Abstract
The outermost cell layer of plants, the epidermis, and its outer (lateral) membrane domain facing the environment are continuously challenged by biotic and abiotic stresses. Therefore, the epidermis and the outer membrane domain provide important selective and protective barriers. However, only a small number of specifically outer membrane-localized proteins are known. Similarly, molecular mechanisms underlying the trafficking and the polar placement of outer membrane domain proteins require further exploration. Here, we demonstrate that ACTIN7 (ACT7) mediates trafficking of the PENETRATION3 (PEN3) outer membrane protein from the trans-Golgi network (TGN) to the plasma membrane in the root epidermis of Arabidopsis (Arabidopsis thaliana) and that actin function contributes to PEN3 endocytic recycling. In contrast to such generic ACT7-dependent trafficking from the TGN, the EXOCYST84b (EXO84b) tethering factor mediates PEN3 outer-membrane polarity. Moreover, precise EXO84b placement at the outer membrane domain itself requires ACT7 function. Hence, our results uncover spatially and mechanistically distinct requirements for ACT7 function during outer lateral membrane cargo trafficking and polarity establishment. They further identify an exocyst tethering complex mediator of outer lateral membrane cargo polarity.
Highlights
The outermost cell layer of plants, the epidermis, and its outer membrane domain facing the environment are continuously challenged by biotic and abiotic stresses
Compared to control cells treated with dimethyl sulfoxide (DMSO) solvent only (Fig. 1A), a functional PEN3-GFP fusion (PEN3-GFP) expressed from its own promoter (Stein et al, 2006), accumulated in endomembrane agglomerations upon brefeldin A (BFA) treatment, while PEN3-GFP polarity was not affected in epidermal cells (Fig. 1B), consistent with previous observations (Łangowski et al, 2010)
We demonstrate an additional strong requirement for actin function in PEN3 endocytic recycling and identify ACT7 as a major isoform required for actin-dependent trafficking from the trans-Golgi network (TGN) to the plasma membrane (PM)
Summary
The outermost cell layer of plants, the epidermis, and its outer (lateral) membrane domain facing the environment are continuously challenged by biotic and abiotic stresses. Outer membrane proteins involved in ion/nutrient uptake include the IRON-REGULATED TRANSPORTER1 (IRT1; Barberon et al, 2014), the boron exporter BOR4 (Miwa et al, 2007), and the boric acid uptake channel NIP5;1 (Alassimone et al, 2010; Takano et al, 2010), and NIP5;1 subcellular localization depends on D-Gal (Uehara et al, 2014) Another boric acid/borate exporter, BOR1, localizes to the inner lateral membrane of root epidermal cells bordering the cortical cell layer, and specific Tyr residues in BOR1 mediate its polar localization and vacuolar targeting (Takano et al, 2010). Components of the exocyst complex, including EXO84b, EXO70A1, and others, are polarly localized at the outer lateral membrane of Arabidopsis root epidermal cells (Fendrych et al, 2013) Their functions in establishment of outer membrane cargo polarity remain to be further explored. We examine mechanisms underlying PEN3 trafficking to and its polar localization at the outer lateral membrane domain
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