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Transport Proteins Can Be Studied Using Artificial Membranes and Recombinant Cells

المؤلف:  Harvey Lodish, Arnold Berk, Chris A. Kaiser, Monty Krieger, Anthony Bretscher, Hidde Ploegh, Angelika Amon, and Kelsey C. Martin.

المصدر:  Molecular Cell Biology

الجزء والصفحة:  8th E , P480

2026-05-21

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Transport Proteins Can Be Studied Using Artificial Membranes and Recombinant Cells There are a variety of approaches to studying the intrinsic properties of transport proteins, such as the Vmax and Km parameters and the key residues responsible for binding. Most cellular membranes contain many different types of transport proteins but a relatively low concentration of any particular one, which makes functional studies of a single protein difficult. To facilitate such studies, researchers use two approaches to enrich a transport protein of interest so that it predominates in the membrane: purification and insertion into artificial membranes, and overexpression in recombinant cells.

In the first approach, a specific transport protein is extracted from its membrane with detergent and purified. Al though transport proteins can be isolated from membranes and purified, their functional properties (i.e., their role in the movement of substrates across membranes) can be studied only when they are associated with a membrane. Thus the purified proteins are usually reincorporated into pure phospholipid bilayer membranes, such as liposomes, across which substrate transport can be readily measured. One good source of GLUT1 is erythrocyte mem branes. Another is recombinant cultured mammalian cells that express a GLUT1 transgene, often one that encodes a modified GLUT1 that contains an epitope tag [a portion of a molecule to which a monoclonal antibody can bind] fused to its N- or C-terminus. All of the integral membrane proteins in either of these two types of cells can be extracted by using a non-ionic detergent such as octylglucoside, which solubilizes the membrane without significantly denaturing the membrane proteins. The glucose uniporter GLUT1 can be purified from the solubilized mixture by antibody affinity chromatography on a column containing either a GLUT1-specific monoclonal antibody or an antibody specific for the epitope tag, then incorporated into liposomes made of pure phospholipids.

Alternatively, the gene encoding a specific transport protein can be expressed at high levels in a cell type that normally does not express it. The difference between the transport rate of a substance by the transfected cells and by control nontransfected cells will be due to the expressed transport protein. In these systems, the functional properties of the various membrane proteins can be examined without ambiguity caused, for instance, by partial protein denaturation during isolation and purification procedures. As an example, over expressing GLUT1 in lines of cultured fibroblasts increases their rate of uptake of glucose severalfold, and expression of mutant GLUT1 proteins with specific amino acid alterations can identify residues important for substrate binding.