Abstract
Leader peptidase, typical of inner membrane proteins of Escherichia coli, does not have an amino-terminal leader sequence. This protein contains an internal signal peptide, residues 51-83, which is essential for assembly and remains as a membrane anchor domain. We have employed site-directed mutagenesis techniques to either delete residues within this domain or substitute a charged amino acid for one of these residues to determine the important properties of the internal signal. The deletion analysis showed that a very small apolar domain, residues 70-76, is essential for assembly, whereas residues that flank it are dispensable for its function. However, point mutations with charged amino acid residues within the polar sequence (residues 77-82) slow or abolish leader peptidase membrane assembly. Thus, a polar region, Arg-Ser-Phe-Ile-Tyr-Glu, is important for the signal peptide function of leader peptidase, unlike other signals identified thus far.
Highlights
Leader peptidase, typical of inner membrane pro- (Dalbey and Wickner, 1985)
The removal of leader peptides teins of Escherichia coli, doesnothaveanaminoby leader peptidase in Escherichia coli is necessary for exterminal leader sequence
In a recent study (Dalbey and Wickner, 1988) we showed that the apolar character of this region is important for membrane assembly of leader peptidase, whereas the basic amino terminusof the internalsignal is not required for efficient assembly
Summary
We have defined residues 51-83 astheinternal signal peptide of leader peptidase because it constitutes an interchangeable element with the leader peptide of OmpA (Dalbey et al, 1987).This signal is comprised of a basic amino terminus and a central apolar domain. Even after 10 min of chase, leader peptidase lacking residues 62-70 (Fig. 5C) was inaccessible to protease This again shows that the apolar core (residues 70-76) is vital for the signal's function. Assembly when this apolar core was completely removed The most striking effects on assembly occurin the hydro- carboxyl-terminal regions that maybe important for their philic region, adjacent to the second apolar domain Position 79, cannot translocate across the membrane even 3) A third possibility is that thepolar region is part after 10 min of chase (Fig.7A) This wasvery surprising of the signal and comprises part of the membrane-spanning because few examples exist where polar sequences,which region.
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