The object of this proposal is to continue the study of two aspects of our recent discovery in the field of membrane biology. The first, is the spontaneous insertion of integral membrane proteins into preformed bilayers and the second is the membrane fusion that is catalyzed by the same methodology. We have observed that the presence of impurities in the bilayer of preformed vesicles triggers both the incorporation of purified integral membrane proteins as well as the fusion of unilamellar proteoliposomes when the phospholipids are in the gel state. The rapid fusion of liposomes and proteoliposomes by this manipulation of the phospholipid bilayer presents an ideal model system for the study of the cellular membrane fusion at the level of the phospholipid bilayer. We propose to utilize this system to investigate several concepts in membrane biology while we extend the applicability of this method for reconstruction studies. We plan to construct proteoliposomes with several different membrane proteins by this procedure; some of the integral membrane proteins for this study will be selected to demonstrate a functional interaction within the newly formed membrane while others will be employed to modify the surface availability and characteristics of the proteoliposome. As a result of this aspect of the work, we expect to be able to construct a model cell membrane composed of several different integral membrane proteins in which many of the current concepts in cellular and membrane biology can be tested. Furthermore, the apparent ability to construct proteoliposomes of cellular dimension (7 microns) by this procedure will be further examined in an effort to design and construct a model cell of predetermined protein composition and lipid/protein ratio. The stability of these very large proteoliposomes of high lipid/protein ratio has exceeded our expectations and we believe that the proposed proteoliposomes of low lipid-to-protein ratio will provide a very stable vesicle which may represent the ideal model cell. The long range goals are two-fold with respect to the phenomenon we have discovered. We propose to determine whether the mechanism by which we have incorporated integral membrane proteins into preformed bilayers represents the in vivo participation of the phospholipid in cellular membranes. We will determine too whether the specificity of membrane fusion among biological membrane is regulated in part by the proposed mechanism for the fusion of proteoliposomes in this procedure.
| Scotto, A W; Gompper, M E (1990) Spontaneous incorporation of bacteriorhodopsin into large preformed vesicles. Biochemistry 29:7244-51 |
