Abstract
Expression of the Mdr2-protein in secretory vesicules (SVs) from the yeast mutant sec6-4 causes a time- and temperature-dependent enhancement of phosphatidylcholine (PC) translocation from the outer to the inner leaflet of the SV lipid bilayer. We show that this activity is independent of changes either in the membrane potential or the pH gradient (inside positive) generated in these SVs by the yeast proton-translocating PMA1 ATPase. However, loading of the SVs with the primary bile salt taurocholate results in an apparent enhancement of Mdr2-mediated PC translocation activity. Reducing the intravesicular taurocholate (TC) concentration by dissipating the electrochemical potential across the SV membranes eliminates the enhancing effect of TC. Three lines of evidence suggest that the enhanced Mdr2-mediated PC translocation activity is not caused by a regulatory effect of TC on Mdr2 but rather reflected the formation of TC/PC aggregates or micelles in the lumen of SVs. First, significantly higher detergent concentrations are required to reveal the fluorescence of (7-nitro-2-1,3-benzoxadiazol-4-yl)amino-PC molecules translocated in Mdr2-SV under conditions of TC stimulation than under control conditions; second, the nonmicelle-forming bile salt taurodehydrocholate does not cause enhancement of PC translocation in Mdr2-SVs; third, enzyme marker studies indicate that TC behaves as a potent lipid solubilizer directly extracting PC molecules out of the bilayer without causing leakage. This results in the formation of intravesicular aggregates or mixed micelles, and provokes the apparent stimulation of Mdr2 activity. These data demonstrate a unique relationship between Mdr2, PC, and TC in the process of bile formation and secretion.
Highlights
Analysis of the biochemical basis of multidrug resistance (MDR)1 led to the discovery of a group of membrane proteins, called P-glycoproteins or P-gps [1], which become overex
Significantly higher detergent concentrations are required to reveal the fluorescence of (7-nitro-2–1,3-benzoxadiazol-4-yl)amino-PC molecules translocated in Mdr2-SV under conditions of TC stimulation than under control conditions; second, the nonmicelle-forming bile salt taurodehydrocholate does not cause enhancement of PC translocation in Mdr2-SVs; third, enzyme marker studies indicate that TC behaves as a potent lipid solubilizer directly extracting PC molecules out of the bilayer without causing leakage
When the vesicle buffer (VB)/NO3 buffer system was used in the translocation assays, the addition of TC had no effect on the PC translocation activity of Mdr2. Since this latter condition significantly decreases the intravesicular accumulation of TC [37], while leaving unaffected the lipid translocation capacity of Mdr2 (Fig. 2), these results indicate that sufficient amounts of TC are required in the luminal space of SVs to cause the apparent enhancement of Mdr2-mediated NBD-PC translocation activity
Summary
Analysis of the biochemical basis of multidrug resistance (MDR) led to the discovery of a group of membrane proteins, called P-glycoproteins or P-gps [1], which become overex-. Smit et al suggested that the Mdr P-gp may act as a flippase for major phospholipid in normal bile transferring PC molecules from the inner to the outer leaflet of the canalicular membrane We tested this hypothesis directly and recently confirmed that Mdr can function as a PC translocator [24]. Since the reducing anion dithionite is membrane impermeable, it was possible to determine accurately the asymmetric distribution of the labeled lipid [27] Using this experimental system, we observed in SVs from Mdr2-expressing yeast cells a time-and temperature-dependent translocation of the marker lipid from the outer to the inner leaflet of the. This enhanced activity was strictly ATP- and magnesium-dependent and completely abolished by the addition of vanadate or verapamil This activity was specific to the Mdr isoform of P-gps and was not detected in SVs from yeast cells expressing Mdr. The Mdr activity was specific for the phospholipid PC since neither phosphatidylethanolamine (PE) nor phosphatidylserine (PS) were substrates for Mdr
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