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Genome-scale prediction of subcellular localization (SCL) is not only useful for inferring protein function but also for supporting proteomic data. In line with the secretome concept, a rational and original analytical strategy mimicking the secretion steps that determine ultimate SCL was developed for Gram-positive (monoderm) bacteria. Based on the biology of protein secretion, a flowchart and decision trees were designed considering (i) membrane targeting, (ii) protein secretion systems, (iii) membrane retention, and (iv) cell-wall retention by domains or post-translocational modifications, as well as (v) incorporation to cell-surface supramolecular structures. Using  Listeria monocytogenes  as a case study, results were compared with known data set from SCL predictors and experimental proteomics. While in good agreement with experimental extracytoplasmic fractions, the secretomics-based method outperforms other genomic analyses, which were simply not intended to be as inclusive. Compared to all other localization predictors, this method does not only supply a static snapshot of protein SCL but also offers the full picture of the secretion process dynamics: (i) the protein routing is detailed, (ii) the number of distinct SCL and protein categories is comprehensive, (iii) the description of protein type and topology is provided, (iv) the SCL is unambiguously differentiated from the protein category, and (v) the multiple SCL and protein category are fully considered. In that sense, the secretomics-based method is much more than a SCL predictor. Besides a major step forward in genomics and proteomics of protein secretion, the secretomics-based method appears as a strategy of choice to generate  in silico  hypotheses for experimental testing.

Subcellular Localization of Extracytoplasmic Proteins in Monoderm Bacteria: Rational Secretomics-Based Strategy for Genomic and Proteomic Analyses