Mutations eliminating the protein export function of a membrane-spanning sequence.

Abstract

Individual membrane protein spanning sequences can promote protein export. To help define the sequence features necessary for this action, we identified mutations disrupting export mediated by the first spanning sequence (TM1) of the Escherichia coli serine chemoreceptor. Mutant spanning sequences were generated and characterized using beta-galactosidase and alkaline phosphatase gene fusions. The protein export function of TM1 was remarkably tolerant of single charged residues, and the introduction of pairs of charged amino acids was necessary to eliminate export. The results are accommodated by a model in which export requires a stretch of uncharged residues whose summed hydrophobicity exceeds a particular threshold value. This threshold approximates the minimum hydrophobicity required for cleavable signal sequence function. In addition, the threshold was near the minimum hydrophobicity observed for wild-type spanning sequences in a collection of topologically characterized membrane proteins.