Asymmetric reconstitution of the glucose transporter from Ehrlich ascites cell plasma membrane: Role of alkali cations

Abstract

Gel chromatography of solubilized Ehrlich cell plasma membranes and preformed asolectin vesicles coupled to a freeze-thaw cycle results in the reconstitution of 3- O methyl- d-glucose transport. The transport activity of the liposomes formed is critically dependent on the cation present during reconstitution. Liposomes formed in K + show high levels of carrier-mediated 3- O-methyl- d-glucose uptake (495 pmol/min/mg protein) while those formed in Na + do not (33 pmol/min/mg protein). The inactivity in Na + is not due to a diminished incorporation of glucose transporter nor is it due to carrier molecules reconstituted with a different orientation from those in K + liposomes. Instead, the low glucose transport level in Na + liposomes is related to the small size of vesicles formed with Na +. A second freeze-thaw cycle in K + causes a two- to threefold increase in the available intravesicular volume of Na + liposomes and results in an eightfold increase in carrier-mediated 3- O-methyl- d-glucose uptake. K + liposomes, treated in an identical manner, show only a twofold increase in uptake. The glucose transporter was identified as a protein with a molecular mass range of 44.7 to 66.8 kDa, by the d-glucose inhibitable photoincorporation of [ 3H]cytochalasin B. The carrier protein is inserted in reconstituted vesicles in a nonrandom manner with at least 80% of the molecules oriented with the cytoplasmic domain accessible to the external medium. In contrast, the neutral Na +-dependent amino acid transport system appears to be randomly reconstituted.