Auto-inhibition of Drs2p, a Yeast Phospholipid Flippase, by Its Carboxyl-terminal Tail

Abstract

Background: Drs2p was known to be stimulated by phosphatidylinositol 4-phosphate (PI(4)P), but the mechanism was unclear.Results: Loss of the C-terminal tail, or presence of PI(4)P, stimulates the activity of Drs2p. Conclusion: Inhibition of Drs2p activity by its C-terminal tail is relieved by PI(4)P. Significance: This is the first evidence for an auto-inhibitory domain in a phospholipid flippase.

On the molecular mechanism of flippase- and scramblase-mediated phospholipid transport

“…5A) and activate its flippase activity in a synergistic manner, the C-terminal tail of Drs2p might contain an auto-inhibitory domain. This hypothesis was confirmed by expressing a variant of Drs2p for which a TEV cleavage site had been inserted close to the end of predicted transmembrane segment M10 [148]. Cleavage by the TEV protease removed the PtdIns(4)P binding site together with the rest of the C-terminus.…”

“…The results by Zhou et al [148] raise new questions of how auto-inhibition is precisely achieved for Drs2p. What is the necessary and sufficient region of the C-terminus that is inhibitory?…”

“…Drs2p was later found to be an effector of Gea2p since addition of the Sec7 domain of Gea2p to isolated TGN membranes stimulated the NBD-PtdSer flippase activity of Drs2p [143]. Interestingly, addition of both Gea2p and PtdIns(4)P to TGN membranes promotes synergistic activation of Drs2p [143], although stimulation by Gea2p of ATP hydrolysis by Drs2p could not be observed with purified and reconstituted Drs2p [148], suggesting that Drs2p activation by Gea2p requires an additional cellular factor.…”

“…Cleavage by the TEV protease removed the PtdIns(4)P binding site together with the rest of the C-terminus. Whereas the full-length Drs2p may be activated ~6-fold by PtdIns(4)P with respect to ATPase activity and NBD-PtdSer transport, the truncated version of Drs2p displays an increased basal activity (measured in the absence of PtdIns(4)P) which is no longer stimulated by PtdIns(4)P [148]. This suggested that the Cterminal tail of Drs2p contains an auto-inhibitory domain that can be displaced by addition of PtdIns(4)P.…”

Inner workings and biological impact of phospholipid flippases

“…Moreover, the basic patch overlaps with a binding site for the Arf GEF Gea2, and this interaction also stimulates Drs2 flippase activity. Analogous to regulation of the Ca 2+ P-type ATPase PMCA1 by calmodulin (Di Leva et al, 2008), the C-terminal tail of Drs2 is an auto-inhibitory regulatory domain, and binding to PI4P relieves the auto-inhibition to stimulate activity (Jacquot et al, 2012;Zhou et al, 2013). Whereas PI4P recruits AP-1, GGA and epsinR adaptors to facilitate assembly of budding clathrin-coated vesicles, Gea2p initiates vesicle biogenesis by promoting membrane recruitment of Arf (Behnia and Munro, 2005;Wang et al, 2007;Wang et al, 2003).…”