Signal sequences are N-terminal extensions of newly synthesized secretory and membrane proteins. They are usually 16 to 30 amino acid residues in length and comprised of a hydrophilic, usually positively charged N-terminal region, a central hydrophobic domain and a C-terminal region with the cleavage site for signal peptidase. Besides these common characteristics, signal sequences do not share sequence similarity and some are more than 50 amino acid residues long.

In eukaryotes, signal sequences direct the insertion of proteins into the membrane of the endoplasmic reticulum and are usually cleaved off by signal peptidase. The resulting signal peptides are presumably rapidly degraded, but some still have functions on their own. Here, we describe examples of post-targeting functions of membrane-integral signal peptides, of signal peptides released from the membrane into either the cytosol or endoplasmic reticulum lumen and of signal peptide fragments generated by intramembrane cleavage. Thus, signal peptides must be considered as an additional resource in the context of the function of secretory and membrane proteins.

Introduction

Signal sequences are N-terminal extensions of nascent polypeptide chains that mediate protein targeting to the membrane of the endoplasmic reticulum (ER).^{1 , 2} They are on average 16 to 30 amino acid residues in length comprising a characteristic tripartite structure: (1) a hydrophilic, usually positively charged n-region, (2) a central hydrophobic h-region of 5-15 residues and (3) a c-region with the cleavage site for signal peptidase (SPase) (Fig. 1A).³ The consensus cleavage site consists of amino acids with short side chains at the −1 and no charged amino acid residues at the −3 position.⁴ Besides these common features, signal sequences can be quite different in sequence and in length, displaying extended n-regions or two hydrophobic regions (Fig. 1A).

Figure 1

Signal sequence structures and signal peptide fates. A) Examples of differently sized signal sequences. Signal sequences can be as small as 16 amino acid residues but some are more then 50 amino acid (aa) residues in length. A characteristic feature of (more...)

When a signal sequence emerges from the ribosome, it is recognized by the signal recognition particle (SRP).^{5 , 6} SRP retards elongation of the nascent polypeptide chain until the ribosome-nascent chain complex has docked onto the SRP receptor at the membrane of the ER⁷ (see chapters by Beckmann and Sinning). Upon docking, the nascent chain is inserted into the ER translocon and polypeptide chain elongation resumes.^{8 , 9} The translocon is a heterotrimeric protein complex consisting of the Sec61α, Sec61β and Sec61γ chains¹⁰ (see chapter by Wagner et al.). Membrane insertion of the signal sequence into the translocation channel proceeds in a loop-like fashion with the n-region remaining on the cytosolic side and the c-region emerging into the ER lumen (Fig. 1B).^{1 , 11 - 13} Typically, signal sequences are cleaved off cotranslationally by SPase, a heterooligomeric complex consisting of five polypeptides (see ref. 14 for review).

Besides targeting proteins for insertion into the ER membrane, signal sequences affect interactions with the translocon or regulate membrane insertion efficiencies. These diverse targeting functions of signal sequences have been discussed previously.^{15 , 16} Here we focus on functions of signal sequences after their insertion into the ER membrane and cleavage from their preprotein. Cleaved signal sequences, termed the signal peptides, are released from the translocation site into the lipid bilayer and spans the ER membrane in a carbonate-resistant manner like integral membrane proteins.^{15 , 17 - 19} Subsequently signal peptides follow various routes ranging from persisting as a stable, membrane-integrated form to proteolytic clearance (Fig. 1B). The signal sequences of serum albumin and vesicular stomatitis virus G-protein (VSV-G) are among the shortest signal sequences (Fig. 1A). They can be considered as minimal signal sequences that may only function in the targeting of the nascent preprotein for insertion into the ER membrane and may rapidly be degraded. Signal peptides for which post-targeting functions have been demonstrated, have extended n-regions or two h-regions (see Fig. 1A and below). These extended signal sequences have post-targeting functions, either as membrane-spanning peptide, as peptide released from the membrane or as signal peptide-fragment, generated by intramembrane proteolysis. Here we review currently known examples and discuss the emerging new paradigms of signal peptide function beyond protein targeting.

Signal Peptides That Have a Function as Membrane-Spanning Molecules

Transmembrane domains (TMDs) of integral membrane proteins typically consist of 20 to 25 residues, however even shorter peptide stretches such as the h-region of signal peptides can be sufficient to form stable TMDs.^{20 , 21} To date all known examples of such stably membrane-integrated signal peptides are of viral origin and are derived from precursors of viral envelope glycoproteins.

Signal Peptides of Arenaviral Glycoproteins

Arenaviral glycoprotein C precursors (pGP-Cs) have unusual signal sequences.^{22 - 24} They are longer than average signal sequences comprising 58 amino acid residues and contain an extended n-region including a myristoylation consensus site and two h-regions separated by a lysine residue (Fig. 1B and 2A). Signal sequences of arenaviral GP-Cs are highly conserved among all known family members reflecting an essential additional post-targeting function in the viral life cycle.^{22 , 25 - 27} As a prototypic member we will discuss here the fate and functions of the signal peptide of the lymphocytic choriomeningitis virus (LCMV) glycoprotein^{22 , 25 , 27} and point out differences to Lassa^{23 , 28 , 29} and Junín viruses.^{24 , 26 , 30}

Figure 2

Signal peptides of arenaviral glycoproteins (GPs) are essential for glycoprotein maturation and viral infectivity. A) Outline of the GP-C precursor protein (pre GP-C) of the lymphocytic choriomeningitis virus (LCMV). Indicated are the signal sequence (more...)

The LCMV GP-C is synthesized as a precursor protein with an N-terminal signal sequence that is cleaved off by SPase during membrane insertion. The resulting signal peptide (SP^GP-C) remains membrane-anchored and associates with GP-C (Fig. 2B).^{22 - 24 , 27} During intracellular transport, GP-C is proteolytically processed into GP1 and GP2. SP^GP-C remains associated with the glycoproteins and is required for GP-C maturation into GP1 and GP2 and virus infectivity.^{23 , 25 - 27 , 30} Besides having an extended n-region, SP^GP-C has two h-regions (Figs. 1B and 2A). While one SP^GP-C h-region is dispensable for targeting and membrane insertion, both h-regions are required for GP-C processing and transport to the cell surface.^{25 , 27 , 29} Similarly, a substitution of the LCMV SP^GP-C with an unrelated minimal (VSV-G) signal sequence prevents GP-C maturation. For the Lassa virus SP^GP-C, it has been shown that its function in glycoprotein maturation can also be provided in trans, that is, when synthesized separately from the glycoprotein.²⁸ The LCMV SP^GP-C adopts an unusual topology in the ER membrane by exposing its n-region on the lumenal side of the membrane (Fig. 2B).²⁷ For the Junn virus SP^GP-C, however, an alternative topology with the n-region and c-region pointing towards the cytosol has been suggested.³¹ In this way, the Junín virus SP^GP-C is able to mask an ER-retrieval signal within the GP-C cytosolic domain, thus ensuring that only fully assembled GP complexes can leave the ER.²⁶ In contrast to Junín virus, the cytosolic portion of LCMV GP-C is not essential for the interaction with SP^GP-C.²⁷ Whether these reported differences indicate alternative topologies of SP^GP-C during the viral life cycle or reflect evolutionary differences in glycoprotein maturation remains to be investigated. Myristoylation, the second unusual feature of the arenaviral SP^GP-C, is not needed for GP-C maturation but is proposed to be required for LCMV and Junín viral fusion with host cell membranes.^{24 , 25 , 27 , 30} Thus, the individual regions of the arenaviral signal sequences play different roles in both, glycoprotein maturation and virus function.

The Signal Peptide of the Foamy Virus Envelope Protein

The envelope proteins (Env or gp130^Env) of Foamy viruses are produced from a precursor protein (Fig. 3A).³² Membrane insertion of the precursor is mediated by an extended signal sequence that is, however, not cleaved off by signal peptidase.^{33 , 34} Thus, pre-gp130^Env is anchored in the ER membrane by the hydrophobic domain of its signal sequence (formally defined as a signal anchor sequence) and the C-terminal TMD and exposes the N- and C-terminus to the cytosol (Fig. 3B). The cleavage of the N-terminal 126 amino acid residues of pre-gp130^Env containing the signal anchor sequence occurs during intracellular transport by a furin-like protease.^{33 , 34} A second cleavage by furin or a furin-like protease produces the SU and the membrane-integrated TM subunit. SP^FV-Env is incorporated into viral particles where it interacts with the viral Gag protein.^{35 , 36} Furthermore, SP^FV-Env is found to be ubiquitinated and its ubiquitination is suggested to regulate the balance between viral and subviral particles released from infected cells.³⁷ Although not processed by SPase, the SP^FV-Env demonstrates the potential to generate a functional membrane integral component from ER-targeting signal sequences.

Figure 3

The signal sequence of Foamy virus (FV) Env protein is cleaved by a furin-like protease (FLP). A) Outline of the precursor of the Foamy virus envelope protein (pre-Env). Indicated are the signal sequence (S), the signal peptide (SP^Env), the glycoprotein (more...)

Signal Peptides Released from the ER Membrane as Soluble Peptides

Although signal peptides initially accumulate in the ER membrane (Fig. 1B), most of them may not adopt a stable conformation in the lipid bilayer and may be released or extracted from the ER membrane.

The Release of the MMTV Derived Signal Peptide to the Cytosol

The mouse mammary tumour virus (MMTV) envelope protein (Env) is synthesized with an extended signal sequence of 98 amino acid residues.³⁸ Env-encoding transcripts contain an intron and are therefore only exported from the nucleus when special export factors are present (see below). When the intron is spliced out, the resulting mRNA encodes for the Rem protein (regulator of export/expression of MMTV mRNA).^{39 , 40} Env and Rem share the same signal sequence, which has an extended n-region (1-80) containing a nuclear localization signal (NLS), (Fig. 4A).⁴¹ When the Env precursors or Rem are inserted into the ER membrane, their signal sequences are cleaved off. The resulting signal peptides, SP^Rem/Env, initially accumulate in the ER membrane, then appear in the cytosol and, finally are found in the nucleus (Fig. 4B).⁴² The membrane-translocated portions of Env and Rem become glycosylated and accumulate in the ER lumen as a type I membrane protein and secretory protein respectively (Fig. 4B). Studies in a cell-free system have shown that SP^Rem is released from ER membranes in a time and temperature-dependent process and that this process is independent of a proteolytic processing step involving signal peptide peptidase (SPP) see ref. 42 and below). In cells transiently expressing Rem, SP^Rem is found in the nucleus. In MMTV infected, murine mammary gland-derived cells and when overexpressed, Rem is not exclusively targeted to the ER but also directly by its uncleaved signal sequence transported into the nucleus. Thus, the presence of two signals, the ER-signn the N-terminus of Rem allows for a dual targeting of the precursor. For nuclear transport, the NLS was found to be essential. Nuclear SP^Rem/Env as well as Rem function in the nuclear export of intron-containing viral transcripts.³⁹ Besides the NLS, SP^Rem/Env contains a putative leucine-rich nuclear-export signal in its hydrophobic region.^{42 - 44} The function of SP^Rem/Env as nuclear export factor appears to be similar to the well-characterized function of human immunodeficiency virus (HIV) Rev protein.^{39 , 44} The ability to generate such a regulatory factor from the ER-targeting signal sequence adds an intriguing layer to the complexity of the retroviral life cycle.

Figure 4

A viral signal peptide functions as nuclear export factor. A) Outline of the mouse mammary tumour virus (MMTV) envelope protein precursor protein (pre-Env), Rem and SP^Rem. Pre-Env is encoded by an intron-containing mRNA, which upon splicing encodes the (more...)

The Release of a Signal Peptide to the ER Lumen

The envelope glycoproteins of the alphaviruses, Semliki Forest Virus (SFV) and Sinbis Virus, are produced as part of a polyprotein that is sequentially processed cotranslationally by both viral and cellular proteases to yield the cytosolic nucleocapsid protein and the membrane proteins p62, 6K and E1 (Fig. 5).^{45 , 46} During cotranslational processing of the polyprotein precursor, the nucleocapsid protein is autoproteolytically cleaved and remains in the cytosol.⁴⁷ A stretch of apolar residues at the N-terminus of p62 functions as a signal sequence that targets p62 to the ER. After its membrane insertion the signal sequence of p62 is not cleaved off by SPase. Instead p62 is N-glycosylated within its signal sequence and subsequently cleaved in a post-Golgi compartment to generate E3 and E2 (Fig. 5).^{45 , 48} The E3 peptide, essentially representing the signal peptide of p62, remains noncovalently associated with the E1-E2 spike glycoproteins and is found in mature virus particles. Replacement of E3 with an artificial signal sequence abolishes spike heterodimerisation and surface expression of E1.⁴⁹ E3 thus performs the function of a signal sequence and after cleavage the resulting peptide acts as soluble glycoprotein with an important role in the viral life cycle.⁴⁹

Figure 5

Release of a signal sequence to the ER lumen. A) Outline of the Semliki Forest virus (SFV) structural proteins. Indicated are the capsid protein, the glycoprotein subunit p62 (which gets further processed into E3 and E2), the 6K peptide and E1. ^, B) (more...)

In addition to the generation of this soluble, signal sequence-derived peptide, alpha viruses also produce a second membrane-integrated signal peptide, the 6K peptide. The coding region for this small 6 kDa membrane protein lies in between those of the p62 and E1 proteins (Fig. 5). The 6K protein results from two SPase cleavages and essentially consists of an N-terminal TMD and the cleaved signal sequence responsible for the insertion of the E1 protein.⁴⁶ Deletion of the 6K protein has revealed that it is required for efficient assembly of virus and is also involved in the viral budding process.^{50 - 54}

Signal Peptide Processing and the Generation of Bioactive Signal Peptide Fragments

After cleavage by SPase some of the resulting signal peptides are further processed within their hydrophobic regions by SPP (signal peptide peptidase) (Fig. 6 and Table 1).^{15 , 55} SPP is a member of the aspartic intramembrane proteases and is related to presenilin, the proteolytically active subunit of the γ-secretase complex.^{56 - 58} SPP is localised to the ER while several SPP-like proteins are localized to different compartments of the cell.^{59 - 62} Substrates thus far identified for SPP are all signal peptides or internal signal sequences of viral proteins that have been generated by SPase cleavage (Table 1).⁵⁵ Comparing signal peptides that are substrates with those that are not, revealed that helix-breaking residues within the h-region are required for efficient processing and flanking regions can affect processing. In order to be cleaved by SPP, substrate peptides or proteins have to be liberated from the nascent chain by SPase.⁵⁵ It was proposed that SPP substrates require flexibility in the lipid bilayer to expose an accessible peptide bond for intramembrane proteolysis, however, the exact substrate specificity is not completely understood.⁶³ Several signal peptides and internal signal sequences of viral polyproteins have been shown by now to be processed by SPP and for some of them specific functions of the generated fragments have either been proposed or demonstrated (Table 1).

Figure 6

Generation of signal peptide fragments and their functions. A) The signal sequence of preprolactin (pre-Prl) is cleaved by signal peptidase (SPase) and the resulting SP^Prl is further processed by the intramembrane protease signal peptide peptidase (SPP). (more...)

Table 1

SPP substrates. Upon liberation by SPase, signal peptides of the following precursor proteins are processed by SPP. Known or suggested post-targeting functions and the bioactive fragments are listed.

Conclusion and Perspectives

We have described here selected examples of signal peptides that perform distinct post-targeting functions in different cellular locations: in a membrane, in the cytosol/nucleus or in the ER lumen (see Fig. 7). Most examples are currently from viral proteins, thus it will now be interesting to see how common post-targeting functions of signal peptides derived from cellular proteins are. It is likely, that many signal peptides contain just the targeting information and are rapidly degraded. Little is known about signal peptide degradation, however, there are hints that SPP may play a role in this respect. The signal peptides of calreticulin, an ER lumenal lectin and the VSV-G protein are apparently substrates for SPP and the resulting fragments are expected to be nonfunctional.⁵⁵ Furthermore, SPP has been implicated in the degradation of misfolded TMD proteins^{82 , 83} supporting the notion of SPP involvement in signal peptide degradation. Also alternative degradation pathway may exist in which full-length signal peptides are ubiquitinylated and dislocated to the cytosolic 26S proteasome like misfolded membrane proteins.^{84 - 87} Intriguingly, it has recently been shown that intracellular peptides similar to those generated by the proteasome have the potential to affect cell signalling,⁸⁸ implying an even bigger complexity of peptide mediated cellular processes. A detailed understanding of the molecular machinery involved in the turn over of signal peptides and the growing number of known post-targeting functions of signal sequences will likely give new insight into functions that signal peptides can perform after their cleavage from the parent protein.

Figure 7

Summary of different fates and functions of signal peptides. A) Signal peptides can function as membrane integrated peptides (e.g., signal peptides of arenavirus and foamy virus glycoproteins). B) Signal peptides can be released from the membrane to the (more...)

Acknowledgements

We apologize to all those colleagues whose work has not been cited owing to space limitations. M.K.L. is supported by the Landesstiftung Baden-Württemberg and B.D. is supported by a grant from the Deutsche Forschungsgemeinschaft, SFB 638. We thank Stephen High and Martin Pool for critically reading of the manuscript and helpful comments.

Protein Transport into the Endoplasmic Reticulum, edited by. Richard Zimmermann ©2009 Landes Bioscience.

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