Orientation of membrane vesicles from Escherichia coli as detected by freeze cleave electron microscopy

Abstract

The application of freeze cleave electron microscopy to whole cells of E. coli revealed that the particles exposed on the resulting two inner membrane faces are asymmetrically distributed. This method can therefore be used to determine the orientation of membrane vesicles from E. coli. Membrane vesicles freshly prepared in potassium phosphate buffer (K+ vesicles) by osmotic lysis of spheroplasts consisted almost entirely of right side out vesicles. Their size suggested that each cell gives rise to one vesicle. When the membrane vesicles were subjected to one cycle of freezing and thawing, the number of inside out vesicles rose to about 25%. However, due to the small size of most of the inside out vesicles, these contribute only 2 to 3% of the total membrane surface area of the preparation. The inside out vesicles appear to arise from infoldings of the membrane of right side out vesicles. They also accumulate within the latter, thus producing multivesicular membrane sacs. Na+ vesicles (vesicles prepared in sodium phosphate buffer) subjected to freezing and thawing appeared to lose structural rigidity more than did K+ vesicles. In contrast to the membrane vesicles prepared by the osmotic lysis of spheroplasts, those obtained by breaking intact cells by a single passage through a French pressure cell were uniformly very small (only 40 to 110 nm in diameter); approximately 60 to 80% were inside out. To reconcile the polarity of the membrane vesicles with the enzymic activities of such preparations, we propose that 'dislocation' of membrane proteins occurs during osmotic lysis of spheroplasts.