Position-specific Asp-Lys pairing can affect signal sequence function and membrane protein topology.

Positively charged amino acids are major determinants of the topology of bacterial inner membrane proteins, whereas negatively charged residues by themselves have little or no influence on the transmembrane orientation.Further, positively charged amino acids can very efficiently block the function of signal sequences when placed immediately downstream, while negatively charged residues are much less potent also in this regard.Here, we show that a negatively charged aspartic acid situated close to a positively charged lysine can attenuate both of these effects in a position-specific manner, suggesting that intraor intermolecular charge pairing can modulate the interactions between positively charged residues in the nascent chain and parts of the secretory machinery or membrane phospholipids.These observations further underscore the importance of charged amino acids during protein translocation and membrane protein as- sembly.Positively charged amino acids (lysine and arginine) are important components of topogenic signals in bacterial secretory and membrane-bound proteins.They are typically found at the N terminus of signal sequences (1) and in cytoplasmic segments of inner membrane proteins (2) but are rare immediately downstream of signal sequences (3) and in regions of membrane proteins facing the periplasm.In general, their presence thus tends to correlate with a cytoplasmic location, and many experimental studies have corroborated this observation (4-6).Negatively charged residues, in contrast, have not been found to play any important roles during protein secretion or membrane protein assembly.However, even if topologically impotent when on their own (7), it is still possible that they could affect the topological effects of nearby positively charged residues in certain contexts.Although slight effects of negatively charged residues, e.g. on protein secretion, have been reported (8-lo), no systematic study of the effects of charge pairs on membrane protein topology or the function of signal sequences has been carried out thus far.Here, we have studied the effects of Lys-Asp pairs, both when present in a critical region just downstream of a signal