Cleavable endoplasmic reticulum (ER) signal peptides (SPs) and other non-cleavable signal sequences target roughly a quarter of the human proteome to the ER. These short peptides, mostly located at the N-termini of proteins, are highly diverse. For most proteins targeted to the ER, it is the interactions between the signal sequences and the various ER targeting and translocation machineries such as the signal recognition particle (SRP), the protein-conducting channel Sec61, and the signal peptidase complex (SPC) that determine the proteins’ target location and provide translocation fidelity.
The secretory pathway is a protein trafficking highway utilized by more than a quarter of the human proteome 
. Soluble secreted proteins such as antibodies and protein hormones rely on this pathway. The pathway also delivers transmembrane proteins (TMPs) to the endoplasmic reticulum (ER), its downstream organelles such as the Golgi apparatus, and the plasma membrane.
All secretory proteins are translated by cytosolic ribosomes and must be first targeted to and then transported across (or inserted into) the ER membrane at the early stage of their life, either co- or post-translationally 
. A complex network of cytosolic and ER membrane-resident macromolecules facilitate and assist the ER targeting and translocation.
Both ER targeting and translocation/insertion critically depend on so-called signal sequences (SSs), short hydrophobic peptide stretches in the amino acid sequence of the newly synthesized proteins that are recognized by the secretory machinery as trafficking signals. While SSs may appear exceedingly simple, they possess a remarkably versatile and complex physiology. Besides the choice of trafficking routes, SSs carry information about translocation efficiency, occurrence and timing of cleavage, and post-targeting functions.
There are four main classes of SSs: (i) cleavable signal peptides (SPs), found on secreted proteins such as insulin and type I membrane proteins such as HLA molecules; (ii) type II signal anchor sequences (SASs), found on single- and multi-pass transmembrane proteins (TMPs) such as the membrane-bound form of tumor necrosis factor (TNF); (iii) type III SASs found, e.g., on Sec61β; and (iv) tail anchors (TAs) found on proteins such as Sec61γ (Figure 1a). Signal peptides (SPs) are by far the most populous class of SSs. In humans alone, there are an estimated >3000 different SP-containing proteins, constituting >10% of the whole proteome. SPs are usually localized within the first 30 amino acids of the coding sequence but can in some cases also be found more internally. The defining trait of SPs is the capacity to be cleaved by the aptly named signal peptidase complex (SPC).
The primary sequence of SPs is only loosely defined. In fact, approximately 20% of randomized sequences can promote protein secretion in yeast 
. SPs are characterized by a tripartite structure (Figure 1
b–d): (i) an often positively charged, N-terminal ‘n-region’ that faces the cytosol; (ii) a short hydrophobic core—most commonly between 7 and 15, but not longer than 18–20 amino acids called ‘h-region’; and (iii) a polar luminal C-terminal ‘c-region’ that contains the scissile bond and must be occupied by short, hydrophobic residues at positions −1 and −3 relative to the cleavage site 
. Initially, SPs are inserted into the ER membrane with the N-terminus facing towards the cytosol (Nin
) and the mature sequence facing the organellar lumen (Cout
). In the case of type I TMPs, the removal of the SP leads to an ‘inverted’ topology of the mature sequence in which the N-terminus is facing ‘outside’ (Nout
), while the C-terminus is facing the cytosol (Cin
) (Figure 1
Types of signal sequences. (a
) Depiction of the four types of SSs with their membrane topology indicated. Hydrophobic segments are depicted in magenta. (b
) Signal peptides have a tripartite structure, consisting of an n- (cyan), h- (magenta), and c-region (yellow) and are cleaved in the ER lumen by the SPC (green flash). (c
) Frequency of residue types relative to the cleavage site 
) Relative length of the respective regions (colored as in (b
)) as a function of total SP length. The bulk of the length variation stems from the n-region. Panels c–d were adapted from 
2. SP Recognition in the Cytosol and ER Targeting
As the first step of their life cycle, secreted proteins and TMPs must be targeted to the ER membrane (Figure 2). The timing of translation, folding, and ER transport is of particular importance: on one hand, nascent chains (NCs) can only cross the ER membrane in an unfolded state, while on the other hand, this prerequisite exposes their hydrophobic segments and makes them prone to aggregation and proteolysis. Therefore, these proteins must be shielded from the hydrophilic cytosol, which is achieved by one of two separate strategies: (i) direct recognition of the nascent protein at the ribosomal exit tunnel by the SRP, leading to co-translational translocation/insertion through the recruitment of the ribosome–nascent chain complex (RNC) to the ER; or (ii) post-translational transport to the ER, which requires the involvement of chaperones to protect the clients from the aqueous environment.
ER delivery pathways for SP-containing proteins. The upper panel shows a schematic of each delivery pathway. The central component of each pathway is underscored. Left
: SRP (blue/brown subunits) recognizes SPs emerging from the ribosomal exit tunnel and shields them through the SRP54 M-domain. SP binding triggers the heterodimerization of the SRP54 NG domain (blue) with that of SRα (green), guiding the RNC to the ER. A large conformational rearrangement partially exposes the SP for handover to Sec61 
: Cytosolic calmodulin or Hsp40-assisted Hsc70 recognize SP-containing proteins and guide them to the ER. Right
: The recently discovered Snd pathway likely consists of a cytosolic component, Snd1, which might act as a chaperone, and two ER membrane-resident components, Snd2/3, which might facilitate handover to Sec61 in some unknown way 
. Lower panels show specifics of each pathway.