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We performed a computational analysis on the first 70 aa atthe N-t (N-terminal analysis) of thewhole proteome and on 70 aa windows starting from allinternal TMDs (Internal TMDs analysis) toidentifypotential SPC cleavage sites not associated with signal peptides (cryptic cleavage sites) byusing a pre-existing a signal peptide prediction softwareSignalP and comparing the output of twonetworks (no-TM andTM modes). Our hits represent membrane protein with type-II oriented TMDpredicted to be cleaved inno-TM network mode but not inTM network mode. Moreover, we rank the hits based on the numberof disease-linked mutations.

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We used the command-line version of theSignalP4.1 program. The program takes a proteins sequence (FASTA formatted) of 70 amino acids length as input and predicts thecleavage site and reports the Y-score (combined cleavage site score) in its output. To extractproteins with cryptic cleavage sites, we created two types of peptide sequences by considering the first 70 amino acids of all:

• proteins that lack a canonical signal peptide (N-terminal analysis)
• transmembrane domains of proteins that lack a canonical signal peptide (Internal TMDs analysis)

We predicted the Y-scores of both types of peptide sequences using the SignalP-TM (TM network mode) and SignalP-noTM (no-TM network mode) version (see Methods for details).A protein/transmembrane domain was considered to have a non-canonical cleavage site ifits corresponding peptide’s Y-score in the SignalP-TM output was less than 0.6 and in SignalP-noTM output was more than 0.5. We retrieved allthe proteins and their associated feature information fromUniProt/Swiss-Prot.We assigned mutational information provided in (a)UniProt,COSMIC, andClinVAR databases.

Validation of the hits (proteins predicted to have non-canonical cleavage sites) were then performed by ectopically expressing the WT (Wild Type) and mutant constructs inWT HEK293T cells or lacking the regulatory SPC subunit SPCS1. Cell lysates were then analyzedvia Western blot to detect possible SPCS1-dependent cleavage fragments (see Methods for details).

Note: The information about the data downloaded from UniProt, COSMIC and ClinVar can be retrieved from Materials and Methods of the accompanying manuscript.

Marius Lemberg:m.lemberg@uni-koeln.de (Lemberg lab, Heidelberg/Cologne)

Matthias Feige:matthias.feige@tum.de (CPB lab, Munich)

Gurdeep Singh:gurdeep.singh@bioquant.uni-heidelberg.de (Russell lab, Heidelberg)

Zanotti A, Coelho JPL, Kaylani D, Singh G, Tauber M, Hitzenberger M, Avci D, Zacharias M, Russell RB, Lemberg MK, Feige MJThe human signal peptidase complex acts as a quality control enzyme for membrane proteins.Science (2022).doi: 10.1126/science.abo5672PMID:36454823