Protein secretion routes in fungi are mostly determined by the length of the hydrophobic helix in the signal peptide

Abstract

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 (SRP) or the alternative Sec63 translocon 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 analyse secretion signals across the fungi. Computational predictions by the Phobius model robustly separate known SRP-dependent from Sec63-dependent proteins in S. cerevisiae. Prior work suggested that this separation is driven by the compound hydropathy of the signal peptides hydrophobic helix, i.e., its length multiplied by maximum hydropathy. Instead, we find that the length of the hydrophobic helix is the major discriminator in native proteins: 8-13 amino acids for Sec63-dependent proteins and 16-27 amino acids for SRP-dependent proteins. Secreted proteins in diverse fungal species also separate into distinct populations by Phobius predictions and by the length of the hydrophobic helix. Our analysis across fungi shows that distinct functional groups of proteins, including fungal cell wall proteins and extracellular proteins, have cleaved signal peptides with short hydrophobic helices, similarly to Sec63-dependent proteins in S. cerevisiae. Our results suggest that the Sec63 translocon is critical for cell wall biogenesis and protein secretion in fungi, including secretion of major virulence factors in fungal pathogens of plants and animals. Author SummaryFungal cells have their "stomachs on the outside" - they secrete proteins to the environment that act as enzymes that digest macromolecules and importers of nutrients back across the cell membrane. For fungi that cause infections, the environment is the infected host animal, plant, or other fungus. Fungal cells are also protected by a cell wall, built by proteins that are secreted across the cell membrane and other proteins that use the cells secretory system and are then retained in the membrane. These secreted proteins are translocated across membranes by cellular machinery called translocons, and different secreted proteins use different translocons. Detailed studies of translocons in brewers yeast (Saccharomyces cerevisiae) identified the features in secreted proteins that make the protein use one translocon compared to another. Specifically, there is a helical structure near the start of secreted proteins, and current understanding is that the hydrophobicity of this helix directs the protein through the matched transposon. Here, we show that the length of the hydrophobic helix, not necessarily its maximum hydrophobicity, matters most in fungal cells: shorter helices using a translocon including the Sec63 component and longer helices using a translocon involving the "Signal Recognition Particle". Because these translocons differ between yeast and animals, and even differ across the wide diversity of fungi, we next asked if the hydrophobic helices that direct secretion have similar properties in diverse fungi including pathogens of humans, frogs, and plants. Indeed, our computational predictions separate these these helices into short and long helix groups across fungi. The conservation of short helices in cleaved signal peptides across fungi is consistent with their using the Sec63 translocon and not the Signal Recognition Particle, which merits further investigation. Secreted proteins with cleaved signal peptides, that likely use the Sec63 translocon, include cell wall proteins, digestive enzymes, and "effectors" that manipulate the infected host to promote fungal infections.