Testing the charge difference hypothesis for the assembly of a eucaryotic multispanning membrane protein

The Glut1 glucose transporter is a glycoprotein whose membrane topology has been verified by a number of experimental observations, all of which are consistent with a 12-transmembrane helix model originally based on hydrophobicity analysis. We used Glut1 as a model multispanning membrane protein to test the Charge Difference Hypothesis (Hartmann, E., Rapoport, T. A., and Lodish, H. F. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5786-5790), which asserts that the topology of a eucaryotic multispanning membrane protein is determined solely by the amino acid charge difference across the first transmembrane segment. The charge difference across the first transmembrane segment of Glut1 was progressively inverted in two independent series of mutants, one series in which only the number of positively charged amino acid residues in the two flanking domains was altered and the other in which only the number of negatively charged residues in the two flanking domains was changed. The results indicate that the charge difference across the first transmembrane segment does affect the topology of the protein, but that contrary to the hypothesis, it only dictates the orientation of the first transmembrane segment and the disposition of the amino terminus and the first linker domain. Charge inversion resulted in the formation of aberrant molecules in which either the first or second transmembrane segment failed to insert into the membrane. The topology of downstream regions of Glut1 was unaffected by charge inversion across the first transmembrane segment, indicating that downstream sequences are important in determining the local topological disposition of the molecule.