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.     

Requirements for mouse mammary tumor virus Rem signal peptide processing and function

Mouse mammary tumor virus (MMTV) encodes a Rev-like protein, Rem, which is involved in the nuclear export and expression of viral RNA. Previous data have shown that all Rev-like functions are localized to the 98-amino-acid signal peptide (SP) at the N terminus of MMTV Rem or envelope proteins. MMTV-SP uses endoplasmic reticulum-associated degradation (ERAD) for protein trafficking. Rem cleavage by signal peptidase in the ER is necessary for MMTV-SP function in a reporter assay, but many requirements for trafficking are not known. To allow detection and localization of both MMTV-SP and the C-terminal cleavage product, we prepared plasmids expressing green fluorescent protein (GFP) tags. N-terminal Rem tagging led to protein accumulation relative to untagged Rem and allowed signal peptidase cleavage but reduced its specific activity. C-terminal tagging also led to Rem accumulation yet dramatically reduced cleavage, GFP fluorescence, and activity relative to N-terminally tagged Rem (GFPRem). Substitutions of an invariant leucine at position 71 between the known RNA-binding and nuclear export sequences interfered with GFPRem accumulation and activity but not cleavage. Similarly, deletion of 100 or 150 C-terminal amino acids from GFPRem dramatically reduced both Rem and MMTV-SP levels and function. Removal of the entire C terminus (203 amino acids) restored both protein levels and activity of MMTV-SP. Only C-terminal GFP tagging, and not other modifications, appeared to trap Rem in the ER membrane. Thus, Rem conformation in both the ER lumen and cytoplasm determines cleavage, retrotranslocation, and MMTV-SP function. These mutants further characterize intermediates in Rem trafficking and have implications for all proteins affected by ERAD.     

Long-lived signal peptide of lymphocytic choriomeningitis virus glycoprotein pGP-C

Signal peptides (SPs) direct nascent secretory and membrane proteins to the membrane of the endoplasmic reticulum. They are usually cleaved from the nascent polypeptide by signal peptidase and then further proteolytically processed. The SP of the pre-glycoprotein (pGP-C) of the lymphocytic choriomeningitis virus SPGP-C (signal peptide of pGP-C) shows different properties: 1) The SPGP-C is unusually long (58 amino acid residues) and contains two hydrophobic segments interrupted by a lysine residue. 2) The SPGP-C is cleaved only from a subset of pGP-C proteins. A substantial portion of pGP-C accumulates that still contains the SPGP-C.3)The cleaved SPGP-C is rather long-lived (t(1/2) of more than 6 h). 4) The cleaved SPGP-C resides in the membrane and is resistant to digestion with proteinase K even in the presence of detergents, suggesting a very compact structure. 5) SPGP-C accumulates in virus particles. These unusual features of the cleaved SPGP-C suggest that SPGP-C not only targets the nascent pGP-C to the endoplasmic reticulum membrane but also has additional functions in lymphocytic choriomeningitis virus life cycle.     

mCLCA4 ER processing and secretion requires luminal sorting motifs

Ca(+)-activated Cl(-) channel (CLCA) proteins are encoded by a family of highly related and clustered genes in mammals that are markedly upregulated in inflammation and have been shown to affect chloride transport. Here we describe the cellular processing and regulatory sequences underlying murine (m) CLCA4 proteins. The 125-kDa mCLCA4 gene product is cleaved to 90- and 40-kDa fragments, and the NH(2)- and COOH-terminal fragments are secreted, where they are found in cell media and associated with the plasma membrane. The 125-kDa full-length protein is only found in the endoplasmic reticulum (ER), and specific luminal diarginine retention and dileucine forward trafficking signals contained within the CLCA4 sequence regulate export from the ER and proteolytic processing. Mutation of the dileucine luminal sequences resulted in ER trapping of the immaturely glycosylated 125-kDa peptide, indicating that proteolytic cleavage occurs following recognition of the trafficking motifs. Moreover, the mutated dileucine and diarginine signal sequences directed processing of a secreted form of enhanced green fluorescent protein in a manner consistent with the effects on mCLCA4.     

Juxtaposition of signal-peptide charge and core region hydrophobicity is critical for functional signal peptides

In Escherichia coli, exported proteins are synthesized as precursors containing an amino-terminal signal peptide which directs transport through the translocase to the proper destination. We have constructed a series of signal peptide mutants, incorporating linker sequences of varying lengths between the amino-terminal charge and core region hydrophobicity, to examine the requirement for the juxtaposition of these two structural features in promoting protein transport. In vivo and in vitro analyses indicated that high transport efficiency via signal peptides with core regions of marginal hydrophobicity absolutely requires the proximity of sufficient charge.     

Functional significance of cleavable signal peptides of G protein-coupled receptors

About 5-10% of the G protein-coupled receptors (GPCRs) contain N-terminal signal peptides that are cleaved off by the signal peptidases of the endoplasmic reticulum (ER) during the translocon-mediated receptor insertion into the ER membrane. The reason as to why only a subset of the GPCRs requires these additional signal peptides was addressed in the past decade only by a limited number of studies. Recent progress suggests that signal peptides of GPCRs do not only serve the classical ER targeting and translocon gating functions as described for the signal peptides of secretory proteins. In the case of GPCRs, uncleaved pseudo signal peptides may regulate receptor expression at the plasma membrane and may also influence G protein coupling. Moreover, signal peptides of GPCRs seem to match functionally with sequences of the mature N tails. In this review, we summarize the current knowledge about cleavable signal peptides of GPCRs and address the question whether these sequences may be future drug targets in pharmacology.     

Targeting and retention of HPV16 E7 to the endoplasmic reticulum enhances immune tumour protection

The endoplasmic reticulum (ER) is where the major histocompatibility complex (MHC) class I molecules are loaded with epitopes to cause an immune cellular response. Most of the protein antigens are degraded in the cytoplasm to amino acids and few epitopes reach the ER. Antigen targeting of this organelle by Calreticulin (CRT) fusion avoids this degradation and enhances the immune response. We constructed a recombinant adenovirus to express the E7 antigen with an ER‐targeting signal peptide (SP) plus an ER retention signal (KDEL sequence). In cell‐culture experiments we demonstrated that this new E7 antigen, SP‐E7‐KDEL, targeted the ER. Infection of mice with this recombinant adenovirus that expresses SP‐E7‐KDEL showed interferon induction and tumour‐protection response, similar to that provided by an adenovirus expressing the E7 antigen fused to CRT. This work demonstrated that just by adding a SP and the KDEL sequence, antigens can be targeted and retained in the ER with a consequent enhancement of immune response and tumour protection. These results will have significant clinical applications.     

Signal Peptide-Binding Drug as a Selective Inhibitor of Co-Translational Protein Translocation

In eukaryotic cells, surface expression of most type I transmembrane proteins requires translation and simultaneous insertion of the precursor protein into the endoplasmic reticulum (ER) membrane for subsequent routing to the cell surface. This co-translational translocation pathway is initiated when a hydrophobic N-terminal signal peptide (SP) on the nascent protein emerges from the ribosome, binds the cytosolic signal recognition particle (SRP), and targets the ribosome-nascent chain complex to the Sec61 translocon, a universally conserved protein-conducting channel in the ER-membrane. Despite their common function in Sec61 targeting and ER translocation, SPs have diverse but unique primary sequences. Thus, drugs that recognise SPs could be exploited to inhibit translocation of specific proteins into the ER. Here, through flow cytometric analysis the small-molecule macrocycle cyclotriazadisulfonamide (CADA) is identified as a highly selective human CD4 (hCD4) down-modulator. We show that CADA inhibits CD4 biogenesis and that this is due to its ability to inhibit co-translational translocation of CD4 into the lumen of the ER, both in cells as in a cell-free in vitro translation/translocation system. The activity of CADA maps to the cleavable N-terminal SP of hCD4. Moreover, through surface plasmon resonance analysis we were able to show direct binding of CADA to the SP of hCD4 and identify this SP as the target of our drug. Furthermore, CADA locks the SP in the translocon during a post-targeting step, possibly in a folded state, and prevents the translocation of the associated protein into the ER lumen. Instead, the precursor protein is routed to the cytosol for degradation. These findings demonstrate that a synthetic, cell-permeable small-molecule can be developed as a SP-binding drug to selectively inhibit protein translocation and to reversibly regulate the expression of specific target proteins.     

Internally located cleavable signal sequences direct the formation of Semliki Forest virus membrane proteins from a polyprotein precursor.

The proteolytic processes involved in the cotranslational production of the Semliki Forest virus proteins p62, 6K, and E1 from a common precursor polypeptide were analyzed by an in vitro translation-translocation assay. By studying the behavior of wild-type and mutant variants of the polyprotein, we show that the signal sequences responsible for membrane translocation of the 6K and E1 proteins reside in the C-terminal regions of p62 and 6K, respectively. We present evidence suggesting that the polyprotein is processed on the luminal side by signal peptidase at consensus cleavage sites immediately following the signal sequences. Our results also lead us to conclude that the 6K protein is a transmembrane polypeptide with its N terminus on the luminal side of the membrane (type I). Thus, the production of all three membrane proteins is directed by alternating signal and stop-transfer (anchor) sequences that function in translocation and cleavage of the virus precursor polyprotein. This also shows conclusively that internally located signal sequences can be cleaved by signal peptidase.     

Analysis of the retention signals of two resident luminal endoplasmic reticulum proteins by in vitro mutagenesis

Protein disulfide isomerase (PDI, ERp59), ERp72, and ERp61 are luminal proteins of the endoplasmic reticulum (ER) that are characterized by the presence of sequences corresponding to the active site regions of PDI. Each one of these proteins possesses a different COOH-terminal tetrapeptide ER retention signal. In order to investigate what other tetrapeptide sequences could serve as retention signals and to determine to what extent the function of the retention signal is modulated by the protein carrying the signal, we have constructed a set of mutants of two of these resident ER proteins, PDI and ERp72. In each of these proteins, the wild type tetrapeptide sequences were replaced by each member of the set of the 12 possible combinations consisting of (K,R,Q)-(D,E)-(D,E)-L. Analysis of the efficiency of retention of the variant proteins when each was transiently expressed in COS cells showed that the retention efficiencies vary with both the COOH-terminal sequence and with the protein that carries this sequence.     

A tripartite structure of the signals that determine protein insertion into the endoplasmic reticulum membrane

Multilineage colony stimulating factor is a secretory protein with a cleavable signal sequence that is unusually long and hydrophobic. Using molecular cloning techniques we exchanged sequences NH2- or COOH-terminally flanking the hydrophobic signal sequence. Such modified fusion proteins still inserted into the membrane but their signal sequence was not cleaved. Instead the proteins were now anchored in the membrane by the formerly cleaved signal sequence (signal-anchor sequence). They exposed the NH2 terminus on the exoplasmic and the COOH terminus on the cytoplasmic side of the membrane. We conclude from our results that hydrophilic sequences flanking the hydrophobic core of a signal sequence can determine cleavage by signal peptidase and insertion into the membrane. It appears that negatively charged amino acid residues close to the NH2 terminal side of the hydrophobic segment are compatible with translocation of this segment across the membrane. A tripartite structure is proposed for signal-anchor sequences: a hydrophobic core region that mediates targeting to and insertion into the ER membrane and flanking hydrophilic segments that determine the orientation of the protein in the membrane.     

Gene fusions of signal sequences with a modified beta-glucuronidase gene results in retention of the beta-glucuronidase protein in the secretory pathway/plasma membrane.

Signal sequences and endoplasmic reticulum (ER) retention signals are known to play central roles in targeting and translocation in the secretory pathway, but molecular aspects about their involvement are poorly understood. We tested the effectiveness of deduced signal sequences from various genes (hydroxyproline-rich glycoprotein [HRGP] from Phaseolus vulgaris; Serpin from Manduca sexta) to direct a modified beta-glucuronidase (GUS) protein into the secretory pathway in transgenic tobacco (Nicotiana tabacum L.). The reporter protein was not secreted to the cell wall/extracellular space as monitored using extracellular fluid analysis (low- or high-ionic-strength conditions) but occurred in membranes with a density of 1.16 to 1.20 g/mL. Membrane-bound GUS equilibrated with the plasma membrane (PM) and the ER on linear sucrose gradients with or without ethylenediaminetetraacetic acid, suggesting that GUS associates with the ER and the PM. Confocal microscopy of fixed cultured cells prepared from GUS control and HRGP signal peptide (SP)-GUS-expressing plants suggested only cytosolic localization in GUS-expressing plants but substantial peripheral localization in HRGP SP-GUS plants, which is consistent with GUS being associated with the PM. Aqueous two-phase partitioning of microsomal membranes from HRGP SP-GUS and Serpin SP-GUS transgenic leaves also indicated that GUS activity was enriched in the ER and the PM. These observations, together with hydrophobic moment plot analysis, suggest that properties of the SP-GUS protein result in its retention in the secretory pathway and PM.     

Ancient, independent evolution and distinct molecular features of the novel human T-lymphotropic virus type 4

Background

The human endogenous retrovirus HERV-K(HML-2) family is associated with testicular germ cell tumors (GCT). Various HML-2 proviruses encode viral proteins such as Env and Rec.

Results

We describe here that HML-2 Env gives rise to a 13 kDa signal peptide (SP) that harbors a different C-terminus compared to Rec. Subsequent to guiding Env to the endoplasmatic reticulum (ER), HML-2 SP is released into the cytosol. Biochemical analysis and confocal microscopy demonstrated that similar to Rec, SP efficiently translocates to the granular component of nucleoli. Unlike Rec, SP does not shuttle between nucleus and cytoplasm. SP is less stable than Rec as it is subjected to proteasomal degradation. Moreover, SP lacks export activity towards HML-2 genomic RNA, the main function of Rec in the original viral context, and SP does not interfere with Rec's RNA export activity.

Conclusion

SP is a previously unrecognized HML-2 protein that, besides targeting and translocation of Env into the ER lumen, may exert biological functions distinct from Rec. HML-2 SP represents another functional similarity with the closely related Mouse Mammary Tumor Virus that encodes an Env-derived SP named p14. Our findings furthermore support the emerging concept of bioactive SPs as a conserved retroviral strategy to modulate their host cell environment, evidenced here by a "retroviral fossil". While the specific role of HML-2 SP remains to be elucidated in the context of human biology, we speculate that it may be involved in immune evasion of GCT cells or tumorigenesis.     

Functional characterization of bacterial signal peptide OmpA in a baculovirus-mediated expression system

Signal sequences are evolutionarily conserved and are often functionally interchangeable between prokaryotes and eukaryotes. However, we have found that the bacterial signal peptide, OmpA, functions incompletely in insect cells. Upon baculovirus-mediated expression of chloramphenicol acetyltransferase (CAT) in insect cells, OmpA signal peptide led to the cytosolic accumulation of the CAT molecules in an aglycosylated, signal-peptide cleaved form, in addition to the secretion of the glycosylated CAT. When green fluorescent protein (GFP) was used as another reporter, the GFP molecules expressed from the OmpA-GFP construct was distributed primarily in the cytosol as aggresome-like structures. These results together suggest that, subsequent to the cleavage of OmpA signal peptide in the ER, some of the processed proteins are returned to the cytoplasm. Since the prototypical insect signal peptide, melittin, did not result in this ER-to-cytosol dislocation of the reporter proteins, we proposed a model explaining the dislocation process in insect cells, apparently selective to the OmpA-directed secretory pathway bypassing the co-translational transport.     

A Conserved Endoplasmic Reticulum Membrane Protein Complex (EMC) Facilitates Phospholipid Transfer from the ER to Mitochondria

Mitochondrial membrane biogenesis and lipid metabolism require phospholipid transfer from the endoplasmic reticulum (ER) to mitochondria. Transfer is thought to occur at regions of close contact of these organelles and to be nonvesicular, but the mechanism is not known. Here we used a novel genetic screen in S. cerevisiae to identify mutants with defects in lipid exchange between the ER and mitochondria. We show that a strain missing multiple components of the conserved ER membrane protein complex (EMC) has decreased phosphatidylserine (PS) transfer from the ER to mitochondria. Mitochondria from this strain have significantly reduced levels of PS and its derivative phosphatidylethanolamine (PE). Cells lacking EMC proteins and the ER–mitochondria tethering complex called ERMES (the ER–mitochondria encounter structure) are inviable, suggesting that the EMC also functions as a tether. These defects are corrected by expression of an engineered ER–mitochondrial tethering protein that artificially tethers the ER to mitochondria. EMC mutants have a significant reduction in the amount of ER tethered to mitochondria even though ERMES remained intact in these mutants, suggesting that the EMC performs an additional tethering function to ERMES. We find that all Emc proteins interact with the mitochondrial translocase of the outer membrane (TOM) complex protein Tom5 and this interaction is important for PS transfer and cell growth, suggesting that the EMC forms a tether by associating with the TOM complex. Together, our findings support that the EMC tethers ER to mitochondria, which is required for phospholipid synthesis and cell growth.     

Primary sequence domains required for the retention of rotavirus VP7 in the endoplasmic reticulum

Rotavirus VP7 is a membrane-associated protein of the endoplasmic reticulum (ER). It is the product of rotavirus gene 9 which potentially encodes a protein of 326 amino acids that contains two amino terminal hydrophobic domains, h1 and h2, each preceded by an initiation codon. Comparison of the size of products derived from altered genes containing coding sequences for both h1 and h2 with those lacking the h1 sequence ('dhl' mutants), indicates that initiation takes place at M30 immediately preceding h2 (residues F32 to L48) and that h2 is cleaved, confirming the studies of others (Stirzaker, S.C., P.L. Whitfeld, D.L. Christie, A.R. Bellamy, and G.W. Both. 1987. J. Cell Biol. 105:2897-2903). Our previous work had shown that deletions in the carboxy end of h2, extending to amino acid 61 in the open reading frame, resulted in secretion of VP7. The region from amino acid number 51-61, present in wild-type VP7 but missing in the secreted mutant delta 47-61, was thus implicated to have a role in ER retention. To test this, a series of chimeric genes were constructed by fusing the first 63 codons of wild-type VP7, delta 1-14 or delta 51-61/dhl, to the mouse salivary alpha-amylase gene, a secretory protein, such that the fusion junction was located at the exact mature terminus of amylase. The chimeric proteins VP7(63)/amylase, delta 1-14(63)/amylase and delta 51-61(63)/dhl/amylase were secreted when expressed in cells and the h2 domain was cleaved when mRNA was translated in vitro. These results imply that the sequence 51-61 is necessary but not sufficient for ER retention. When a second series of VP7/amylase chimera were constructed extending the VP7 contribution to amino acid 111, the product expressed by delta 1-14(111)/amylase was not secreted whereas that of delta 47-61(111)/amylase was. Significantly, the intracellular delta 1-14(111)/amylase product exhibited an amylase enzymatic specific activity that was similar to that of the wild-type amylase product. We conclude that two regions of VP7 mediate its retention in the ER, the first lies within the sequence 51-61 and the second within the sequence 62-111, which contains the glycosylation site for VP7. Both regions are necessary for retention, though neither is sufficient alone.     

The signal peptide of the rotavirus glycoprotein VP7 is essential for its retention in the ER as an integral membrane protein

The rotavirus glycoprotein VP7 has a cleavable signal peptide and is normally resident as an integral membrane protein in the ER of infected cells. A gene was constructed in which the VP7 H2 signal peptide was replaced by one from influenza hemagglutinin. COS cells transfected with this gene produced VP7 with the correct amino terminus, but the protein was rapidly secreted. Uncleaved VP7 from either precursor was not detected in cells after brief pulse-labeling, suggesting that the signal peptide was not acting as a temporary anchor; rather, it exerted its effect despite rapid cleavage. By splicing the H2 signal peptide onto another reporter protein, the malaria S-antigen, we demonstrated that H2 was necessary, but not itself sufficient, for targeting and retention. We propose that an interaction between the cleaved signal peptide and other downstream sequences in VP7 is required for retention of this protein in the ER as an integral membrane polypeptide.     

Estradiol stabilizes estrogen receptor messenger ribonucleic acid in sheep endometrium via discrete sequence elements in its 3'-untranslated region

The preovulatory surge of estrogen up-regulates estrogen receptor-alpha (ER) gene expression in the uterus during the estrous/menstrual cycles of female mammals. Previously, we demonstrated that the 5-fold increase in ER mRNA levels in endometrium of ovariectomized ewes treated with a physiological dose of estradiol (E2) is entirely due to an increase in ER mRNA stability. Our current work confirms that the E2 effect is specific to ER mRNA. The sequence of ER mRNA, cloned from sheep endometrium, shows a high degree of conservation with those of other species, even in the 5'- and the very long 3'-untranslated regions. In a cell-free assay, ER mRNA demonstrates greater stability with endometrial extracts from E2-treated ewes compared with those from untreated ovariectomized ewes. The E2-enhanced stability of ER mRNA was ablated by prior treatment of the extracts with proteinase K, 70 C heat, and oxidizing and alkylating reagents, indicating that a protein is responsible for stabilization of the message. The 3'-untranslated region of ER mRNA contains discrete sequences required for E2-enhanced stability, four of which were identified by extensive deletion mutant analyses. Transfer of two of the four minimal E2-modulated stability sequences conferred E2-enhanced stability to a heterologous RNA. These minimal E2-modulated stability sequences contain a common 10-base, uridine-rich sequence that is predicted to reside in a loop structure. Throughout our studies, estrogen stabilization of ER mRNA in sheep endometrium resembled that of vitellogenin mRNA in frog liver, indicating conservation of this ancient mechanism for enhancing gene expression in response to estrogen.