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Frequently Asked Questions

Changes from version 4.0 to 4.1
Changes from version 3 to 4
Biological background, signal peptides
Biological background, other sorting signals
Biological background, organism groups
History

Changes from version 4.0 to 4.1

— What's new?

Please see the Version history page.

— Why do you present a choice between two cutoff settings? Can't you just decide on one?

The optimal cutoff really depends on what you want to use the method for. If it is important to find all signal peptides, use the sensitive cutoff. If you want an estimate of the number of signal peptides in a genome, use the default cutoff.

— Why have you imposed a minimum length?

Because we believe that predictions of signal peptides shorter than ten residues made by SignalP 4.1 are false. The shortest known signal peptides are 11 residues long (with one exception, SP23_TENMO, which does not look like a signal peptide at all). Click here for an updated list of experimentally confirmed signal peptides from UniProt of length 11 or shorter.

— What happened to the Background page?

It's here! The important material from the Background page has been integrated into this FAQ, we hope you like the new format. If you prefer to see the old Background page, it is still available under SignalP 4.0.

Changes from version 3 to 4

— What's new?

Please see the Version history page.

— What happened to the HMM part?

While making SignalP 4.0, we did retrain the Hidden Markov Model (HMM) part of SignalP. However, we found that it did not perform better than the neural networks in any of the performance parameters we tested. Therefore, we decided not to include it. If the HMM output is important for you, you can still use SignalP 3.0.

— Why is my favourite signal peptide no longer predicted correctly? SignalP 3.0 could do it!

As explained on the Performance page, SignalP 4 with the default cutoff has a lower sensitivity than SignalP 3. Please try again with the new "Sensitive" setting.

— What happened to the Yes/No answers for max C score etc.?

SignalP 3.0 provided five Yes/No answers for the NN part. We found that this was confusing for users and obscured the fact that the D-score is the best score for discriminating between signal peptides and non-signal peptides.

Biological background, signal peptides

— What are signal peptides?

The term "signal peptide" is used with two meanings: In the broad sense (used in many textbooks), a signal peptide is any sorting signal embedded in the amino acid sequence of a protein. In the narrow sense (used in most of the scientific literature), a signal peptide is an N-terminal signal that directs the protein across the ER membrane in eukaryotes and across the plasma membrane in prokaryotes. Signal peptides in the narrow sense are also known as ER signal peptides or secretory signal peptides. Read more in UniProt, in Wikipedia, and in the Sequence feature ontology.

It is important to emphasize that SignalP predicts signal peptides in the narrow sense only.

— Are signal peptides always N-terminal?

In the narrow sense: Yes, per definition. In the broad sense: No, there are several sorting signal that are C-terminal (e.g. the PTS1 signal for peroxisomal import) or internal (e.g. the nuclear localization signal).

— Are signal peptides (in the narrow sense) always cleaved?

No, there are rare cases of uncleaved signal peptides. For an updated list of such proteins annotated in UniProt, click here. These should not be confused with signal anchors, see below. In SignalP, these typically get high S-scores but low C- and Y-scores.

— Which protease is responsible for signal peptide cleavage?

In bacteria, it is Signal Peptidase I (SPase I), also known as Leader Peptidase (Lep). In eukaryotes, it is the signal peptidase complex (SPC), which consists of four subunits in yeast and five in mammals. Read more in MEROPS.

— My protein has a signal peptide. Can I then safely conclude that it is secreted?

No. You can only conclude that it enters the secretory pathway.

In eukaryotes, there are several opportunities for a protein with a signal peptide to escape secretion. It could:

  • It could be retained in the endoplasmic reticulum (ER). Soluble ER-resident proteins have a C-terminal retention signal with the consensus sequence KDEL, see PROSITE.
  • be retained in the Golgi apparatus,
  • be directed to the lysosome (vacuole in plants and fungi),
  • have one or more transmembrane helices and therefore be retained in either the plasma membrane, or one of the membranes of the secretory pathway (ER, Golgi, lysosome/vacuole), or
  • have a signal for GPI-anchoring, a C-terminal cleaved peptide which functions as a signal for attachment of a Glycophosphatidylinositol (GPI) group that anchors the protein to the outer face of the plasma membrane.
In Gram-positive bacteria, a protein with a signal peptide could:
  • have one or more transmembrane helices, or
  • be attached to the cell wall.

In Gram-negative bacteria, a protein with a signal peptide could:

  • have one or more transmembrane helices,
  • be retained in the periplasm, or
  • be inserted into the outer membrane as a β-barrel transmembrane protein.

— Does SignalP predict signal peptides of bacterial lipoproteins?

No. Bacterial lipoproteins have special signal peptides which are cleaved by Signal Peptidase II (SPase II), also known as Lipoprotein signal peptidase (Lsp). A diacylglyceryl group is attached to a Cysteine residue in position +1 relative to the cleavage site, which bears no resemblance to the SPase I cleavage site. See also MEROPS and PROSITE.

For prediction of prokaryotic lipoproteins we recommend using the LipoP server.

— Does SignalP predict TAT (Twin-arginine translocation) signal peptides?

Not very well. Bacterial TAT signal peptides, which direct their proteins through an alternative translocon (TatABC), have a special motif containing two Arginines in the n-region. Additionally, they are in general longer and less hydrophobic than normal (Sec) signal peptides. As a consequence, they are sometimes missed by SignalP, even though they are cleaved by SPase I (Lep). See also PROSITE and InterPro.

For prediction of TAT signal peptides we recommend using the TatP server.


Biological background, other sorting signals

— What are signal anchors?

A signal anchor is a transmembrane helix located close to the N-terminus of a protein with an N-in orientation (i.e. the N-terminus is on the cytoplasmic side of the membrane). It functions much like a signal peptide since it is recognized by the Signal Recognition Particle (SRP) and inserted into the translocon; but instead of being cleaved and degraded it remains in the membrane and anchors the protein to it. Proteins anchored in this way are known as Type II transmembrane proteins.
Signal peptides versus signal anchors Signal peptides (above) versus
signal anchors (below)
It is important to realize that the difference between signal peptides and signal anchors is not a question of presence or absence of a cleavage site. Instead, the most important difference seems to be the length of the hydrophobic domain. It has been shown experimentally that it is possible to convert a cleaved signal peptide to a signal anchor merely by lengthening the h-region, without altering the cleavage site (Chou & Kendall 1990; Nilsson, Whitley, & von Heijne 1994).

The introduction of the Hidden Markov Model (HMM) method in SignalP version 2 made it possible to some extent to distinguish signal peptides from signal anchors (in that version, only in eukaryotes). However, SignalP 4 (based entirely on the Neural Network (NN) method), does a better job, since its negative set is not confined only to transmembrane helices annotated as signal anchors, but includes all types of transmembrane segments close to the N-terminus.

— What should I use for predicting signal peptides in the broad sense?

For mitochondrial and plastid import signals, also known as transit peptides, we recommend TargetP. For other sorting signals, we refer to our paper "Locating proteins in the cell using TargetP, SignalP, and related tools".

— What should I use for predicting non-classical (leaderless) secreted proteins?

Not all secretory proteins carry signal peptides. Some proteins enter a non-classical secretory pathway without any currently known sequence motif. In eukaryotes, these proteins are mostly growth factors and extracellular matrix binding proteins. In Gram-negative bacteria, the type I, III, IV and VI secretion systems function without signal peptides. For prediction of such proteins we recommend the SecretomeP server.

Biological background, organism groups

— Why is there no version for Archaea?

Because there are too few experimentally confirmed signal peptides from this organism group in the UniProt database (click here for an updated list).

— Which version should I use for vira and bacteriophages?

You should use the version corresponding to the host organism. There are some indications that viral signal peptides differ from those of the host organism, but SignalP currently does not take that into account.

— Which version should I use for Tenericutes/Mollicutes (Mycoplasma and related genera)?

You shouldn't use SignalP at all for these organisms, since they seem to lack a type I signal peptidase completely!

— Which version should I use for metagenomic sequences of unknown origin?

This is an unsolved question. Please use all three versions to search for signal peptides in such data.

— Is one version enough for all eukaryotic organisms, or are there differences within the eukaryotes?

It is known that some yeast signal peptides are not recognized by mammalian cells (Bird et al., 1987 and 1990). Therefore, it would be natural to assume that separate SignalP versions for yeast and Mammalia would provide better predictions than a common eukaryotic version. While developing SignalP 4.0 we tried dividing the eukaryotic data into animals, fungi, and plants and training separate methods for these three groups. However, this did not give any improvement, and performance for all three groups was better when using the method trained on all eukaryotic sequences together.

— Are two versions enough for all bacteria, or are there differences within the Gram-positive/Gram-negative bacterial groups?

The Gram-negative version of SignalP is almost certainly biased towards E. coli and other γ-proteobacteria, since these constitute the bulk of the experimentally annotated bacterial proteins in UniProt. Unpublished results suggest that some bacteria have very divergent cleavage site motifs. Future versions of SignalP might therefore divide the Gram-negative bacteria into several classes, if data are available.

Gram-positive bacteria probably constitute a more homogenous group, but it is an open question whether there are differences in signal peptides between Actinobacteria (high G+C Gram-positive bacteria) and Firmicutes (low G+C Gram-positive bacteria). More data on Actinobacteria are needed before that can be answered.


History

— How are the various versions of SignalP related?

Please see the Version history page.

— Was there ever a Nobel prize awarded for signal peptides?

Yes, for signal peptides in the broad sense. The importance of signal peptides was emphasized in 1999 when G√ľnter Blobel received the Nobel Prize in physiology or medicine for his discovery "proteins have intrinsic signal that govern their transport and localization in the cell". The press release can be read here.

— Was SignalP the first signal peptide predictor?

No, but it was, to our knowledge, the first to be implemented as a web server (in 1996). Among the earlier methods were McGeoch (1985) and von Heijne (1986), both of which have been included in PSORT.

— How many times have the SignalP papers been cited?

This information is available on Henrik Nielsen's ResearcherID and Google Scholar pages.


CORRESPONDENCE

Henrik Nielsen,