phenylindole (DAPI) staining of the nuclei of the NT-1 cells shown in (A) and (B).
Bar in (A) = 20 μm; bars in (D) and (F) = 10 μm.
- Figure 6.
MAF1 Is Conserved among Higher Plants.
(A) Alignment of deduced protein sequences of full-length cDNAs and ESTs from different plant species. Identical amino acids are shown by reverse lettering. The two most highly conserved domains are indicated by black bars above the sequence. Dashes indicate gaps in the alignment. Numbers at left refer to amino acid position in each sequence. Asterisks indicate the ends of the ESTs. L.e., Lycopersicon esculentum; A.t., Arabidopsis thaliana; G.m., Glycine max; Z.m., Zea mays; C.e., Canna edulis; P.p., Picramnia pentandra; T.a., Triticum aestivum. The GenBank accession numbers are AF118113, AB008267, AF118115, AF118114, AF118116, AF118117, and AF118118, respectively.
(B) Comparison of the positions of the most highly conserved sequences (black lines) and the basic domains (filled bars) in tomato MAF1. Numbers indicate amino acid positions. L.e., Lycopersicon esculentum.
