Protein X is an unknown membrane protein found in erythrocytes that can be extracted primarily from plasma membranes using a concentrated salt solution. What type of membrane protein is protein X? Since protein X is extracted from the membrane by treatment with a concentrated salt solution, it is an integral membrane protein with a large number of polar or charged amino acids. A salt solution is required to extract protein X from the membrane; therefore, it is an amphitropic membrane protein that is reversibly attached to the membrane. Treatment with a concentrated salt solution will extract integral, peripheral, and amphitropic membrane proteins; thus, the type of membrane protein cannot be determined. Protein X is an integral membrane protein because it is extracted from the membrane using a concentrated salt solution. Since protein X is extracted from the membrane by salt treatment, it is a peripheral membrane protein. Continued below... To determine the location of protein X in the plasma membrane, erythrocytes were subjected to two different treatments. First, intact erythrocytes were treated with proteolytic enzymes. After the cells were lysed and membrane components extracted, protein X was intact. In the second experiment, the erythrocyte cells were permeabilized prior to treatment with proteolytic enzymes. When the plasma membrane components were then extracted, protein X was extensively fragmented. What do these observations suggest about the location of protein X in the plasma membrane? Proteolytic enzymes cannot fragment protein X in intact erythrocytes; thus, protein X is embedded in the lipid bilayer and does not have any extracellular domains. Since proteolytic enzymes cannot digest protein X unless the plasma membrane is disrupted, protein X is polytopic and embedded in the lipid bilayer with several intracellular domains. Since proteolytic enzymes cannot digest protein X unless the plasma membrane of intact cells is disrupted, protein X associates with the inner surface of the membrane. Since proteolytic enzymes cannot digest protein X unless the plasma membrane is disrupted, protein X is covalently attached to the inner leaflet of the lipid bilayer. Proteolytic enzymes cannot fragment protein X in intact erythrocytes; therefore, protein X associates with the membrane transiently and is primarily located in the cytosol.