Surface plasmon resonance-based interaction studies reveal competition of Streptomyces lividans type I signal peptidases for binding preproteins.
Type I signal peptidases (SPases) are responsible for the cleavage of signal peptides from secretory proteins. Streptomyces lividans contains four different SPases, denoted SipW, SipX, SipY and SipZ, having at least some differences in their substrate specificity. In this report in vitro preprotein binding/processing and protein secretion in single SPase mutants was determined to gain more insight into the substrate specificity of the different SPases and the underlying molecular basis. Results indicated that preproteins do not preferentially bind to a particular SPase, suggesting SPase competition for binding preproteins. This observation, together with the fact that each SPase could process each preprotein tested with a similar efficiency in an in vitro assay, suggested that there is no real specificity in substrate binding and processing, and that they are all actively involved in preprotein processing in vivo. Although this seems to be the case for some proteins tested, high-level secretion of others was clearly dependent on only one particular SPase demonstrating clear differences in substrate preference at the in vivo processing level. Hence, these results strongly suggest that there are additional factors other than the cleavage requirements of the enzymes that strongly affect the substrate preference of SPases in vivo.
Geukens
Nick
N
Laboratory of Bacteriology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
Rao C V
Smitha
S
Laboratory of Bacteriology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
Mellado
Rafael P
RP
Department of Microbial Biotechnology, Centro Nacional de Biotecnologia, Campus de la Universidad Autonoma, Cantoblanco, 28049 Madrid, Spain.
Frederix
Filip
F
IMEC, MCP-ART, Kapeldreef 75, B-3001 Leuven, Belgium.
Reekmans
Gunter
G
IMEC, MCP-ART, Kapeldreef 75, B-3001 Leuven, Belgium.
De Keersmaeker
Sophie
S
Laboratory of Bacteriology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
Vrancken
Kristof
K
Laboratory of Bacteriology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
Bonroy
Kristien
K
IMEC, MCP-ART, Kapeldreef 75, B-3001 Leuven, Belgium.
Van Mellaert
Lieve
L
Laboratory of Bacteriology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
Lammertyn
Elke
E
Laboratory of Bacteriology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
Anné
Jozef
J
Laboratory of Bacteriology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D003470:Culture Media / Q000737:chemistry; D017353:Gene Deletion; D008565:Membrane Proteins / Q000378:metabolism*; D011485:Protein Binding; D011498:Protein Precursors / Q000378:metabolism*; D021382:Protein Sorting Signals; D021381:Protein Transport; D012697:Serine Endopeptidases / Q000378:metabolism*; D048170:Streptomyces lividans / Q000201:enzymology*; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance
D001426:Bacterial Proteins; D003470:Culture Media; D008565:Membrane Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D012697:Serine Endopeptidases; C026515:type I signal peptidase