Abstract
Escherichia coli-derived phosphatidylethanolamine (PE) or PE with fully saturated fatty acids was able to correct in vitro a defect in folding in the lipid-dependent epitope 4B1 of lactose permease (LacY) resulting from in vivo assembly in the absence of PE. PE plasmalogen, PE with two unsaturated fatty acids, and lyso-PE, which all do not favor bilayer organization, did not support proper refolding. Proper refolding occurred when these latter lipids were mixed with a bilayer-forming lipid (phosphatidylglycerol), which alone could not support refolding. L-Phosphatidylserine (PS; natural diastereomer) did support proper refolding. PE derivatives of increasing degrees of methylation were progressively less effective in supporting refolding, with phosphatidylcholine being completely ineffective. Therefore, the properties of nonmethylated aminophospholipids capable of organization into a bilayer configuration are essential for the recovery of the native state of epitope 4B1 after misassembly in vivo in the absence of PE. Neither D-PS (sn-glycero-1-phosphate backbone) nor P-D-S (D-serine in the head group) is competent in supporting proper refolding unless used in binary mixtures with phosphatidylglycerol. The detailed characterization of phospholipid-assisted refolding reported here further supports a specific rather than nonspecific role for PE in structural maturation of lactose permease in vivo (Bogdanov, M., and Dowhan, W. (1998) EMBO J. 17, 5255-5264).
Highlights
Escherichia coli-derived phosphatidylethanolamine (PE) or PE with fully saturated fatty acids was able to correct in vitro a defect in folding in the lipid-dependent epitope 4B1 of lactose permease (LacY) resulting from in vivo assembly in the absence of PE
We proposed as a general principle that phospholipids can act as molecular chaperones of non-protein origin that mediate the folding of multispanning polytopic membrane proteins, thereby extending the definition of chaperones to other biomolecules in addition to proteins
Effect of Lamellar Versus Non-lamellar Forming Phospholipids on LacY Refolding—We previously demonstrated [8] that LacY assembled in vivo in PE-containing membranes, but not in PE-deficient membranes, subjected to SDS-PAGE and Western blot analysis is recognized by a conformation-specific monoclonal antibodies (mAbs) 4B1 directed against the continuous epitope 4B1 (Fig. 1) within the periplasmic domain P7 [8]
Summary
Escherichia coli-derived phosphatidylethanolamine (PE) or PE with fully saturated fatty acids was able to correct in vitro a defect in folding in the lipid-dependent epitope 4B1 of lactose permease (LacY) resulting from in vivo assembly in the absence of PE. Phosphatidylethanolamine as a Molecular Chaperone in vitro a LacY folding defect caused by either in vivo or in vitro assembly in PE-deficient membranes, but once this epitope is formed in vivo, PE is not required to maintain its conformation Based on these results, we proposed as a general principle that phospholipids can act as molecular chaperones of non-protein origin that mediate the folding of multispanning polytopic membrane proteins, thereby extending the definition of chaperones to other biomolecules in addition to proteins. The proper conformation of epitope 4B1 is required for full function of LacY as a transporter [5]
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