TESTING THE CHARGE DIFFERENCE HYPOTHESIS FOR THE ASSEMBLY OF A EUKARYOTIC MULTISPANNING MEMBRANE-PROTEIN

The Glut1 glucose transporter is a glycoprotein whose membrane topolog y has been verified by a number of experimental observations, all of w hich are consistent with a 12-transmembrane helix model originally bas ed 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 m ultispanning membrane protein is determined solely by the amino acid c harge difference across the first transmembrane segment. The charge di fference across the first transmembrane segment of Glut1 was progressi vely inverted in two independent series of mutants, one series in whic h only the number of positively charged amino acid residues in the two flanking domains was altered and the other in which only the number o f negatively charged residues in the two flanking domains was changed. The results indicate that the charge difference across the first tran smembrane segment does affect the topology of the protein, but that co ntrary to the hypothesis, it only dictates the orientation of the firs t 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 import ant in determining the local topological disposition of the molecule.