Fig 1.
Identification and subcellular localization of DMP1 isoforms DMP1.1 and DMP1.2.
(A) Western blot analysis of native and mutant DMP1 proteins transiently expressed in Nicotiana benthamiana abaxial leaf epidermis cells, in leaves of a transgenic Arabidopsis thaliana DMP1 overexpressor line, and in senescing leaves of wild-type A. thaliana Col-0 plants. Substitutions and deletions of the DMP1 open reading frame resulting in loss of the larger or the smaller isoform are indicated in red characters. Proteins were expressed from the 35S promoter (black characters) or the native Arabidopsis DMP1 promoter (blue characters; ~20-times more protein was applied than in the other lanes). (B) Amino acid sequence of the DMP1.1 N-terminus with the second methionine in position 20 highlighted in red and a putative TP-targeting dileucine signal marked in blue letters (top line), the DMP1.2 N-terminus (center line), and the common C-terminus with putative ER-export signals highlighted in green letters (bottom line). TMD, transmembrane domain. (C-E) Determination by CLSM of (C) DMP1.1-eGFP, (D) DMP1.2-eGFP and (E) DMP1ΔL6L7-eGFP subcellular localization in coexpression experiments in transiently transfected tobacco abaxial epidermis cells (2 dpi) and transgenic Arabidopsis plants. The TP-located fusion protein TPK1-mRFP was used as TP marker and the PM-associated fusion protein mRFP-MUB2 as PM marker in tobacco. Staining of the PM in Arabidopsis plants was performed by incubating the cells for 10–15 min with the fluorescent dye FM4-64. Enlarged details in insets. Scale bars: 10 μm.
Fig 2.
DMP1loop2-eGFP labels the TP and eGFP-DMP1 is mistargeted to the PM.
Determination by CLSM of DMP1loop2-eGFP and eGFP-DMP1 subcellular localization. (A) DMP1loop2-eGFP colocalizes with TPK1-mRFP in the TP of tobacco epidermis cells. (B) In Arabidopsis hypocotyl, young cotyledon and adult leaves, DMP1loop2-eGFP localizes in the TP and vacuolar structures such as tonoplastic bulbs and transvacuolar strands but not in the ER membrane. (C) The N-terminal fusion protein eGFP-DMP1 colocalizes with mRFP-MUB2 in the PM of tobacco lower epidermis cells. (D) In Arabidopsis epidermis cells eGFP-DMP1 colocalizes with the fluorescent dye FM4-64 (10–15 min staining time) in the PM and additionally decorates endosomes (inset). Scale bars: 10 μm.
Fig 3.
Labeling of the PM by DMP1-eGFP is mostly undetectable by CLSM.
(A) Coexpression of DMP1-eGFP and PM-associated mRFP-MUB2 in tobacco lower epidermis cells and (B) expression of DMP1-eGFP in transgenic Arabidopsis cells that were briefly incubated (10–15 min) with FM4-64 to stain the PM shows segregation of fluorescence signals (enlarged details in insets). (C) Protoplasts were prepared from late adult Arabidopsis leaves expressing DMP1-eGFP or (D) eGFP-DMP1 for clear distinction between the fluorescence signals originating from the PM, the TP and the ER at this developmental stage. (C and D) Top rows show cross sections through the center plane of the protoplasts (z = 0 μm). DMP1-eGFP decorates the TP, whereas eGFP-DMP1 decorates the PM. Bottom rows show cross sections through the cortical region (z = -20 μm). They reveal labeling of endomembranes, presumably both the ER membrane and the TP only for DMP1-eGFP but not eGFP-DMP1. (E) Sporadically occurring DMP1-eGFP fluorescence signals at the PM in abaxial tobacco epidermis cells. DMP1-eGFP fluorescence signals from the plasma membranes of adjacent cells (arrows in insets 1 and 2) are weaker than those from the two vacuolar membranes (arrowheads in insets 1 and 2), indicating weaker accumulation of DMP1-eGFP in the PM compared to the TP. (F) Sporadical co-labeling of the TP and the PM was also observed with DMP1loop2-eGFP. Fluorescence intensity of labeled membranes was quantified along the 4.5 μm paths indicated by arrows in insets 1–4. TP versus PM fluorescence ratios (TP/PM) were calculated as the combined fluorescence values of the two TP signals divided by those of the two PM signals (merged in inset 1–3) for each cross section. Scale bars: 10 μm.
Fig 4.
Distribution of DMP1 isoforms in fractionated membranes from different transgenic Arabidopsis DMP1 expression lines.
(A) Microsomal membranes from 8 days old DMP1-eGFP, eGFP-DMP1, DMP1, DMP1.1 and DMP1.2 seedlings were fractionated on linear sucrose gradients and 10 μl of each fraction were analysed by Western blotting. Marker protein-specific antibodies (indicated below the three top panels) were used to visualize the distribution of microsomes originating from the ER, TP and PM. Anti-BiP2 detects the ER-resident protein BiP2, anti-v-ATPase detects tonoplast-derived vesicles and anti-H+-ATPase detects PM-derived vesicles. Depicted are representative distributions observed in all lines. Anti-DMP1 antibodies were used to detect all DMP1 isoforms and fusion proteins in the transgenic plants (five bottom panels). (B) Microsomal membranes from 8 days old DMP1, DMP1.1, DMP1.2, DMP1-eGFP, DMP1.1-eGFP and DMP1.2-eGFP seedlings were separated using an aqueous two-phase system. The PM-enriched upper phases U3 and endomembrane-enriched lower phases L3 were extracted three times to maximize PM and endomembranes separation. Separation of PM vesicles (U3) from endomembranes (L3) was monitored against the initial, unfractionated microsomal fraction (I) on Western Blots using anti-H+-ATPase and anti-BiP2 antibodies in each line (left panels). DMP1 proteins were detected using anti-DMP1 antibodies (right panels). 1 μg total protein was loaded for each fraction.
Fig 5.
Analysis of DMP1 isoform interactions by the split-ubiquitin assay in yeast.
DMP1.1-DMP1.1 and DMP1.1-DMP1.2 interactions were investigated using the split-ubiquitin system in yeast. Cub-DMP1.1 fusion protein was used as bait and appropriate growth and selection conditions were established using co-expression with NubG, NubG-KAT1, NubG-SUT1 (negative controls) and NubI (positive control). Cub-DMP1.1 interacts with NubG-DMP1.1 and NubG-DMP1.2, respectively, but not with the DMP1-homologs DMP2 or DMP7.
Fig 6.
Rerouting of DMP1.2 to the TP by DMP1.1.
(A-C) Left panels: Fluorescence signals of the indicated coexpressed fusion proteins containing the two YFP moieties (nYFP and cYFP) at 2 dpi in tobacco epidermis cells. Center panels: mRFP fluorescence signals visualizing the cytoplasm and the lumen of the nucleus. Right panels: superimposed YFP and mRFP signals. The three proteins for each assay were encoded on the same vector to ensure synchronized expression and equimolar protein levels. (D-E) DMP1.2-mRFP subcellular localization was investigated in coexpression experiments with (D) DMP1.2-eGFP and (E) DMP1.1-eGFP. The enlarged insets highlight residual DMP1.2-eGFP in the PM in presence of DMP1.1-eGFP. To exclude protein-protein interaction between the two fluorophores, fluorescence patterns of (F) DMP1.2-mRFP expressed individually, (G) DMP1.2-mRFP expressed in the presence of unfused DMP1.2, and (H) DMP1.2-mRFP expressed in the presence of unfused DMP1.1 were investigated. (E) and (H): Arrows indicate transvacuolar strands; filled arrowheads indicate the nucleus; open arrowheads indicate other TP-enclosed smaller organelles. Scale bars: 20 μm.
Fig 7.
(A) Summary of subcellular location results. (B) Model for the localization of DMP1.1 and DMP1.2 proteins when individually or co-expressed. Individually expressed DMP1.1 is targeted to the TP and DMP1.2 to the PM. DMP1.2 is efficiently rerouted to the TP upon protein-protein interaction with DMP1.1 and only a minor fraction is targeted to the PM. Additional marginal accumulation of DMP1.1-eGFP and DMP1.2-eGFP in the ER membrane observed in some tissues is artifactual and not depicted. As both DMP1.1 and DMP1.2 trafficking to the TP or the PM is Brefeldin A-sensitive, DMP1.1-DMP1.2 protein-protein interaction presumably takes place in the ER membrane, Golgi and/or prevacuolar compartments before transit to either the TP or the PM.