Abstract
Membrane proteins play important roles in various cellular processes. Methods that can retain their structure and membrane topology information during their characterization are desirable for understanding their structure-function behavior. Here, we use giant plasma membrane vesicles (GPMVs) to form the supported cell membrane and develop a blotting method to control the orientation of the deposited cell membrane in order to study membrane proteins from either the extracellular or the cytoplasmic sides. We show that the membrane orientation can be retained in the directly-deposited membrane and the deposited membrane on mica can be blotted onto glass to reverse the membrane orientation. We used Aquaporin 3 (AQP3), an abundant native transmembrane protein in Hela cells, as a target to examine the cell membrane orientation in the directly-deposited and reversed membrane platforms. The immunostaining of antibodies targeting either the cyto-domain or ecto-domain of AQP3 shows that the intracellular side of the cell membrane faced the bulk aqueous environment when the GPMVs spontaneously ruptured on the support and that the membrane orientation was reversed after blotting. With this blotting method, we can thus control the orientation of the supported cell membrane to study membrane protein functions and structures from either side of the cell plasma membrane.
Highlights
When membrane proteins are studied and characterized, it is important that their structures be kept intact and that their natural topologies be maintained
We examined the membrane orientation of the giant plasma membrane vesicles (GPMVs) by immunostaining of an asymmetric transmembrane protein, Aquaporin 3 (AQP3)
We used the cell membrane from Hela cells without any gene overexpression or modification, and the native AQP3 in the membrane should be in their native topology and we use the known topological information of AQP3 to postulate the membrane orientation
Summary
When membrane proteins are studied and characterized, it is important that their structures be kept intact and that their natural topologies be maintained. Some studies have shown that the outer leaflets of the cell membrane vesicles faced toward the bulk solution (that is, the outer leaflets were still facing outside-out) after they ruptured on the support[8,9,10,14,16], whereas other studies have shown the opposite result – that is, that the inner leaflets faced toward the bulk solution[13,20]. These different results might be due to the different membrane vesicle sizes used, as suggested by Tutus et al.[9], or to the different preparation methods used to prepare the membrane vesicles. The results show that we can construct cell membrane platforms with controllable membrane orientation
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