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UBC Theses and Dissertations

The TMEM16 tethering protein Ist2 directly interacts with the ORP lipid transporters Osh6 and Osh7 to maintain aminophospholipid metabolism Wong, Andrew King On

Abstract

Membrane contact sites are sites of close apposition between two subcellular membranes that are believed to facilitate calcium signaling and lipid transport between organelles. The endoplasmic reticulum (ER) forms contacts with many organelles as well as the plasma membrane (PM). ER-PM contacts have been proposed to be held together by many families of protein tethers, and in the budding yeast Saccharomyces cerevisiae, proposed tethering proteins include the vesicle-associated membrane protein-associated protein homologues Scs2/22, the extended synaptotagmin homologues Tcb1/2/3, transmembrane protein Ice2, the lipin Pah1, and the transmembrane protein 16 (TMEM16) homologue Ist2. While these proteins are believed to function redundantly in creating ER-PM contacts, many of these proteins also possess functional modules such as lipid transfer domains, suggesting that they may also have specific functions in addition to tethering, with the identification of these functions being an area of current research. Here, by leveraging existing high-throughput datasets as a starting point, we identified Ist2 as a yeast ER-PM tether that participates in transport of the lipid phosphatidylserine (PS) from the ER to the PM through a physical interaction with the oxysterol-binding protein-related protein homologues and lipid transfer proteins Osh6 and Osh7. We found that Ist2 binds to both Osh6/7 through a binding site located in its disordered linker region between its ER-anchored TMEM16 domain and its C-terminal PM-binding helix. As well, we uncovered genetic evidence that Ist2 and Osh6/7 function in the same pathway as the PS decarboxylase Psd2, with loss of Ist2 or both Osh6/7 resulting in a strong growth defect and decreased cellular phosphatidylethanolamine levels when combined with loss of a redundant PS decarboxylase Psd1. Thus, these findings identify a physiologically relevant molecular link between members of the ORP and TMEM16 family of proteins and lays the groundwork for future studies to uncover the function of these proteins at membrane contact sites.

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