Sequences beyond the cleavage site influence signal peptide function

The earliest events in protein secretion include targeting to and translocation across the endoplasmic reticulum membrane. To dissect the mechanism by which signal sequences mediate translocation in eukaryotes, we are examining the behavior of fusion proteins and deletion mutants in cell-free systems. We demonstrate that the protein domain being translocated can have profound impact on the efficiency of the translocation process. Specifically, deletions in the mature prolactin "passenger" domain, beyond the signal cleavage site, reduce the efficiency of signal function. The effect of these deletions on signal function is observed when this signal sequence is in its normal position, at the amino terminus, and when internalized by the addition of 117 amino acids of chimpanzee alpha-globin. Alterations in the interaction of the deletion mutants with the signal recognition particle and with another component of the translocation system, signal peptidase, were observed. Our results suggest that subtle changes in sequences beyond the signal cleavage site can alter the efficiency of co-translational translocation by affecting various signal-receptor interactions.     

Mutations in the signal sequence of prepro-alpha-factor inhibit both translocation into the endoplasmic reticulum and processing by signal peptidase in yeast cells.

The effects of five single-amino-acid substitution mutations within the signal sequence of yeast prepro-alpha-factor were tested in yeast cells. After short pulse-labelings, virtually all of the alpha-factor precursor proteins from a wild-type gene were glycosylated and processed by signal peptidase. In contrast, the signal sequence mutations resulted in the accumulation of mostly unglycosylated prepro-alpha-factor after a short labeling interval, indicating a defect in translocation of the protein into the endoplasmic reticulum. Confirming this interpretation, unglycosylated mutant prepro-alpha-factor in cell extracts was sensitive to proteinase K and therefore in a cytosolic location. The signal sequence mutations reduced the rate of translocation into the endoplasmic reticulum by as much as 25-fold or more. In at least one case, mutant prepro-alpha-factor molecules were translocated almost entirely posttranslationally. Four of the five mutations also reduced the rate of proteolytic processing by signal peptidase in vivo, even though the signal peptide alterations are not located near the cleavage site. This study demonstrates that a single-amino-acid substitution mutation within a eucaryotic signal peptide can affect both translocation and proteolytic processing in vivo and may indicate that the recognition sequences for translocation and processing overlap within the signal peptide.     

PrlC, a suppressor of signal sequence mutations in Escherichia coli, can direct the insertion of the signal sequence into the membrane

The prlC gene product of Escherichia coli can be altered by mutation so that it restores export of proteins with defective signal sequences. The strongest suppressor, prlC8, restores processing of a mutant signal sequence to a rate indistinguishable from the wild-type. Data obtained by changing gene dosage of the dominant suppressor and its specificity for different signal sequence mutations suggest that PrlC8 interacts directly with the hydrophobic core of the signal sequence. Despite the fact that signal sequence processing appears to be mediated by leader peptidase, the processed mature protein is not translocated efficiently from the cytoplasm. Results obtained with various double mutants indicate that PrlC8-mediated processing of mutant signal sequences does not require components of the cellular export machinery such as SecA, SecB or PrlA (SecY) and that the block in translocation from the cytoplasm occurs because PrlA (SecY) fails to recognize the defective signal sequence. We suggest that PrlC8 directs insertion of the mutant signal sequence into the membrane bilayer to an extent that processing by leader peptidase can occur. This reaction is novel in that it has not been observed previously in vivo.     

Functional limits of conformation, hydrophobicity, and steric constraints in prokaryotic signal peptide cleavage regions. Wild type transport by a simple polymeric signal sequence

These experiments examine the role of conformation, hydrophobicity, and steric constraints in the function of the prokaryotic signal peptide cleavage region. The experimental strategy involves replacement of the wild type Escherichia coli alkaline phosphatase signal peptide cleavage region with a series of idealized model sequences designed to epitomize the particular structural and physical variables under study. By analyzing model sequences whose conformations have been determined by physical studies, we have demonstrated that efficient transport does not depend on the structural preference of the cleavage region. Although previous studies based on Chou-Fasman analysis have suggested that the cleavage region forms a beta-turn which is required for transport, our results demonstrate that either a beta-turn- or alpha-helix-fostering sequence in the cleavage region functions indistinguishably from wild type. Furthermore, the presence of a proline residue between the core and cleavage region, although common in natural sequences, is not essential for export. Cleavage regions of varying hydrophobicities can support translocation across the inner membrane, but the placement of bulky residues at positions -1 and -3 upstream of the cleavage site abolishes processing and transport to the periplasm. By reducing the signal peptide to simplified, idealized segments, this study has identified a largely polymeric sequence, MKQST(L10)-(A6), that functions equivalently to the wild type alkaline phosphatase signal peptide. This work starts to provide a basis for the design of a universal prokaryotic signal peptide that incorporates all the critical physical and structural characteristics required for transport function.     

A part of the transmembrane domain of pro-TNF can function as a cleavable signal sequence that generates a biologically active secretory form of TNF.

To determine the minimum requirement in the 76-residue leader sequence of pro-tumor necrosis factor (TNF) for membrane translocation across the endoplasmic reticulum (ER) and for the maturation of pro-TNF, we constructed pro-TNF mutants in which a part of the transmembrane domain of pro-TNF was directly linked to the N-terminus of the mature domain, and evaluated their translocational behavior across the ER-membrane and their secretion from the transfected cells. The in vitro translation/translocation assay involving a canine pancreatic microsomal membrane system including a mutant, Delta-75-47, -32-1, revealed that the N-terminal half of the transmembrane domain of pro-TNF consisting of 14 residues functioned as a cleavable signal sequence; it generated a cleaved form of TNF having a molecular mass similar to that of mature TNF. Analysis of the cleavage site by site-directed mutagenesis indicated that the site was inside the leader sequence of this mutant. When the mutant, Delta-75-47, -32-1, was expressed in COS-1 cells, efficient secretion of a biologically active soluble TNF was observed. Further deletion of the hydrophobic domain from this mutant inhibited the translocation, indicating that some extent of hydrophobicity is indispensable for the membrane translocation of the mature domain of TNF. Thus, the N-terminal half of the transmembrane domain of pro-TNF could function as a cleavable signal sequence when linked to the mature domain of TNF, and secretion of a biologically active secretory form of TNF could be achieved with this 14-residue hydrophobic segment. In intact pro-TNF, however, this 14-residue sequence could not function as a cleavable signal sequence during intracellular processing, indicating that the remainder of the 76-residue leader sequence of pro-TNF inhibits the signal peptide cleavage and thus enables the leader sequence to function as a type II signal-anchor sequence that generates a transmembrane form of TNF.     

Structural features in the NH2-terminal region of a model eukaryotic signal peptide influence the site of its cleavage by signal peptidase

The 20-amino acid signal peptide of human pre (delta pro)apolipoprotein A-II contains the tripartite domain structure typical of eukaryotic prepeptides, i.e. a positively charged NH2-terminal (n) region, a hydrophobic core (h) region, and a COOH-terminal polar domain (c region). This signal sequence has multiple potential sites for cotranslational processing making it an attractive model for assessing the consequences of systematic structural alterations on the site selected for signal peptidase cleavage. We previously analyzed 40 mutant derivatives of this model preprotein using an in vitro translation/canine microsome processing assay. The results showed that the position of the boundary between the h and c regions and properties of the -1 residue are critical in defining the site of cotranslational cleavage. To investigate whether structural features in the NH2-terminal region of signal peptides play a role in cleavage specificity, we have now inserted various amino acids between the positively charged n region (NH2-Met-Lys) and the h region of a "parental" pre(delta pro)apoA-II mutant that has roughly equal cleavage between Gly18 decreases and Gly20 decreases. Movement of the n/h boundary toward the NH2 terminus results in a dramatic shift in cleavage to Gly18 decreases. Replacement of the Lys2 residue with hydrophilic, negatively charged residues preserves the original sites of cleavage. Replacement with a hydrophobic residue causes cleavage to shift "upstream." Simultaneous alteration of the position of n/h and h/c boundaries has an additive effect on the site of signal peptidase cleavage. None of these mutations produced a marked decrease in the efficiency of in vitro cotranslational translocation or cleavage. However, in sequence contexts having poor signal function, introduction of hydrophobic residues between the n and h regions markedly improved the efficiency of translocation/processing. We conclude that the position of the n/h boundary as well as positioning of the h/c boundary affects the site of cleavage chosen by signal peptidase.     

Characterization of the cleavage of signal peptide at the C-terminus of hepatitis C virus core protein by signal peptide peptidase

Production of hepatitis C virus (HCV) core protein requires the cleavages of polyprotein by signal peptidase and signal peptide peptidase (SPP). Cleavage of signal peptide at the C-terminus of HCV core protein by SPP was characterized in this study. The spko mutant (mutate a.a. 189–193 from ASAYQ to PPFPF) is more efficient than the A/F mutant (mutate a.a 189 and 191 from A to F) in blocking the cleavage of signal peptide by signal peptidase. The cleavage efficiency of SPP is inversely proportional to the length of C-terminal extension of the signal peptide: the longer the extension, the less efficiency the cleavage is. Thus, reducing the length of C-terminal extension of signal peptide by signal peptidase cleavage could facilitate further cleavage by SPP. The recombinant core protein fused with signal peptide from the C-terminus of p7 protein, but not those from the C-termini of E1 and E2, could be cleaved by SPP. Therefore, the sequence of the signal peptide is important but not the sole determinant for its cleavage by SPP. Replacement of the HCV core protein E.R.-associated domain (a.a. 120–150) with the E.R.-associated domain (a.a.1–50) of SARS-CoV membrane protein results in the failure of cleavage of this recombinant protein by SPP, though this protein still is E.R.-associated. This result suggests that not only E.R.-association but also specific protein sequence is important for the HCV core protein signal peptide cleavage by SPP. Thus, our results suggest that both sequences of the signal peptide and the E.R.-associated domain are important for the signal peptide cleavage of HCV core protein by SPP. Electronic Supplementary MaterialThe online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Deletion of the amino-terminal domain of asialoglycoprotein receptor H1 allows cleavage of the internal signal sequence

Human asialoglycoprotein receptor H1 is a single-spanning membrane protein with an amino-terminal domain of 40 residues exposed to the cytoplasm and the carboxyl-terminal domain translocated to the exoplasmic side of the membrane. It has been shown earlier that the transmembrane segment functions as an internal uncleaved signal sequence for insertion into the endoplasmic reticulum. In a deletion protein lacking almost the entire cytoplasmic domain, the signal sequence is cleaved at the carboxyl-terminal end of the transmembrane segment. All available criteria suggest that the protein is processed by signal peptidase. The cytoplasmic domain of the receptor does not directly inhibit signal cleavage since it does not detectably hinder cleavage of the normally amino-terminal signal sequence of influenza hemagglutinin in fusion proteins. We suggest that by its size or structure it affects the position of the receptor in the membrane and thus the accessibility of the potential cleavage site to signal peptidase.     

Importance of the propeptide sequence of human preproparathyroid hormone for signal sequence function

The function of amino-terminal pro-specific peptides (propeptides), sequences often found on intermediate precursor forms of secreted proteins, is poorly understood. Human preproparathyroid hormone (prepro-PTH), a precursor protein containing such a propeptide, is initially synthesized as a precursor containing a 25-amino acid signal sequence, a 6-amino acid propeptide, and the 84-amino acid mature secreted peptide. Cloned cDNA encoding prepro-PTH and synthetic oligonucleotides were used to generate a mutant missing precisely the pro-specific sequences. The effects of this deletion on signal sequence function and on secretion per se were assessed after expression of the mutant cDNA in intact cells and in a cell-free translation system using synthetic mRNA in the presence of microsomal membranes. The mutant precursor protein was inefficiently translocated and cleaved, and cleavage occurred both at the normal site and within the signal sequence. Thus, for the eukaryotic protein prepro-PTH, sequences immediately downstream and separate from the classically defined signal sequence facilitate accurate and efficient signal function.     

The role of the N region in signal sequence and signal-anchor function.

To determine the role of sequences other than the hydrophobic core in mediating signal sequence function, we examined the behavior of fusion proteins and deletion mutants in cell-free systems. We demonstrate that neither the N nor the C region of the preprolactin signal sequence is necessary for translocation. However, insertion of sequences with either a net charge of +2.5 or -6.0 between the N region and the hydrophobic core of the signal converted it into a signal-anchor. The topologies adopted (types I and II, respectively) were opposite those predicted from the distribution of charges surrounding the hydrophobic core of the signals. When these mutant signals were located in the interior of an otherwise secreted protein, both sequences functioned as stop-transfer sequences. Related mutations were assayed in fusion proteins in which the IgM transmembrane domain functioned as an amino-terminal signal-anchor. For these molecules, the distribution of charged residues surrounding the hydrophobic core had no influence on the topology adopted. Our results suggest that features other than simple charge distribution play an important role in determining membrane topology in vitro.     

Effects of neighboring sequence environment in predicting cleavage sites of signal peptides

Signal peptide has a pivotal role in the translocation of secretory protein. Some models have been designed to predict its cleavage site. It is reported that the cleavage site has relationship with the neighboring sequence environment, i.e., hydrophobic core h-region, and the specific patterns in c-region. In some studies, this finding does facilitate the prediction of cleavage site. However, in these models, sequence environment information is merely taken account of as model inputs and no detailed investigation into its effect on the prediction of cleavage site has been made. In this work, we analyze the constraint on cleave site placed by the hydrophobic core of signal peptide and then use it to improve the performance of the signal peptide cleavage site prediction. Our model is designed as follows: firstly, a sliding window is used to scan sample and artificial neural network (ANN) is employed to give cleavage site/non-cleavage site scores. Then, based on an estimated hydrophobic h-region a correcting function is proposed to improve the prediction result, in which the sequence environment is taken into account. A trend of cleavage site is indicated by our analysis for each position, which is consistent with experimental findings. Through this correcting step, the improvement of prediction accuracy is over 7%. It therefore demonstrates the neighboring sequence environment is helpful for determination of cleavage site. Program written in Matlab can be downloaded from http://www.scucic.cn/combined model/source code.html.     

A new method for predicting signal sequence cleavage sites.

A new method for identifying secretory signal sequences and for predicting the site of cleavage between a signal sequence and the mature exported protein is described. The predictive accuracy is estimated to be around 75-80% for both prokaryotic and eukaryotic proteins.     

The topogenic contribution of uncharged amino acids on signal sequence orientation in the endoplasmic reticulum

Signal sequences for insertion of proteins into the endoplasmic reticulum induce translocation of either the C- or the N-terminal sequence across the membrane. The end that is translocated is primarily determined by the flanking charges and the hydrophobic domain of the signal. To characterize the hydrophobic contribution to topogenesis, we have challenged the translocation machinery in vivo in transfected COS cells with model proteins differing exclusively in the apolar segment of the signal. Homo-oligomers of hydrophobic amino acids as different in size and shape as Val(19), Trp(19), and Tyr(22) generated functional signal sequences with similar topologies in the membrane. The longer a homo-oligomeric sequence of a given residue, the more N-terminal translocation was obtained. To determine the topogenic contribution of all uncharged amino acids in the context of a hydrophobic signal sequence, two residues in a generic oligoleucine signal were exchanged for all uncharged amino acids. The resulting scale resembles a hydrophobicity scale with the more hydrophobic residues promoting N-terminal translocation. In addition, the helix breakers glycine and proline showed a position-dependent effect, which raises the possibility of a conformational contribution to topogenesis.     

Preprotein transfer to the Escherichia coli translocase requires the co-operative binding of SecB and the signal sequence to SecA

In Escherichia coli , precursor proteins are targeted to the membrane-bound translocase by the cytosolic chaperone SecB. SecB binds to the extreme carboxy-terminus of the SecA ATPase translocase subunit, and this interaction is promoted by preproteins. The mutant SecB proteins, L75Q and E77K, which interfere with preprotein translocation in vivo , are unable to stimulate in vitro translocation. Both mutants bind proOmpA but fail to support the SecA-dependent membrane binding of proOmpA because of a marked reduction in their binding affinities for SecA. The stimulatory effect of preproteins on the interaction between SecB and SecA exclusively involves the signal sequence domain of the preprotein, as it can be mimicked by a synthetic signal peptide and is not observed with a mutant preprotein (Δ8proOmpA) bearing a non-functional signal sequence. Δ8proOmpA is not translocated across wild-type membranes, but the translocation defect is suppressed in inner membrane vesicles derived from a prlA4 strain. SecB reduces the translocation of Δ8proOmpA into these vesicles and almost completely prevents translocation when, in addition, the SecB binding site on SecA is removed. These data demonstrate that efficient targeting of preproteins by SecB requires both a functional signal sequence and a SecB binding domain on SecA. It is concluded that the SecB–SecA interaction is needed to dissociate the mature preprotein domain from SecB and that binding of the signal sequence domain to SecA is required to ensure efficient transfer of the preprotein to the translocase.     

The role of the membrane-spanning domain of type I signal peptidases in substrate cleavage site selection

Type I signal peptidase (SPase I) catalyzes the cleavage of the amino-terminal signal sequences from preproteins destined for cell export. Preproteins contain a signal sequence with a positively charged n-region, a hydrophobic h-region, and a neutral but polar c-region. Despite having no distinct consensus sequence other than a commonly found c-region "Ala-X-Ala" motif preceding the cleavage site, signal sequences are recognized by SPase I with high fidelity. Remarkably, other potential Ala-X-Ala sites are not cleaved within the preprotein. One hypothesis is that the source of this fidelity is due to the anchoring of both the SPase I enzyme (by way of its transmembrane segment) and the preprotein substrate (by the h-region in the signal sequence) in the membrane. This limits the enzyme-substrate interactions such that cleavage occurs at only one site. In this work we have, for the first time, successfully isolated Bacillus subtilis type I signal peptidase (SipS) and a truncated version lacking the transmembrane domain (SipS-P2). With purified full-length as well as truncated constructs of both B. subtilis and Escherichia coli (Lep) SPase I, in vitro specificity studies indicate that the transmembrane domains of either enzyme are not important determinants of in vitro cleavage fidelity, since enzyme constructs lacking them reveal no alternate site processing of pro-OmpA nuclease A substrate. In addition, experiments with mutant pro-OmpA nuclease A substrate constructs indicate that the h-region of the signal peptide is also not critical for substrate specificity. In contrast, certain mutants in the c-region of the signal peptide result in alternate site cleavage by both Lep and SipS enzymes.     

Selection of functional signal peptide cleavage sites from a library of random sequences.

The export of proteins to the periplasmic compartment of bacterial cells is mediated by an amino-terminal signal peptide. After transport, the signal peptide is cleaved by a processing enzyme, signal peptidase I. A comparison of the cleavage sites of many exported proteins has identified a conserved feature of small, uncharged amino acids at positions -1 and -3 relative to the cleavage site. To determine experimentally the sequences required for efficient signal peptide cleavage, we simultaneously randomized the amino acid residues from positions -4 to +2 of the TEM-1 beta-lactamase enzyme to form a library of random sequences. Mutants that provide wild-type levels of ampicillin resistance were then selected from the random-sequence library. The sequences of 15 mutants indicated a bias towards small amino acids. The N-terminal amino acid sequence of the mature enzyme was determined for nine of the mutants to assign the new -1 and -3 residues. Alanine was present in the -1 position for all nine of these mutants, strongly supporting the importance of alanine at the -1 position. The amino acids at the -3 position were much less conserved but were consistent with the -3 rules derived from sequence comparisons. Compared with the wild type, two of the nine mutants have an altered cleavage position, suggesting that sequence is more important than position for processing of the signal peptide.     

Insertion of a multispanning membrane protein occurs sequentially and requires only one signal sequence

To study the insertion of multispanning membrane proteins into the endoplasmic reticulum, we constructed novel proteins on the cDNA level by repeating, up to four times, the internal signal-anchor domain of the asialoglycoprotein receptor H1. Upon in vitro translation in the presence of microsomes, these polypeptides are indeed inserted as polytopic membrane proteins. The first hydrophobic domain functions as a signal and the second as a stop-transfer sequence, while the third initiates a second translocation process, halted again by the fourth. We were able to demonstrate that insertion occurs sequentially, starting with the first apolar segment from the amino terminus. By replacing the original signal-anchor domains by a mutant sequence not recognized by signal recognition particle (SRP), it was shown that only the first hydrophobic domain needs to be a signal sequence and that the second translocation event does not require SRP.     

An internal signal sequence directs intramembrane proteolysis of a cellular immunoglobulin domain protein

Precursor proteolysis is a crucial mechanism for regulating protein structure and function. Signal peptidase (SP) is an enzyme with a well defined role in cleaving N-terminal signal sequences but no demonstrated function in the proteolysis of cellular precursor proteins. We provide evidence that SP mediates intraprotein cleavage of IgSF1, a large cellular Ig domain protein that is processed into two separate Ig domain proteins. In addition, our results suggest the involvement of signal peptide peptidase (SPP), an intramembrane protease, which acts on substrates that have been previously cleaved by SP. We show that IgSF1 is processed through sequential proteolysis by SP and SPP. Cleavage is directed by an internal signal sequence and generates two separate Ig domain proteins from a polytopic precursor. Our findings suggest that SP and SPP function are not restricted to N-terminal signal sequence cleavage but also contribute to the processing of cellular transmembrane proteins.     

Structural determinants for signal sequence function in the mammalian endoplasmic reticulum

Signal sequences function in protein targeting to and translocation across the endoplasmic reticulum membrane. To investigate the structural requirements for signal sequence function, chimeras of the Escherichia coli LamB signal peptide and prolactin were prepared. The LamB signal peptide was chosen by virtue of the extensive biophysical and biological characterization of its activity. In vitro, nascent prolactin chains bearing the LamB signal peptide (LamB) were targeted in a signal recognition particle (SRP)-dependent manner to rough microsomes but remained protease- and salt-sensitive and translocated at low efficiency. Full translocation activity was obtained in a gain of function mutant (LamB*) in which three hydrophobic residues in the LamB hydrophobic core were converted to leucine residues. Cross-linking studies demonstrated that the LamB* signal sequence displayed markedly enhanced interactions with SRP and integral membrane proteins. In contrast, chemically denatured LamB and LamB*-precursors bound with identical efficiencies and in a salt-resistant manner to rough microsomes, suggesting that during de novo synthesis the signal sequence of LamB-bearing precursors assumes a conformation refractory to translocation. These data indicate that a leucine-rich signal sequence is necessary for optimal interaction with SRP and suggest that SRP, by maintaining the signal sequence in a conformation suitable for membrane binding, performs a chaperone function.