Glycophorin A transmembrane domain dimerization in plasma membrane vesicles derived from CHO, HEK 293T, and A431 cells

Abstract

Membrane protein interactions, which underlie biological function, take place in the complex cellular membrane environment. Plasma membrane derived vesicles are a model system which allows the interactions between membrane proteins to be studied without the need for their extraction, purification, and reconstitution into lipid bilayers. Plasma membrane vesicles can be produced from different cell lines and by different methods, providing a rich variety of native-like model systems. With these choices, however, questions arise as to how the different types of vesicle preparations affect the interactions between membrane proteins. Here we address this question using the glycophorin A transmembrane domain (GpA) as a model system. We compare the dimerization of GpA in six different vesicle preparations derived from Chinese hamster ovary (CHO), Human Embryonic Kidney 293T (HEK 293T) and A431 cells. We accomplish this with the use of a FRET-based method which yields the FRET efficiency, the donor concentration, and the acceptor concentration in each vesicle. We show that the vesicle preparation protocol has no statistically significant effect on GpA dimerization. Based on these results, we propose that any of the six plasma membrane preparations investigated here can be used as a model system for studies of membrane protein interactions.

Figure 1

FRET efficiencies measured for GpA in plasma membrane derived vesicles, as a function of acceptor concentration. Genes encoding GpA attached to either YFP or mCherry at its C-terminus via a (GGS)₅ linker were expressed in cells. After GpA was trafficked to the plasma membrane, the cells were vesiculated using three different methods. (A) CHO, HEK293T and A431 vesicles, produced with the DTT/formaldehyde method of Scott (17). (B) CHO vesicles, produced with the DTT/formaldehyde method and the new chloride salt osmotic method that we have developed (21). (C) A431 cells vesiculated using the DTT/formaldehyde method, the chloride salt method, and the salt cocktail method developed by Cohen at al (20). Each data point corresponds to a single vesicle.

Figure 2

Dimeric GpA fractions as a function of total GpA concentration. The dimeric fractions were averaged in bins of 5 × 10 ⁻⁴ receptors/nm². The solid lines are the fits of the dimerization model to the data points prior to their binning. (A) CHO, HEK293T and A431 vesicles, produced with the DTT/formaldehyde method. (B) CHO vesicles, produced with the DTT/formaldehyde method and the chloride salt osmotic method. (C) A431 cells vesiculated using the three different methods.