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. Complete conversion of wild-type (wt) preMBP (greater than 90%) to mature protein required 4300-fold more enzyme than substrate during a 15 min reaction. Most preMBP species with alterations in the signal peptide processing region that were efficiently processed (greater than 85%) in vivo were also processed in vitro, although the efficiency of processing was usually lower than the corresponding in vivo value. Increasing the level of SPase I in the in vitro reaction often increased the extent of preMBP processing. A number of amino acid substitutions in the processing region that drastically reduced or eliminated processing in vivo also eliminated processing in vitro. Processing occurred at an alternate site in some mutant preMBP species in vivo, but this event occurred very inefficiently in vitro. Amino acid substitutions in the hydrophobic core or in the charged regions at the N-terminus of the signal peptide and early mature region of preMBP slightly reduced in vitro processing as compared to processing of wt preMBP, regardless of their effect on secretion in vivo.
Talarico
T L
TL
Department of Molecular Genetics and Microbiology, Burroughs Wellcome Co., Research Triangle Park, NC 27709.
Barkocy-Gallagher
G A
GA
Ray
P H
PH
Bassford
P J
PJ
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000302:isolation & purification / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism*; D012697:Serine Endopeptidases; D013379:Substrate Specificity
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; C461599:maltose transport system, E coli; C059025:preMBP protein, E coli; D008320:Maltose; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase