The hypothesis that the Sec complex is critical for cell wall biogenesis and protein secretion across fungi is supported, and the Phobius model is used to analyze secretion signals across the fungal kingdom.
Abstract
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 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.
The secretory (Sec) pathway is the major pathway of Bacillus subtilis for protein export from the cytoplasm to the extracellular milieu. An amino‐terminal signal peptide is required to initiate protein translocation across the cytoplasmic membrane via Sec. Generally, Sec pathway signal peptides of B. subtilis are around 20–30 amino acids long and tend to be relatively hydrophobic compared to signal peptides from other organisms. In view of the importance of signal peptides for high‐level secretory protein production, understanding how signal peptide structure and length influence the efficiency of protein secretion is crucial. Accordingly, the present study was aimed at investigating the effects of signal peptide size and, in particular the subdomain sizes of a signal peptide, in relation to protein secretion efficiency. To this end, a rational engineering approach was employed for the design of shortened signal peptides that can still drive effective protein secretion. This involved in silico interaction studies with the signal recognition particle, which is involved in protein targeting from the ribosome to the membrane. Our results show that the hydrophobic H‐domain of a B. subtilis signal peptide can be shortened from seventeen to seven hydrophobic amino acid residues without a detectable decrease in protein secretion.
D. A. Pranoto, Danilo Milo, Lorenzo de la Parra Soto et al.· Microbial Biotechnology· 0 citations
Proteins destined for secretion typically contain N-terminal signal sequences that target nascent chains to the endoplasmic reticulum (ER). Following targeting, these sequences are cleaved by the heterotetrameric signal peptidase complex (SPC). Despite a conserved N-terminal (n), hydrophobic core (h) and C-terminal (c) tripartite organization, their sequences are highly variable. How SPC subunits contribute to recognition of these diverse sequences remains poorly understood. Sec11, the catalytic subunit of the SPC, contains an N-terminal transmembrane (TM) domain and a C-terminal hydrophobic region. The latter was unresolved in human SPC cryo-EM structures, likely due to intrinsic flexibility, yet AlphaFold predictions of the yeast SPC suggest that this region forms a C-terminal short helix (CTS) strategically positioned within the presumed signal sequence-binding site. This structural arrangement led us to hypothesize its possible role in substrate handling during signal peptide processing and we undertook to investigate its function using biochemical assays combined with molecular dynamics (MD) simulations. Topology mapping confirmed that the Sec11 CTS traverses the ER membrane and MD simulations showed that removal of the Sec11 CTS does not substantially alter overall SPC architecture or the proximal membrane environment. While N-terminal signal sequences of varying hydrophobicity were efficiently cleaved, internal signal sequences with extended n-region were selectively defective in cleavage in the absence of the Sec11 CTS. These data suggest that the Sec11 CTS specifically stabilizes internal signal sequences for productive engagement with SPC.
Yeonji Chung, Mariia Borbuliak, Sanghun Hwang et al.· Journal of Biological Chemis...· 0 citations
The results indicate that −1PRF and spike protein maturation in alphaviruses is tuned by the dynamic remodeling of the ER translocon, which could allow polyprotein biogenesis to adapt to different stages of viral replication and to distinct host or vector environments.
Antonio Bonifasi, Gayani Apsara Ranasinghe, Ashish Jhangiani et al.· bioRxiv· 0 citations
The Sec61 complex plays a critical role in protein translocation into the endoplasmic reticulum (ER), enabling the passage of soluble proteins into the lumen and the lateral insertion of membrane proteins. The Sec complex, comprising Sec61, Sec63, Sec62, and other auxiliary proteins, is especially important for the translocation of precursor proteins with inefficient signal sequences (SSs) that require additional assistance to trigger the opening of the Sec61 channel. In this study, we investigated how the hydrophobicity of SSs affects the interaction with the Sec complex at the single-molecule level using optical tweezers. We analyzed three SSs representing low, moderate, and high hydrophobicity in the h-regions. Our results show that the highly hydrophobic SS from DPAP-B pαF forms a less stable bond with the Sec complex, evidenced by shorter interaction time and reduced energy barrier to the unbound state. In contrast, SSs with lower hydrophobicity establish more stable interactions, characterized by extended interaction times, and elevated energy barriers to reach the unbound state. These findings suggest that the Sec complex may selectively recognize and interact with SSs based on their hydrophobicity, offering insights into the mechanisms by which defects in SS translocation contribute to ER-associated diseases.
W. Sánchez, Luka Robeson, Hilda M. Alfaro-Valdés et al.· Biochemical and Biophysical...· 0 citations
It is proposed that the faster folding of TPs, relative to downstream MPs, plays an important role in maintaining preproteins in an import-competent state, and that TPs need to fold into a specific conformation to function properly.
The structure suggests that the ribosome alone, even without a nascent chain, is sufficient for lateral opening of Sec61 in a lipid environment, independent of its functional state.
Stefan Pfeffer, L. Burbaum, Pia Unverdorben et al.· 0 citations
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