Arabidopsis Synaptotagmin 1 Is Required for the Maintenance of Plasma Membrane Integrity and Cell Viability

Abstract

Plasma membrane repair in animal cells uses synaptotagmin 7, a Ca 2+ -activated membrane fusion protein that mediates delivery of intracellular membranes to wound sites by a mechanism resembling neuronal Ca 2+ -regulated exocytosis. Here, we show that loss of function of the homologous Arabidopsis thaliana Synaptotagmin 1 protein (SYT1) reduces the viability of cells as a consequence of a decrease in the integrity of the plasma membrane. This reduced integrity is enhanced in the syt1-2 null mutant in conditions of osmotic stress likely caused by a defective plasma membrane repair. Consistent with a role in plasma membrane repair, SYT1 is ubiquitously expressed, is located at the plasma membrane, and shares all domains characteristic of animal synaptotagmins (i.e., an N terminus-transmembrane domain and a cytoplasmic region containing two C2 domains with phospholipid binding activities). Our analyses support that membrane trafficking mediated by SYT1 is important for plasma membrane integrity and plant fitness.

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

“…A high-resolution subcellular localization for SYT1 was obtained by immunogold Transmission Electron Microscopy (immunoTEM) of high-pressure frozen, freezesubstituted wild-type roots probed with the anti-SYT1 antibody. Similar to previous immunoTEM results (Schapire et al, 2008), the anti-SYT1 signal was observed at both the ER and PM (Supplemental Fig. S4).…”

“…Moreover, cell biological and biochemical analyses have reported SYT1 being at the ER, PM, and plasmodesmata (Schapire et al, 2008;Lewis and Lazarowitz, 2010;Yamazaki et al, 2010). To unequivocally establish the SYT1 subcellular localization, we developed new polyclonal anti-SYT1 244-541 antibodies (hereafter, anti-SYT1), and demonstrated their specificity by the absence of signal in the null syt1-2 mutant (hereafter, syt1) via western blot and immunolocalization (Supplemental Fig.…”

“…According to the tensegrity model, the SYT1-labeled ER-PM contact sites might function as deformable platforms that coordinate the cortical ER and cytoskeleton responses to minimize the mechanical loads at the PM. This model would not require the activation of transcriptional programs and can explain the instantaneous PM instability observed upon exposure to multiple stresses, including freezing, osmotic, and mechanical challenge (Yamazaki et al, 2008;Schapire et al, 2008;this study). This model could also explain the enhanced cytoskeleton depolymerization observed in the syt1 mutant due to the limited ability of SYT1-depleted contact sites to properly transfer mechanical cues to the cortical cytoskeleton (Fig.…”

“…To assess the accuracy of these predictions and to distinguish between the two possible SYT1 orientations (TM anchored at the ER and C2 domains tethering the PM, or TM anchored at the PM and C2 domains tethering the ER), we first analyzed where isolated SYT1 C2 domains were targeted. For this purpose, we generated stable transgenic lines harboring the SYT1 244-541 peptide containing the GFP-tagged SYT1 C2 domains C2A and C2B (SYT1-C2AB; Schapire et al, 2008) fused to the 35S promoter. We also used a 35SproGFP and the SYT1-GFP lines as controls.…”

“…In previous reports, SYT1 has been described as an essential component for PM integrity maintenance, especially under conditions of high potential for membrane disruption such as freezing or salt stresses (Schapire et al, 2008(Schapire et al, , 2009Yamazaki et al, 2008). In these reports, SYT1 was proposed to act as a Ca 2+ -dependent regulator of membrane fusion, in analogy to the classical animal SYTs that mediate Ca 2+ -triggered vesicle fusion during neurotransmission (Carr and Munson, 2007).…”