title: A proteomic view on genome-based signal peptide predictions.
abstract: The availability of complete genome sequences has allowed the prediction of all exported proteins of the corresponding organisms with dedicated algorithms. Even though numerous studies report on genome-based predictions of signal peptides and cell retention signals, they lack a proteomic verification. For example, 180 secretory and 114 lipoprotein signal peptides were predicted recently for the Gram-positive eubacterium Bacillus subtilis. In the present studies, proteomic approaches were used to define the extracellular complement of the B. subtilis secretome. Using different growth conditions and a hyper-secreting mutant, approximately 200 extracellular proteins were visualized by two-dimensional (2D) gel electrophoresis, of which 82 were identified by mass spectrometry. These include 41 proteins that have a potential signal peptide with a type I signal peptidase (SPase) cleavage site, and lack a retention signal. Strikingly, the remaining 41 proteins were predicted previously to be cell associated because of the apparent absence of a signal peptide (22), or the presence of specific cell retention signals in addition to an export signal (19). To test the importance of the five type I SPases and the unique lipoprotein-specific SPase of B. subtilis, the extracellular proteome of (multiple) SPase mutants was analyzed. Surprisingly, only the processing of the polytopic membrane protein YfnI was strongly inhibited in Spase I mutants, showing for the first time that a native eubacterial membrane protein is a genuine Spase I substrate. Furthermore, a mutation affecting lipoprotein modification and processing resulted in the shedding of at least 23 (lipo-)proteins into the medium. In conclusion, our observations show that genome-based predictions reflect the actual composition of the extracellular proteome for approximately 50%. Major problems are currently encountered with the prediction of extracellular proteins lacking signal peptides (including cytoplasmic proteins) and lipoproteins.
authors.affiliation: Institut für Mikrobiologie und Molekularbiologie, Ernst-Moritz-Arndt-Universiät Greifswald, D-17487 Greifswald, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Antelmann
authors.forename: H
authors.initials: H
authors.lastname: Tjalsma
authors.forename: B
authors.initials: B
authors.lastname: Voigt
authors.forename: S
authors.initials: S
authors.lastname: Ohlmeier
authors.forename: S
authors.initials: S
authors.lastname: Bron
authors.forename: J M
authors.initials: JM
authors.lastname: van Dijl
authors.forename: M
authors.initials: M
authors.lastname: Hecker
mesh_terms: D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism; D001412:Bacillus subtilis / Q000737:chemistry / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D016680:Genome, Bacterial; D008074:Lipoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics*; D020543:Proteome / Q000737:chemistry / Q000235:genetics*; D012697:Serine Endopeptidases / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008074:Lipoproteins; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D020543:Proteome; D012697:Serine Endopeptidases; C026515:type I signal peptidase; D016282:Aspartic Acid Endopeptidases; C095652:signal peptidase II