Protein secretion routes in fungi are predicted by the length of the hydrophobic helix in the signal sequence.

Secreted proteins are translocated across membranes through multiple routes. In eukaryotes, secreted proteins with N-terminal signal sequences can use either the signal recognition particle and its receptor or the alternative Sec complex to cross the endoplasmic reticulum membrane. Large-scale experiments on the substrates of these pathways are primarily from the model yeast Saccharomyces cerevisiae, but less is known about conservation of translocation pathways. Here, we take a computational approach to analyze secretion signals across the fungal kingdom. Computational predictions by the Phobius model separate secreted proteins in diverse fungal species into distinct populations: cleaved signal peptides with short hydrophobic helices of 8 to 13 amino acids and transmembrane proteins with long hydrophobic helices of 16 to 27 amino acids, similarly to S. cerevisiae. These computational predictions also robustly distinguish translocation routes in S. cerevisiae: Sec-dependent translocation of native proteins is accurately predicted by the presence of a cleaved signal peptide, while conversely signal recognition particle-dependent translocation is predicted by a retained signal-anchor. Analysis of multiple hydrophobicity scales and signal peptide prediction algorithms shows that the Phobius-predicted length of the hydrophobic helix alone is an effective predictor of translocation route. Our results support the hypothesis that the Sec complex is critical for cell wall biogenesis and protein secretion across fungi.