In a screen for senescence-associated genes in Arabidopsis thaliana, a novel, highly up-regulated membrane protein was identified. It is a member of an uncharacterised, strictly plant-specific gene family and was named AtDMP1 (Arabidopsis thaliana DUF679 domain membrane protein 1). The AtDMP proteins are predicted to have four transmembrane spans, with cytosolic amino- and carboxy-termini. In this study, we investigated the phylogenetic distribution of DMP proteins, their tissue-specific expression and subcellular localisation in A. thaliana. The Chlamydomonas reinhardtii and Physcomitrella patens genomes in dicots contain only a single DMP gene copy, whereas there are five to 13 DMP genes and 11-16 in monocots, many of which supposedly result from recent gene duplications. The ubiquitous occurrence of DMP proteins in green plants and their absence from other kingdoms suggest a role in plant-specific processes. In A. thaliana, expression of nine out of ten DMP genes was detected. The expression patterns were found to be markedly tissue- and development-specific; thus, functional redundancy of most proteins is unlikely. The occurrence of several AtDMPs in tissues undergoing senescence (AtDMP1, -3, -4), dehiscence (AtDMP7) or abscission (AtDMP2, -4, -7) suggests involvement of DMPs in different types of programmed cell death. AtDMP-eGFP fusion proteins were found to localise either to the endoplasmic reticulum, the tonoplast or, under certain conditions, to both membrane systems. Further investigations are in progress to elucidate functions of the AtDMP proteins.

BackgroundArabidopsis 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.ResultsExpression 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.ConclusionsOur 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.

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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.