HECTAR: a method to predict subcellular targeting in heterokonts

Abstract

Background: The heterokonts are a particularly interesting group of eukaryotic organisms; they include many key species of planktonic and coastal algae and several important pathogens. To understand the biology of these organisms, it is necessary to be able to predict the subcellular localisation of their proteins but this is not straightforward, particularly in photosynthetic heterokonts which possess a complex chloroplast, acquired as the result of a secondary endosymbiosis. This is because the bipartite target peptides that deliver proteins to these chloroplasts can be easily confused with the signal peptides of secreted proteins, causing currently available algorithms to make erroneous predictions. HECTAR, a subcellular targeting prediction method which takes into account the specific properties of heterokont proteins, has been developed to address this problem.

Results: HECTAR is a statistical prediction method designed to assign proteins to five different categories of subcellular targeting: Signal peptides, type II signal anchors, chloroplast transit peptides, mitochondrion transit peptides and proteins which do not possess any N-terminal target peptide. The recognition rate of HECTAR is 96.3%, with Matthews correlation coefficients ranging from 0.67 to 0.95. The method is based on a hierarchical architecture which implements the divide and conquer approach to identify the different possible target peptides one at a time. At each node of the hierarchy, the most relevant outputs of various existing subcellular prediction methods are combined by a Support Vector Machine.

Conclusion: The HECTAR method is able to predict the subcellular localisation of heterokont proteins with high accuracy. It also efficiently predicts the subcellular localisation of proteins from cryptophytes, a group that is phylogenetically close to the heterokonts. A variant of HECTAR, called HECTARSEC, can be used to identify signal peptide and type II signal anchor sequences in proteins from any eukaryotic organism. Both HECTAR and HECTARSEC are available as a web application at the following address: http://www.sb-roscoff.fr/hectar/.

Figure 1

Primary and secondary endosymbiosis. part A: Primary endosymbiosis is proposed to have involved the capture of a cyanobacterium (green elipse) by a eukaryotic heterotroph (red elipse). The cyanobacterium would then have been modified during evolution to give rise to a plastid with two surrounding membranes. part B: The secondary endosymbiotic event that gave rise to the heterokonts is proposed to have involved the engulfment of a red algae with a chloroplast (green elipse inside a red elipse) by a eukaryotic heterotroph (brown elipse). The red alga would have become the heterokont plastid with four surrounding membranes.

Figure 2

Comparison of the chloroplast target peptides of red algae and heterokonts. part A: To be imported into a red algal chloroplast, proteins require only a single N-terminal transit peptide (blue bar). The transit peptide is cleaved when the protein passes through the outer chloroplast membrane. The mature protein (yellow) is then transferred into the chloroplast. part B: In heterokonts, proteins which are targeted to the chloroplast possess a bipartite target peptide which is made up of an N-terminal signal peptide (red) followed by a chloroplast transit peptide (blue). The signal peptide is cleaved when the protein passes through the outermost of the four heterokont chloroplast membranes. This places the transit peptide at the new N-terminus of the protein where it can mediate transfer across the two innermost membranes. The transit peptide is cleaved during this second step of the transfer. part C: Sequence logo of the conserved ASAFAP motif surrounding the predicted signal peptide cleavage site based on 55 chloroplast targeted proteins from heterokonts. The logo was built with WebLogo [53] and is based on manually improved alignments of the sequence neighbouring the predicted cleavage site.

Figure 3

Hierarchical architecture of HECTAR. Five categories of subcellar targeting can be predicted by the HECTAR method: signal peptides, type II signal anchors, chloroplast transit peptides, mitochondrion transit peptides and proteins with no detectable N-terminal target peptide. Each decision module (yellow boxes) runs several selected methods (white boxes) to detect specific target peptides. Selected outputs from these methods are then submitted to a SVM which combines these predictors to determine whether a particular target peptide is present in the sequence being analysed. Protein sequences are first analysed by the "signal peptide/anchor" module where a multi-class SVM determines whether a signal peptide or a type II signal anchor is present. If a signal peptide is detected, this sequence is removed from the N-terminal end of the protein sequence and the modified sequence is analysed by the "chloroplast targeted" module which determines whether the signal peptide is followed by a chloroplast transit peptide. In this module the result of a search for the ASAFAP motif is included in the decision process. If a chloroplast transit peptide is present the protein is classified as chloroplastic, otherwise it is classified as having either a signal peptide or a type II signal anchor. When the "signal peptide/anchor" module did not predict either a signal peptide or a type II signal anchor, the protein sequence is analysed by the "mitochondrion targeted" module to determine whether a potential mitochondrion target peptide is present. If a mitochondrion target peptide is found, the protein is assigned as being targeted to the mitochondrion, otherwise it is classified as having no N-terminal target peptide.

Figure 4

Architecture of HECTAR^SEC. HECTAR^SEC is a variant of HECTAR that is dedicated to identifying signal peptides and type II signal anchors in proteins from any eukaryotic organism. This method implements the HECTAR "signal peptide/anchor" module.