Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Kasaras A

PROVIDER: S-EPMC5383025 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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

Kasaras Alexis A Kunze Reinhard R

PloS one 20170406 4

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 Membr ...[more]

PMID: 28384172

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Project description: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.

Project description:Tethering of the plasma membrane (PM) and many organelles to the endoplasmic reticulum (ER) for communication and lipid exchange has been widely reported. However, despite growing interest in multi-vesicular bodies (MVBs) as possible sources of exosomes, tethering of MVBs to the PM has not been reported. Here we show that MVBs and the vacuolar membrane (tonoplast) could be tethered to the PM (PM-MVB/TP tethering) by artificial protein fusions or bimolecular fluorescence complementation (BiFC) complexes that contain a peripheral membrane protein that binds the PM and also a protein that binds MVBs or the tonoplast. PM-binding proteins capable of participating in PM-MVB/TP tethering included StRem1.3, BIK1, PBS1, CPK21, and the PtdIns(4)-binding proteins FAPP1 and Osh2. MVB/TP-binding proteins capable of participating in tethering included ARA6, ARA7, RHA1, RABG3f, and the PtdIns(3)P-binding proteins Vam7p and Hrs-2xFYVE. BiFC complexes or protein fusions capable of producing PM-MVB/TP tethering were visualized as large well-defined patches of fluorescence on the PM that could displace PM proteins such as AtFlotillin1 and also could displace cytoplasmic proteins such as soluble GFP. Furthermore, we identified paralogous ubiquitin E3 ligase proteins, SAUL1 (AtPUB44), and AtPUB43 that could produce PM-MVB/TP tethering. SAUL1 and AtPUB43 could produce tethering in uninfected tissue when paired with MVB-binding proteins or when their E3 ligase domain was deleted. When Nicotiana benthamiana leaf tissue was infected with Phytophthora capsici, full length SAUL1 and AtPUB43 localized in membrane patches consistent with PM-MVB/TP tethering. Our findings define new tools for studying PM-MVB/TP tethering and its possible role in plant defense.Significance statementAlthough not previously observed, the tethering of multi-vesicular bodies to the plasma membrane is of interest due to the potential role of this process in producing exosomes in plants. Here we describe tools for observing and manipulating the tethering of multi-vesicular bodies and the tonoplast to the plant plasma membrane, and describe two plant proteins that may naturally regulate this process during infection.

Dataset Information

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

Publications

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

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