Dual-targeting of Arabidopsis DMP1 isoforms to the tonoplast and the plasma membrane

The reports of dual-targeted proteins in plants have steadily increased over the past years. The vast majority of these proteins are soluble proteins distributed between compartments of the non-secretory pathway, predominantly chloroplasts and mitochondria. In contrast, dual-targeted transmembrane proteins, especially of the secretory pathway, are rare and the mechanisms leading to their differential targeting remain largely unknown. Here, we report dual-targeting of the Arabidopsis DUF679 Membrane Protein 1 (DMP1) to the tonoplast (TP) and the plasma membrane (PM). In Arabidopsis and tobacco two equally abundant DMP1 isoforms are synthesized by alternative translation initiation: a full length protein, DMP1.1, and a truncated one, DMP1.2, which lacks the N-terminal 19 amino acids including a TP-targeting dileucine motif. Accumulation of DMP1.1 and DMP1.2 in the TP and the PM, respectively, is Brefeldin A-sensitive, indicating transit via the Golgi. However, DMP1.2 interacts with DMP1.1, leading to extensive rerouting of DMP1.2 to the TP and “eclipsed” localization of DMP1.2 in the PM where it is barely visible by confocal laser scanning microscopy but clearly detectable by membrane fractionation. It is demonstrated that eGFP fusion to either DMP1 terminus can cause mistargeting artifacts: C-terminal fusion to DMP1.1 or DMP1.2 results in altered ER export and N-terminal fusion to DMP1.1 causes mistargeting to the PM, presumably by masking of the TP targeting signal. These results illustrate how the interplay of alternative translation initiation, presence or absence of targeting information and rerouting due to protein-protein interaction determines the ultimate distribution of a transmembrane protein between two membranes

Arabidopsis senescence-associated protein DMP1 is involved in membrane remodeling of the ER and tonoplast

Background: Arabidopsis DMP1 was discovered in a genome-wide screen for senescence-associated membrane proteins. DMP1 is a member of a novel plant- specific membrane protein family of unknown function. In rosette leaves DMP1 expression increases from very low background level several 100fold during senescence progression. Results: Expression of AtDMP1 fused to eGFP in Nicotiana benthamiana triggers a complex process of succeeding membrane remodeling events affecting the structure of the endoplasmic reticulum (ER) and the vacuole. Induction of spherical structures (“bulbs”), changes in the architecture of the ER from tubular to cisternal elements, expansion of smooth ER, formation of crystalloid ER, and emergence of vacuolar membrane sheets and foamy membrane structures inside the vacuole are proceeding in this order. In some cells it can be observed that the process culminates in cell death after breakdown of the entire ER network and the vacuole. The integrity of the plasma membrane, nucleus and Golgi vesicles are retained until this stage. In Arabidopsis thaliana plants expressing AtDMP1-eGFP by the 35S promoter massive ER and vacuole vesiculation is observed during the latest steps of leaf senescence, whereas earlier in development ER and vacuole morphology are not perturbed. Expression by the native DMP1 promoter visualizes formation of aggregates termed “boluses” in the ER membranes and vesiculation of the entire ER network, which precedes disintegration of the central vacuole during the latest stage of senescence in siliques, rosette and cauline leaves and in darkened rosette leaves. In roots tips, DMP1 is strongly expressed in the cortex undergoing vacuole biogenesis. Conclusions: Our data suggest that DMP1 is directly or indirectly involved in membrane fission during breakdown of the ER and the tonoplast during leaf senescence and in membrane fusion during vacuole biogenesis in roots. We propose that these properties of DMP1, exacerbated by transient overexpression, may cause or contribute to the dramatic membrane remodeling events which lead to cell death in infiltrated tobacco leaves

Untersuchung des seneszenzspezifischen Membranproteins DMP1 und der DMP- Genfamilie in Arabidopsis thaliana

Leaf senescence is the final phase of leaf development through which nutrient remobilization from leaves to sink organs, especially developing seeds in Arabidopsis, is achieved. Leaf senescence is a genetically programmed process in which leaf cells undergo orderly changes in gene expression, metabolism and morphology before they eventually die. Although organelles and cellular membrane systems are strongly reorganized during senescence, hardly any transporters and membrane proteins with senescence-specific functions are known. In Arabidopsis thaliana approximately 2000 genes are significantly upregulated during natural senescence, among them many membrane proteins. In this thesis a novel senescence-associated membrane protein gene was identified and characterized. It belongs to a completely unknown plant-specific gene family which comprises ten members in Arabidopsis thaliana and was named DMP1 (DUF679 domain membrane protein 1). All AtDMP proteins are predicted to have four transmembrane domains, with cytosolic amino- and carboxy-termini. In chapter one, the investigation of AtDMP family is presented. The phylogenetic distribution of DMP proteins revealed that DMPs are ubiquitous in green plants and absent from other kingdoms suggesting an implication in plant-specific processes. Only one DMP copy was found in Chlamydomonas reinhardtii and Physcomitrella patens genomes whereas their number ranged from five to 13 in dicots and 11 to 16 in monocots. The expression patterns of AtDMPs were found to be markedly tissue- and development-specific, excluding functional redundancy for most DMP proteins. DMPs are expressed in tissues undergoing senescence (DMP1, -3, -4), dehiscence (DMP1) and abscission (DMP1, -2, -4, -7) suggesting an involvement of DMP proteins in different types of programmed cell death. When fused to eGFP, all DMP proteins localize to the tonoplast or the ER. Some fusion proteins localized in both membrane systems suggesting competitive targeting and retention signals. In chapter two, the complex membrane reorganization events triggered by overexpression of DMP1-eGFP are described and discussed. In Nicotiana benthamiana DMP1-eGFP induces a range of membrane fusion, fission and remodeling events affecting the architecture of the ER and the vacuole. Induction of tonoplastic invaginations known as “bulbs”, changes in the architecture of the endoplasmic reticulum (ER) from tubular to cisternal elements, expansion of smooth ER, formation of crystalloid ER, emergence of vacuolar sheets and foamy structures inside the vacuole were observed. In a fraction of cells, this process culminates in cell death after breakdown of the entire ER network and the vacuole. In transgenic Arabidopsis DMP1-eGFP overexpression did not perturb ER and vacuole morphology, but expression from the endogenous promoter highlighted formation of “boluses” at the ER and vesiculation of the entire ER network preceding fragmentation of the central vacuole during the latest steps of natural senescence and dark-induced senescence in siliques, rosette and cauline leaves. This suggests that DMP1 has direct or indirect membrane fission properties involved in breakdown of the ER and the vacuole during programmed cell death (PCD). In contrast, in roots tips DMP1 is expressed in the cortex undergoing vacuole biogenesis, suggesting an involvement in membrane fusion. These inherent properties, exacerbated by transient overexpression, are proposed to be at least partially responsible for the dramatic membrane remodeling events which led to cell death in tobacco. A discrepancy between the subcellular localization of the tonoplast-localized DMP1-eGFP and the plasma membrane-localized eGFP-DMP1, initiated the investigation described in chapter 3. A range of mutated fusion proteins were generated and their expression and subcellular localization was analyzed in tobacco and Arabidopsis. It turned out that, due to leaky ribosome scanning at the first translation initiation site, two protein isoforms are synthesized, DMP1.1 and DMP1.2 which lacks the 19 amino terminal residues. DMP1.1-eGFP is targeted to the tonoplast whereas DMP1.2-eGFP is located in the plasma membrane. By mutating amino acids 2 and 3 of DMP1.1 or truncating the four N-terminal amino acids, DMP1.1-eGFP is redirected to the plasma membrane. This suggests that the plasma membrane is the default pathway. The occurrence of DMP1.1 and DMP1.2 was verified in Arabidopsis WT plants using an antibody raised against DMP1. 5’-RACE-PCR and sequencing confirmed that the two protein isoforms are translated from a single transcript. Co-expression studies with DMP1.1-eGFP and DMP1.2-mRFP revealed interaction of the two isoforms. Dimerization of DMP1 was confirmed using the split-ubiquitin system and chemical cross-linking in planta. Thus, DMP1.2 is redirected to the tonoplast by interacting with DMP1.1. This finding is the first demonstration of dual targeting of a plant membrane protein to the tonoplast and plasma membrane displaying an “eclipsed” distribution. In chapter four, DMP1 function was investigated by using different reverse genetic approaches, performing genome-wide transcriptome analyses, screening for protein interactors and analyzing DMP1 promoter. DMP1 senescence-specific transcriptional activation was shown to be governed by WRKY transcription factors. Mutation of two W-boxes, the cognate binding site of WRKY proteins, in the DMP1 promoter led to loss of DMP1 expression during senescence. A dmp1 T-DNA insertion mutant (dmp1-ko) and DMP1 overexpressor plants both display precocious senescence without other phenotypical alterations, reinforcing a specific function of DMP1 during senescence. By RNA gel blot analysis, truncated transcripts were detected in dmp1-ko plants that potentially could give rise to truncated and possibly dysfunctional proteins. These might be responsible for the phenotype since suppression of DMP1 expression using artificial microRNA did not lead to a comparable phenotype. The effects of DMP1 overexpression were investigated by transcriptomics. Strikingly, CYP94B3 which is involved in catabolism of the active jasmonate form JA-Ile, showed the strongest downregulation. This might result in JA-Ile accumulation and lead to early senescence. Moreover, the level of OPDA, a precursor of jasmonic acid considered as intracellular marker for senescence, was quantified by GC-MS and found to be more than twice as high in the mutant than in the WT. To gain more insight in DMP1 function, a split-ubiquitin screen was carried out in yeast. DMP1 was found to interact with Bax Inhibitor-1 (BI-1) and the Cytochrome b5 isoforms E and D. These proteins interact with each other in the context of cell death, corroborating an involvement of DMP1 in programmed cell death during late senescence.Seneszenz ist die letzte Stufe der Blattentwicklung. Während dieses Vorgangs werden in den alternden Blättern gebundene Nährstoffe verfügbar gemacht und zu anderen Pflanzenteilen, wie z.B. jungen Samen, transportiert. Die Alterung ist ein genetisch streng gesteuerter Prozess, während dessen sich die Genexpression, der Metabolismus und die Morphologie der Blattzellen verändern, bis die Zellen schließlich absterben. Obwohl sich Organellen und Zellmembransysteme während der Seneszenz stark verändern, sind kaum seneszenzspezifische Membranproteine bzw. Transporter bekannt. In Arabidopsis thaliana werden während der natürlichen Seneszenz ca. 2000 Gene signifikant hochreguliert, darunter viele Membranproteine. Im Rahmen dieser Arbeit wurde ein neues seneszenzassoziiertes Membranprotein identifiziert und charakterisiert. Das Gen wurde DMP1 (DUF679 domain membrane protein) genannt und gehört zu einer bislang unbekannten pflanzenspezifischen Genfamilie, die in Arabidopsis mit 10 Mitgliedern vertreten ist. Für alle AtDMP Proteine werden vier Transmembranbereiche vorhergesagt, wobei sich sowohl der Amino- als auch der Carboxyterminus auf der cytosolischen Seite der Membran befinden. Im ersten Kapitel dieser Arbeit wurde die AtDMP Genfamilie untersucht. Eine phylogenetische Untersuchung ergab, dass DMPs ausschließlich in Pflanzen vorkommen, was vermuten lässt, dass diese Proteine in pflanzenspezifische Prozesse involviert sind. In den Genomen von Chlamydomonas reinhardtii und Physcomitrella patens kommt jeweils nur ein DMP Gen vor, wohingegen Dicotylen zwischen 11 und 16 und Monocotylen zwischen 5 und 13 Gene besitzen. Es zeigte sich, dass die Expressionsmuster der AtDMPs deutlich gewebe- und entwicklungsspezifisch sind, was eine funktionelle Redundanz der Proteine unwahrscheinlich macht. DMPs sind in verschiedenen Stadien der Blattalterung aktiv, im Einzelnen während der Seneszenz (DMP1, -3, -4), der Dehiszenz (DMP1) und dem Blattwurf (DMP1, -2, -4, -7). Dieses Expressionsverhalten lässt vermuten, dass DMPs in verschiedenen Typen des programmierten Zelltods involviert sind. Proteinfusionen mit eGFP zeigten, dass alle DMPs entweder im Tonoplasten oder in der ER-Membran lokalisiert sind. Manche Fusionsproteine konnten in beiden Membransystemen detektiert werden, was auf kompetitive Ziel- bzw. Rückhaltesignale hindeutet. Die komplexen Vorgänge während der Membranumstrukturierung, hervorgerufen durch die Überexpression von DMP1-eGFP, sind Thema des zweiten Kapitels. In Nicotiana benthamiana induziert transient exprimiertes DMP1-eGFP eine Reihe von Membranfusionen und -teilungen, sowie Veränderungen der ER- und Vakuolenarchitektur. Einstülpungen des Tonoplasten (sogenannte „bulbs“), Umwandlung von tubulären ER-Bereichen in ER-Zisternen, Vergrößerung des glatten ERs, Bildung von kristallartigem ER sowie Veränderungen des Tonoplasten, die zu einer schaumartigen Morphologie der Vakuole führen, wurden beobachtet. In einigen Zellen führen diese Veränderungen zu einem Zusammenbruch des gesamten ER-Netzwerkes und der Vakuole und damit zum Zelltod. In transgenen 35S:DMP1-eGFP Arabidopsispflanzen ist der Aufbau von ER und Vakuole nicht verändert. Wird DMP1-eGFP unter dem eigenen Promoter exprimiert, zeigt sich eine deutliche Aktivität von DMP1 während der letzten Phasen sowohl der natürlichen als auch der dunkelinduzierten Seneszenz. Zunächst ist durch das eGFP die Ausbildung von Aggregaten im ER sowie die Aufspaltung des gesamten ERs in Vesikel zu beobachten. Anschließend fragmentiert die Vakuole. DMP1 scheint also eine Rolle im programmierten Zelltod zu spielen, konkret im Abbau der Membranen von Vakuole und ER. Im Gegensatz dazu steht die Beobachtung, dass DMP1 während der Vakuolenbiogenese im Cortex der Wurzelspitze, aktiv ist. DMP1 scheint folglich auch bei Membranfusionen relevant zu sein. Wird DMP1 transient in Tabak exprimiert, führen diese spezifischen Proteineigenschaften vermutlich zu den beobachteten dramatischen Membranveränderungen sowie zum Zelltod. Kapitel Drei beschäftigt sich mit der subzellulären Lokalisation verschiedener DMP1-GFP- Fusionsproteine. Während DMP1-eGFP im Tonoplasten lokalisiert ist, befindet sich eGFP-DMP1 in der Plasmamembran. Daher wurden eine Reihe mutierter Fusionsproteine hergestellt und ihre Expression und subzelluläre Lokalisation sowohl in Tabak als auch Arabidopsis untersucht. Es zeigte sich, dass aufgrund eines alternativen Startcodons zwei Proteinisoformen, DMP1.1 und DMP1.2 translatiert werden. DMP1.1-eGFP wird zum Tonoplast geleitet, wohingegen DMP1.2-eGFP, dem 19 N-terminale Aminosäuren fehlen, zur Plasmamembran transportiert wird. Wenn die Aminosäuren zwei und drei mutiert oder die ersten vier N-terminalen Aminosäuren entfernt werden, ist auch DMP1.1-eGFP in der Plasmamembran lokalisiert. Diese Beobachtungen legen nahe, dass die Plasmamembran als „default“ pathway für DMP1 angesehen werden kann. Mittels eines DMP1-Antikörpers wurden sowohl DMP1.1 als auch DMP1.2 in Wildtyp- Arabidopsis nachgewiesen. Durch 5‘-RACE-PCR und anschließende Sequenzierung wurde nachgewiesen, dass beide Proteinisoformen von demselben Transkript translatiert werden. Co-Expressionsstudien mit DMP1.1-eGFP und DMP1.2-mRFP zeigten, dass beide Isoformen miteinander interagieren. Die Dimerbildung wurde mit dem Split-Ubiquitin-System und durch chemisches Vernetzen in planta nachgewiesen. Demzufolge wird DMP1.2 durch die Interaktion mit DMP1.1. zur Vakuole umgeleitet. Damit konnte zum ersten Mal duales Targeting mit einer sogenannten „verfinsterten Verteilung“ bei einem pflanzlichen Membranprotein gezeigt werden. In Kapitel Vier wurde mit Hilfe revers-genetischer Ansätze die Funktion von DMP1 untersucht. Dazu wurden Transkriptomanalysen durchgeführt und nach Proteininteraktoren gesucht. Sowohl die dmp1 T-DNA Insertionsmutante (dmp1-ko) als auch DMP1 Überexpressionspflanzen seneszieren früher als der Wildtyp. Andere phänotypische Veränderungen konnten nicht beobachtet werden, was die Seneszenzspezifität von DMP1 weiter untermauert. In einem Gel Blot Test mit dmp1-ko RNA wurden verkürzte Transkripte detektiert, die möglicherweise Vorlage für verkürzte und womöglich dysfunktionale Proteine sind. Da eine Abregulation der DMP1 Expression durch künstliche microRNAs nicht zu einem dmp1-ko vergleichbaren Phänotyp führte, ist es denkbar, dass verkürzte Porteine Ursache des Knockoutphänotyps sind. Die Auswirkungen der DMP1 Überexpression wurden mit Hilfe einer Transkriptomanalyse untersucht. CYP94B3, das an der Inaktivierung der biologisch aktivsten Form von Jasmonsäure (JA-Ile) beteiligt ist, zeigte interessanterweise die stärkste Abregulation. Dies könnte zu einer Akkumulation von JA-Ile und dadurch zu verfrühter Seneszenz führen. OPDA ist eine Vorstufe von Jasmonsäure und gilt als intrazelluläres Seneszenzmerkmal. Der OPDA-Gehalt wurde mittels GC-MS untersucht. Wie sich zeigte, ist die OPDA-Konzentration in den transgenen Pflanzen mehr als zweimal höher als im Wildtyp. Um weitere Einblicke in die Funktion von DMP1 zu erhalten, wurde ein Split-Ubiquitin Screen in Hefe durchgeführt. Es zeigte sich, dass DMP1 mit Bax Inhibitor-1 (BI-1) und den Cytochrom b5 Isoformen E und D interagiert. Diese Proteine wirken während des Zelltods zusammen, was eine mögliche Funktion von DMP1 im programmierten Zelltod in der späten Seneszenz bekräftigt. Es wurde ferner demonstriert, dass die seneszenzspezifische Aktivierung von DMP1 durch WRKY Transkriptionsfaktoren erfolgt. Wurden im DMP1-Promoter zwei Bindungsstellen von WRKY Proteinen, sogenannte W-Boxen, mutiert, konnte DMP1 während der Blattalterung nicht mehr aktiviert werden

<it>Arabidopsis</it> senescence-associated protein DMP1 is involved in membrane remodeling of the ER and tonoplast

Abstract Background Arabidopsis DMP1 was discovered in a genome-wide screen for senescence-associated membrane proteins. DMP1 is a member of a novel plant-specific membrane protein family of unknown function. In rosette leaves DMP1 expression increases from very low background level several 100fold during senescence progression. Results Expression of AtDMP1 fused to eGFP in Nicotiana benthamiana triggers a complex process of succeeding membrane remodeling events affecting the structure of the endoplasmic reticulum (ER) and the vacuole. Induction of spherical structures (“bulbs”), changes in the architecture of the ER from tubular to cisternal elements, expansion of smooth ER, formation of crystalloid ER, and emergence of vacuolar membrane sheets and foamy membrane structures inside the vacuole are proceeding in this order. In some cells it can be observed that the process culminates in cell death after breakdown of the entire ER network and the vacuole. The integrity of the plasma membrane, nucleus and Golgi vesicles are retained until this stage. In Arabidopsis thaliana plants expressing AtDMP1-eGFP by the 35S promoter massive ER and vacuole vesiculation is observed during the latest steps of leaf senescence, whereas earlier in development ER and vacuole morphology are not perturbed. Expression by the native DMP1 promoter visualizes formation of aggregates termed “boluses” in the ER membranes and vesiculation of the entire ER network, which precedes disintegration of the central vacuole during the latest stage of senescence in siliques, rosette and cauline leaves and in darkened rosette leaves. In roots tips, DMP1 is strongly expressed in the cortex undergoing vacuole biogenesis. Conclusions Our data suggest that DMP1 is directly or indirectly involved in membrane fission during breakdown of the ER and the tonoplast during leaf senescence and in membrane fusion during vacuole biogenesis in roots. We propose that these properties of DMP1, exacerbated by transient overexpression, may cause or contribute to the dramatic membrane remodeling events which lead to cell death in infiltrated tobacco leaves.</p

Analysis of DMP1 isoform interactions by the split-ubiquitin assay in yeast.

<p>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.</p

Identification and subcellular localization of DMP1 isoforms DMP1.1 and DMP1.2.

<p>(<b>A</b>) Western blot analysis of native and mutant DMP1 proteins transiently expressed in <i>Nicotiana benthamiana</i> abaxial leaf epidermis cells, in leaves of a transgenic <i>Arabidopsis thaliana</i> DMP1 overexpressor line, and in senescing leaves of wild-type <i>A</i>. <i>thaliana</i> 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 <i>Arabidopsis DMP1</i> promoter (blue characters; ~20-times more protein was applied than in the other lanes). (<b>B</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. (<b>C-E</b>) Determination by CLSM of (C) DMP1.1-eGFP, (D) DMP1.2-eGFP and (E) DMP1_ΔL6L7-eGFP subcellular localization in coexpression experiments in transiently transfected tobacco abaxial epidermis cells (2 dpi) and transgenic <i>Arabidopsis</i> 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 <i>Arabidopsis</i> 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.</p

Rerouting of DMP1.2 to the TP by DMP1.1.

<p>(<b>A-C</b>) 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. (<b>D-E</b>) DMP1.2-mRFP subcellular localization was investigated in coexpression experiments with (<b>D</b>) DMP1.2-eGFP and (<b>E</b>) 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 (<b>F</b>) DMP1.2-mRFP expressed individually, (<b>G</b>) DMP1.2-mRFP expressed in the presence of unfused DMP1.2, and (<b>H</b>) DMP1.2-mRFP expressed in the presence of unfused DMP1.1 were investigated. (<b>E</b>) and (<b>H</b>): Arrows indicate transvacuolar strands; filled arrowheads indicate the nucleus; open arrowheads indicate other TP-enclosed smaller organelles. Scale bars: 20 μm.</p

Labeling of the PM by DMP1-eGFP is mostly undetectable by CLSM.

<p>(<b>A</b>) Coexpression of DMP1-eGFP and PM-associated mRFP-MUB2 in tobacco lower epidermis cells and (<b>B</b>) expression of DMP1-eGFP in transgenic <i>Arabidopsis</i> cells that were briefly incubated (10–15 min) with FM4-64 to stain the PM shows segregation of fluorescence signals (enlarged details in insets). (<b>C</b>) Protoplasts were prepared from late adult <i>Arabidopsis</i> leaves expressing DMP1-eGFP or (<b>D</b>) eGFP-DMP1 for clear distinction between the fluorescence signals originating from the PM, the TP and the ER at this developmental stage. (<b>C</b> and <b>D</b>) 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. (<b>E</b>) 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. (<b>F</b>) Sporadical co-labeling of the TP and the PM was also observed with DMP1_loop2-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.</p

DMP1_loop2-eGFP labels the TP and eGFP-DMP1 is mistargeted to the PM.

<p>Determination by CLSM of DMP1_loop2-eGFP and eGFP-DMP1 subcellular localization. (<b>A</b>) DMP1_loop2-eGFP colocalizes with TPK1-mRFP in the TP of tobacco epidermis cells. (<b>B</b>) In <i>Arabidopsis</i> hypocotyl, young cotyledon and adult leaves, DMP1_loop2-eGFP localizes in the TP and vacuolar structures such as tonoplastic bulbs and transvacuolar strands but not in the ER membrane. (<b>C</b>) The N-terminal fusion protein eGFP-DMP1 colocalizes with mRFP-MUB2 in the PM of tobacco lower epidermis cells. (<b>D</b>) In <i>Arabidopsis</i> 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.</p