The EXO70 inhibitor Endosidin2 alters plasma membrane protein composition in Arabidopsis roots

Xiaohui Li; Peipei Zhu; Yen-Ju Chen; Lei Huang; Diwen Wang; David T Newton; Chuan-Chih Hsu; Guang Lin; W Andy Tao; Christopher J Staiger; Chunhua Zhang

doi:10.3389/fpls.2023.1171957

The EXO70 inhibitor Endosidin2 alters plasma membrane protein composition in Arabidopsis roots

Front Plant Sci. 2023 May 31:14:1171957. doi: 10.3389/fpls.2023.1171957. eCollection 2023.

Authors

Xiaohui Li^{1

2}, Peipei Zhu^{3

4}, Yen-Ju Chen^{1

2}, Lei Huang^{1

2}, Diwen Wang^{1

2}, David T Newton⁵, Chuan-Chih Hsu³, Guang Lin^{6

7}, W Andy Tao^{3

4}, Christopher J Staiger^{1

2

8}, Chunhua Zhang^{1

2}

Affiliations

¹ Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN, United States.
² Center for Plant Biology, Purdue University, West Lafayette, IN, United States.
³ Department of Biochemistry, Purdue University, West Lafayette, IN, United States.
⁴ Department of Chemistry, Purdue University, West Lafayette, IN, United States.
⁵ Department of Statistics, Purdue University, West Lafayette, IN, United States.
⁶ Department of Mathematics, Purdue University, West Lafayette, IN, United States.
⁷ School of Mechanical Engineering, Purdue University, West Lafayette, IN, United States.
⁸ Department of Biological Sciences, Purdue University, West Lafayette, IN, United States.

Abstract

To sustain normal growth and allow rapid responses to environmental cues, plants alter the plasma membrane protein composition under different conditions presumably by regulation of delivery, stability, and internalization. Exocytosis is a conserved cellular process that delivers proteins and lipids to the plasma membrane or extracellular space in eukaryotes. The octameric exocyst complex contributes to exocytosis by tethering secretory vesicles to the correct site for membrane fusion; however, whether the exocyst complex acts universally for all secretory vesicle cargo or just for specialized subsets used during polarized growth and trafficking is currently unknown. In addition to its role in exocytosis, the exocyst complex is also known to participate in membrane recycling and autophagy. Using a previously identified small molecule inhibitor of the plant exocyst complex subunit EXO70A1, Endosidin2 (ES2), combined with a plasma membrane enrichment method and quantitative proteomic analysis, we examined the composition of plasma membrane proteins in the root of Arabidopsis seedlings, after inhibition of the ES2-targetted exocyst complex, and verified our findings by live imaging of GFP-tagged plasma membrane proteins in root epidermal cells. The abundance of 145 plasma membrane proteins was significantly reduced following short-term ES2 treatments and these likely represent candidate cargo proteins of exocyst-mediated trafficking. Gene Ontology analysis showed that these proteins play diverse functions in cell growth, cell wall biosynthesis, hormone signaling, stress response, membrane transport, and nutrient uptake. Additionally, we quantified the effect of ES2 on the spatial distribution of EXO70A1 with live-cell imaging. Our results indicate that the plant exocyst complex mediates constitutive dynamic transport of subsets of plasma membrane proteins during normal root growth.

Keywords: exocyst; exocytosis; plasma membrane protein enrichment; proteomics; small molecule inhibitors.

Grants and funding

GL gratefully acknowledges the support from National Science Foundation (DMS-1555072, DMS-1736364 and DMS-1821233). XL, LH, DW, and CZ were supported by Start-up funds from the Office of the Provost at Purdue University. XL was also partially supported by a research assistantship from the Purdue Center for Plant Biology.