Membrane Protein Misfolding Enforces the Positive-Inside Rule

Abstract

Inner membrane proteins have long been known to follow the “positive-inside rule”, where cytoplasmic loops tend to have a greater number of cationic residues than periplasmic or extracellular loops. This effect can be seen dramatically in dual-topology proteins, such as the small multidrug transporter EmrE, where relatively balanced charge between the protein faces leads to mixed insertion in the membrane; some molecules are inserted with a cytoplasmic N-terminus, and some with a periplasmic. Addition or removal of positive charge can bias the orientation of the inserted protein. Surprisingly, this is true even if the mutation does not occur until after the synthesis of several transmembrane helices. Here, we examine how positive charges bring about orientation bias. We use a GFP-based assay to examine both the orientation of the protein as well as rates of misinsertion and degradation. We find a significant pool of degraded or misfolded protein to be present even with wild-type EmrE. In addition to the redistribution of well-folded protein, the amount, type, and orientation of degradation products changes dramatically upon the introduction of mutations that bias EmrE orientation. While early mutations, especially those surrounding the first transmembrane helix, appear capable of biasing the initial orientation, the effects of later mutations can be explained by altering the rate of misfolding events in accordance with the positive-inside rule. Positive charges thus appear to act as folding signals, enhancing proper folding and insertion when positioned in agreement with the positive-inside rule, and enhancing misfolding when placed in violation.