The Plant Cytoskeleton, NET3C, and VAP27 Mediate the Link between the Plasma Membrane and Endoplasmic Reticulum

Abstract

The cortical endoplasmic reticulum (ER) network in plants is a highly dynamic structure, and it contacts the plasma membrane (PM) at ER-PM anchor/contact sites. These sites are known to be essential for communication between the ER and PM for lipid transport, calcium influx, and ER morphology in mammalian and fungal cells. The nature of these contact sites is unknown in plants, and here, we have identified a complex that forms this bridge. This complex includes (1) NET3C, which belongs to a plant-specific superfamily (NET) of actin-binding proteins, (2) VAP27, a plant homolog of the yeast Scs2 ER-PM contact site protein, and (3) the actin and microtubule networks. We demonstrate that NET3C and VAP27 localize to puncta at the PM and that NET3C and VAP27 form homodimers/oligomers and together form complexes with actin and microtubules. We show that F-actin modulates the turnover of NET3C at these puncta and microtubules regulate the exchange of VAP27 at the same sites. Based on these data, we propose a model for the structure of the plant ER-PM contact sites.

The Arabidopsis Synaptotagmin1 Is Enriched in Endoplasmic Reticulum-Plasma Membrane Contact Sites and Confers Cellular Resistance to Mechanical Stresses

“…FRAP and time-lapse experiments showed that these structures were remarkably stationary, providing a clear distinction between the pools of highly dynamic intracellular SYT1 and stable cortical SYT1. Interestingly, a previous study has shown that VAP27 and NET3C associate tightly to the cortical MTs and F-actin cables, directly connecting the ER to the cortical cytoskeleton at ER-PM contact sites (Wang et al, 2014). Our data support and expand this view and suggest that the cortical tubulin cytoskeleton also provides positional cues for the establishment of ER-PM contact sites.…”

“…2B). Since the SYT1 localization at the ER-PM interface strongly resembled that of the recently reported Arabidopsis VAP27 and NET3C ER-PM markers (Wang et al, 2014), we next performed transient colocalization studies using the RFP-tagged VAP27 fused to the Ubiquitin-10 promoter (UBQ10proVAP27-RFP) and SYT1proSYT1-GFP markers in N. benthamiana leaves. As shown in Figure 2, C and D, the markers colocalized to a great extent in both intracellular and cortical regions of the cell.…”

“…Similar to the Arabidopsis ER-PM contact site markers VAP27 and NET3C, the SYT1-GFP signal appears as a highly punctuated structure reminiscent of ER-PM anchor sites in both N. benthamiana and Arabidopsis epidermal cells (Wang et al, 2014). FRAP and time-lapse experiments showed that these structures were remarkably stationary, providing a clear distinction between the pools of highly dynamic intracellular SYT1 and stable cortical SYT1.…”

“…Recently, a plant homolog of the yeast Scs2 protein, VAP27, was reported to localize at Arabidopsis ER-PM contact sites, identifying the first molecular components of such sites in plants (Wang et al, 2014). The Arabidopsis SYT1 contains the modular structure typical of tricalbins and E-Syts (Craxton, 2010;Yamazaki et al, 2010), is enriched at ER-PM subdomains, and similar to yeast, where Scs2 and Tcbs interact (Manford et al, 2012), it colocalizes with the ER-PM contact sites marker VAP27.…”

“…In plants, these ER-PM contact sites have been morphologically described for decades (Staehelin, 1997), but their physiological roles have not been thoroughly characterized. Furthermore, the identity of molecular components at these sites has remained elusive until the recent characterization of the ER-PM localized VesicleAssociated Protein27-1 (VAP27) and NETWORKED 3C (NET3C) markers in Arabidopsis (Wang et al, 2014).…”