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Characterization of phospholipid binding by the exocyst complex using lipid nanodiscs
Author(s) -
Feyder Michael J.,
Munson Mary
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.04375
Subject(s) - exocyst , phospholipid , chemistry , biochemistry , membrane , exocytosis
Proper regulation of exocytosis is crucial for cell function. Post‐Golgi vesicles require precise localization and temporal specification to fuse at correct sites on the plasma membrane to secrete their cargo 1. Secretion is essential for cell survival and is involved in intercellular communication and cell migration 2. Multisubunit tethering complexes (MTCs) are protein assemblies that coordinate interactions required for membrane fusion. Exocyst is a hetero‐octameric MTC responsible for targeting secretory vesicles to the plasma membrane by recognizing SNAREs, small GTPases, and phospholipid 2. Two exocyst subunits, Sec3 and Exo70, have been shown to bind phosphatidylinositol 4,5 biphosphate (PI(4,5)P 2 ). Sec3 contains an N‐terminal Pleckstrin homology (PH) domain and there is a positively charged patch in the C‐terminus of Exo70 3, 4. These two domains mediate interactions with PI(4,5)P 2 . However, these interactions have only been shown using recombinantly purified individual subunits or domains. Combining exocyst purified from cryo‐milled yeast lysate with lipid nanodiscs, we are able to study binding of the wildtype and mutant octameric complex to various phospholipids. This has allowed us to characterize differential contributions of Sec3 and Exo70 to PI(4,5)P 2 binding. Further, nanodiscs allow us to investigate interactions of the exocyst complex with a wide range of phospholipids, demonstrating the specificity of exocyst‐PI(4,5)P 2 binding. This research is crucial to understanding the mechanism by which exocytic machinery is targeted to sites of exocytosis.1 Finger , Fern P. , and Peter Novick . “ Spatial regulation of exocytosis: lessons from yeast .” The Journal of cell biology 142 . 3 ( 1998 ): 609 – 612 . 2 Wu , Bin , and Wei Guo . “ The exocyst at a glance .” J Cell Sci 128 . 16 ( 2015 ): 2957 – 2964 . 3 Zhang , Xiaoyu , et al. “ Membrane association and functional regulation of Sec3 by phospholipids and Cdc42 .” The Journal of cell biology 180 . 1 ( 2008 ): 145 – 158 . 4 He , Bing , et al. “ Exo70 interacts with phospholipids and mediates the targeting of the exocyst to the plasma membrane .” The EMBO journal 26 . 18 ( 2007 ): 4053 – 4065 .