RT Journal
A1 Antelmann, Haike
A1 Tjalsma, Harold
A1 Voigt, Birgit
A1 Ohlmeier, Steffen
A1 Bron, Sierd
A1 van Dijl, Jan Maarten
A1 Hecker, Michael
T1 A Proteomic View on Genome-Based Signal Peptide Predictions
JF Genome Research 
JO Genome Research 
YR 2001 
FD September 01 
VO 11 
IS 9 
SP 1484 
OP 1502 
DO 10.1101/gr.182801 
UL http://genome.cshlp.org/content/11/9/1484.abstract 
AB 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, ∼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 ∼50%. Major problems are currently encountered with the prediction of extracellular proteins lacking signal peptides (including cytoplasmic proteins) and lipoproteins.