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.

The male gamete membrane protein DMP9/DAU2 is required for double fertilization in flowering plants

“…Considering the default localization of DMP9 in sperm cells, it is suggested that DMP9 on the sperm surface functions prior to or simultaneously with GCS1/HAP2 and GEX2. A recent study reported that two isoforms of DMP1 (DMP1.1 and 1.2) showed different destinations, the tonoplast (TP) and the plasma membrane (PM), within a cell (Kasaras and Kunze, 2017). The fact that DMP9 has no signal sequence at the N terminus and shows no TP localization in onion epidermal cells also strongly suggests that DMP9 localization is restricted to the plasma membrane in sperm cells.…”

Broad-Spectrum Amino Acid Transporters ClAAP3 and ClAAP6 Expressed in Watermelon Fruits

“…Subcellular localization of ClAAP3 and ClAAP6 were performed by using translational fusions with GFP in Nicotiana benthamiana leaves. Each AAP gene was translationally fused with GFP on either N’- and C’- side, since previous studies have shown that depending on the location of targeting motifs, both C or N terminal fusions can mislocalize [26,27]. The results showed that both fusions localize to the structure resembling perinuclear ER membrane and the periphery of the cells, presumably the plasma membrane (Figure 6a,f,k,p).…”

Differential protein abundance of vetiver grass in response to acid mine drainage

“…PDIs rearrange disulfide bridges in ER‐associated proteins to restore protein structure (Nawkar et al, 2018). DMP5 of the plant DMP1 family is part of the secretory pathway and is targeted to the plasma membrane and tonoplast (Kasaras & Kunze 2017). PDI and DMP5 were differentially regulated early on, likely to maintain ER homeostasis.…”