Control of topology and mode of assembly of a polytopic membrane protein by positively charged residues

Abstract

POSITIVELY charged amino acids have been shown to be important elements in targeting-peptides that direct proteins into mitochon-dria, nuclei, and the secretory pathways of both prokaryotic and eukaryotic cells¹. The 'positive-inside' rule, which observes that regions of polytopic (multi-spanning) membrane proteins facing the cytoplasm are generally enriched in arginyl and lysyl residues whereas translocated regions are largely devoid of these residues^2,3, implies that the distribution of positively charged amino acids may also be a major determinant of the transmembrane topology of integral membrane proteins. If this is indeed the case, it should be possible to predictably alter the topology of a polytopic protein by site-directed insertions and/or deletions of positively charged residues in critical locations. I now describe a derivative of Escherichia coli leader peptidase, a polytopic inner-membrane protein, that switches from sec-gene-dependent membrane insertion with a N_out−C_out transmembrane topology to sec-gene-independent insertion with a N_in−C_in topology in response to the addition of four positively charged lysines to its N terminus.