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Biochemical study of Ist2, a yeast ER-to-plasma membrane tether involved in phosphatidylserine transport at membrane contact sites
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International audience. Ist2 is a yeast transmembrane protein localized in the endoplamic reticulum (ER). It is a structuralhomolog of TMEM16 proteins, a family of transporters described as scramblases, i.e. proteins thatcatalyze facilitated diffusion of lipids in a non-specific manner, and/or anion channels.Ist2 is made of a N-terminal transmembrane domain, as well as a long cytosolic C-terminus that bindsto the PM and associates with Osh6, a yeast cytosolic lipid-transfer protein (LTP) (Figure). LTPs achievethe transport of phosphatidylserine (PS) against its concentration gradient, between the ER and theplasma membrane (PM), by exchanging it with phosphatidylinositol-4-phosphate (PI4P) [1]. Given thatseveral TMEM16 family members function as lipid scramblases, our hypothesis is that Ist2, byscrambling PS across the ER membrane cooperates with Osh6 for PS transport from the ER to the PM.The first step is to express, isolate and determine whether Ist2 indeed functions as a lipid scramblase.For this, it is necessary to reconstitute purified Ist2 in proteoliposomes in order to investigate its abilityto transport lipids across the membrane using well established fluorescence-based assays.Toward that goal, we expressed several versions of the Ist2 protein in the yeast S. cerevisiae, using abiotin acceptor domain (BAD) for affinity purification, as previously defined in our laboratory [2]. TheBAD tag has been placed at either the N- or the C- terminus of the protein and a mCherry fluorescentreporter protein has been inserted between Ist2 and the BAD tag. Moreover, for some of theconstructs, the Ist2 protein has been deleted for its C-terminal part, a deletion of about 300 aminoacids corresponding to the unstructured cytosolic tail of the protein (Figure). This tail contains the sitefor the interaction with Osh6. To date, expression of Ist2 in yeast membranes proved successful,solubilization of Ist2 by detergents has been tested, and a preliminary purification trial has beenperformed, giving encouraging results
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