Ability of MBP or RBP signal peptides to influence folding and in vitro translocation of wild-type and hybrid precursors.

Maltose-binding protein (MBP), whose export in E. coli is dependent upon the chaperone SecB, and ribose-binding protein (RBP), whose export is SecB-independent, have been used to generate hybrid secretory proteins. Here, in vitro techniques were used to analyze MBP, RBP, RBP-MBP (RBP signal and MBP mature), and MBP-RBP (MBP signal and RBP mature).

Thirty-three amino acids of the mature moiety of an unprocessed maltose-binding protein are sufficient for export in Escherichia coli.

Maltose-binding protein (MBP) is translocated across the cytoplasmic membrane of Escherichia coli; successful export depends on information in both the signal peptide and the mature moiety of the protein. To determine the shortest portion of the mature region that would maintain detectable entry of MBP into the export pathway, we took advantage of the properties of an MBP species with proline substituted in the +1 position relative to the cleavage site (MBP27-P). This protein efficiently crosses the cytoplasmic membrane but is not processed and acts as a competitive inhibitor of signal peptidase I (leader peptidase).

Beta-turn formation in the processing region is important for efficient maturation of Escherichia coli maltose-binding protein by signal peptidase I in vivo.

Signal peptidase I (also called leader peptidase) is the endopeptidase that removes the signal peptides of most secreted proteins during or after translocation in Escherichia coli. Precursor recognition is contingent in part on the presence of small, uncharged residues in the -3 and -1 positions relative to the cleavage site, and may also depend on the structure of the processing region. Most precursor processing regions include residues likely to form a beta-turn.

In vitro processing by signal peptidase I of precursor maltose-binding protein species with alterations in and around the signal peptide.

Processing of 37 precursor maltose-binding protein (preMBP) species by purified signal peptidase I (SPase I) was assayed. The in vitro reaction was inefficient compared to processing in Escherichia coli cells. The extent of preMBP processing in vitro was higher when SPase I was present during translation as compared to processing after translation was arrested by chloramphenicol.

Regions of maltose-binding protein that influence SecB-dependent and SecA-dependent export in Escherichia coli.

In Escherichia coli, the efficient export of maltose-binding protein (MBP) is dependent on the chaperone SecB, whereas export of ribose-binding protein (RBP) is SecB independent. To localize the regions of MBP involved in interaction with SecB, hybrids between MBP and RBP in SecB mutant cells were constructed and analyzed. One hybrid consisted of the signal peptide and first third of the mature moiety of MBP, followed by the C-terminal two-thirds of RBP (MBP-RBP112).