Arabidopsis EXO70A1 recruits Patellin3 to the cell membrane independent of its role as an exocyst subunit

Abstract

The exocyst is a well-known complex which tethers vesicles at the cell membrane before fusion. Whether an individual subunit can execute a unique function is largely unknown. Using yeast-two-hybrid (Y2H) analysis, we found that EXO70A1 interacted with the GOLD domain of Patellin3 (PATL3). The direct EXO70A1-PATL3 interaction was supported by in vitro and in vivo experiments. In Arabidopsis, PATL3-GFP colocalized with EXO70A1 predominantly at the cell membrane, and PATL3 localization was insensitive to BFA and TryA23. Remarkably, in the exo70a1 mutant, PATL3 proteins accumulated as punctate structures within the cytosol, which did not colocalize with several endomembrane compartment markers, and was insensitive to BFA. Furthermore, PATL3 localization was not changed in the exo70e2, PRsec6 or exo84b mutants. These data suggested that EXO70A1, but not other exocyst subunits, was responsible for PATL3 localization, which is independent of its role in secretory/recycling vesicletethering/fusion. Both EXO70A1 and PATL3 were shown to bind PI4P and PI(4,5)P 2 in vitro. Evidence was obtained that the other four members of the PATL family bound to EXO70A1 as well, and shared a similar localization pattern as PATL3. These findings offered new insights into exocyst subunitspecific function, and provided data and tools for further characterization of PATL family proteins.

Arabidopsis Trichome Contains Two Plasma Membrane Domains with Different Lipid Compositions Which Attract Distinct EXO70 Subunits

“…To address the biological relevance of the trichome lipid domain distribution, we observed multiple EXO70 proteins under the control of the EXO70H4 promoter. EXO70A1 was previously found to bind to PIP 2 [24] and to localize to the PIP 2 -rich region of the pollen tube [20]. Corresponding with its lipid affinity, EXO70A1 localized preferentially to the basal trichome domain and, in many cases, the apical trichome domain was completely devoid of EXO70A1, although there was a certain degree of variability and it was often seen also within the Ortmannian ring area (Figure 3a, Supplemental Figure S1).…”

Identification and Characterization of the EXO70 Gene Family in Polyploid Wheat and Related Species

“…The bacterial cells were centrifuged and resuspended in an infiltration solution (10 mM MES pH 5.6, 0.1 mM Acetosyringone, 10 mM MgCl 2 ) to a final OD 600 = 1.5. Bacterial suspensions were infiltrated into five- to six-week growing stage leaves of N. benthamiana by depressing the plunger of a 1-mL disposable needleless syringe into the abaxial side of leaves [85,86]. The fluorescence signals were observed 48–60 h after injection and images were captured using a confocal laser scanning microscope (LSM780; Carl Zeiss, Jena, Germany) according to the methods described by Wang et al [87].…”

Remove, Recycle, Degrade: Regulating Plasma Membrane Protein Accumulation

“…ES2 affects EXO70A1 localization at the PM, but not EXO84B localization, supporting their independent assembly and function in the complex. EXO70A1 interacts with PtdIns(4)P and PtdIns(4,5)P2 at the PM, showing protein enrichment at apical and basal domains of epidermal root cells (Figures 6A and C;Wu et al, 2017). Mutants defective in phosphoinositol synthesis show alterations in PIN trafficking and localization (Ischebeck et al, 2013;Tejos et al, 2014;Ugalde et al, 2016).…”