Intrinsic disorder is more abundant in eukaryotic than prokaryotic proteins.Methods predicting intrinsic disorder are based on the amino acid sequence of a protein.Therefore, there must exist an underlying difference in the sequences between eukaryotic and prokaryotic proteins causing the (predicted) difference in intrinsic disorder.By comparing proteins, from complete eukaryotic and prokaryotic proteomes, we show that the difference in intrinsic disorder emerges from the linker regions connecting Pfam domains.Eukaryotic proteins have more extended linker regions, and in addition, the eukaryotic linkers are significantly more disordered, 38% vs. 12-16% disordered residues.Next, we examined the underlying reason for the increase in disorder in eukaryotic linkers, and we found that the changes in abundance of only three amino acids cause the increase.Eukaryotic proteins contain 8.6% serine; while prokaryotic proteins have 6.5%, eukaryotic proteins also contain 5.4% proline and 5.3% isoleucine compared with 4.0% proline and � 7.5% isoleucine in the prokaryotes.All these three differences contribute to the increased disorder in eukaryotic proteins.It is tempting to speculate that the increase in serine frequencies in eukaryotes is related to regulation by kinases, but direct evidence for this is lacking.The differences are observed in all phyla, protein families, structural regions and type of protein but are most pronounced in disordered and linker regions.The observation that differences in the abundance of three amino acids cause the difference in disorder between eukaryotic and prokaryotic proteins raises the question: Are amino acid frequencies different in eukaryotic linkers because the linkers are more disordered or do the differences cause the increased disorder? Author SummaryIntrinsic disorder is essential for various functions in eukaryotic cells and is a signature of eukaryotic proteins.Here, we try to understand the origin of the difference in disorder between eukaryotic and prokaryotic proteins.We show that eukaryotic proteins contain more extended linker regions and that these linker regions are significantly more disordered.Further, we show, for the first time, that the difference in disorder originates from a systematic difference in amino acid frequencies between eukaryotic and prokaryotic

MOTIVATION: Knowledge of the correct protein subcellular localization is necessary for understanding the function of a protein. Unfortunately large-scale experimental studies are limited in their accuracy. Therefore, the development of prediction methods has been limited by the amount of accurate experimental data. However, recently large-scale experimental studies have provided new data that can be used to evaluate the accuracy of subcellular predictions in human cells. Using this data we examined the performance of state of the art methods and developed SubCons, an ensemble method that combines four predictors using a Random Forest classifier. RESULTS: SubCons outperforms earlier methods in a dataset of proteins where two independent methods confirm the subcellular localization. Given nine subcellular localizations, SubCons achieves an F1-Score of 0.79 compared to 0.70 of the second best method. Furthermore, at a FPR of 1% the true positive rate (TPR) is over 58% for SubCons compared to less than 50% for the best individual predictor. AVAILABILITY AND IMPLEMENTATION: SubCons is freely available as a webserver (http://subcons.bioinfo.se) and source code from https://bitbucket.org/salvatore_marco/subcons-web-server. The golden dataset as well is available from http://subcons.bioinfo.se/pred/download. CONTACT: [email protected]. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

De novo creation of protein coding genes involves the formation of short ORFs from noncoding regions; some of these ORFs might then become fixed in the population. These orphan proteins need to, at the bare minimum, not cause serious harm to the organism, meaning that they should for instance not aggregate. Therefore, although the creation of short ORFs could be truly random, the fixation should be subjected to some selective pressure. The selective forces acting on orphan proteins have been elusive, and contradictory results have been reported. In Drosophila young proteins are more disordered than ancient ones, while the opposite trend is present in yeast. To the best of our knowledge no valid explanation for this difference has been proposed. To solve this riddle we studied structural properties and age of proteins in 187 eukaryotic organisms. We find that, with the exception of length, there are only small differences in the properties between proteins of different ages. However, when we take the GC content into account we noted that it could explain the opposite trends observed for orphans in yeast (low GC) and Drosophila (high GC). GC content is correlated with codons coding for disorder promoting amino acids. This leads us to propose that intrinsic disorder is not a strong determining factor for fixation of orphan proteins. Instead these proteins largely resemble random proteins given a particular GC level. During evolution the properties of a protein change faster than the GC level causing the relationship between disorder and GC to gradually weaken.