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Recent evidence that a herpes virus protein lacking a classical secretory signal sequence can spread between cells in culture draws attention to a class of proteins that are transported into and out of cells by unconventional means.
The corticotropin-releasing factor receptor type 2a (CRF(2(a)) receptor) belongs to the family of G protein-coupled receptors. The receptor possesses a putative N-terminal signal peptide that is believed to be cleaved-off after mediating the endoplasmic reticulum targeting/insertion process, like the corresponding sequence of the homologous CRF(1) receptor. Here, we have assessed the functional significance of the putative signal peptide of the CRF(2(a)) receptor and show that it is surprisingly completely incapable of mediating endoplasmic reticulum targeting, despite meeting all sequence criteria for a functional signal by prediction algorithms. Moreover, it is uncleaved and forms part of the mature receptor protein. Replacement of residue Asn(13) by hydrophobic or positively charged residues converts the sequence into a fully functional and cleaved signal peptide demonstrating that conventional signal peptide functions are inhibited by a single amino acid residue. Deletion of the domain leads to an increase in the amount of immature, intracellularly retained receptors demonstrating that the sequence has adopted a new function in receptor trafficking through the early secretory pathway. Taken together, our results identify a novel hydrophobic receptor domain in the family of the heptahelical G protein-coupled receptors and the first pseudo signal peptide of a eukaryotic membrane protein. Our data also show that the extreme N termini of the individual CRF receptor subtypes differ substantially.
We have previously shown that the peroxisomal targeting signal in firefly luciferase consists of the COOH-terminal three amino acids of the protein, serine-lysine-leucine (Gould, S.J., G.A. Keller, N. Hosken, J. Wilkinson, and S. Subramani, 1989. J. Cell Biol. 108:1657-1664). Antibodies were raised against a synthetic peptide that contained this tripeptide at its COOH terminus. Immunofluorescence and immunocryoelectron microscopy revealed that the anti-peptide antibodies specifically detected peroxisomes in mammalian cells. Further characterization revealed that the antibodies were primarily directed against the COOH-terminal three amino acids of the peptide. In Western blot experiments, the antibodies recognized 15-20 rat liver peroxisomal proteins, but reacted with only a few proteins from other subcellular compartments. These results provide independent immunological evidence that the peroxisomal targeting signal identified in firefly luciferase is present in many peroxisomal proteins.
The carboxylated (C)-terminus of proteins, which includes the single terminal alpha-carboxyl group and preceding residues, is uniquely positioned to serve as a recognition signature for a variety of cell-biological processes, including protein targeting, subcellular anchoring and the static and dynamic formation of macromolecular complexes. The terminal sequence motifs can be processed by posttranslational modifications, thereby providing a means to increase sequence diversity and to regulate interactions. Several classes of protein domains have been identified that are either designed for or are capable of interacting with protein C-termini - these include PDZ and TPR domains. The interactions between these protein domains and various terminal epitopes play an important role in specifying cell-biological functions. The combination of diversity and the plasticity of the chemistry of C-termini provides mechanisms for spatial and temporal specificity that are exploited by a variety of biological processes, ranging from specifying prokaryotic protein degradation to nucleating mammalian neuronal signaling complexes. Understanding the diverse functions of protein C-termini might also provide an important indexing criterion for functional proteomics.
A group of integral membrane proteins, known as C-tail anchored, is defined by the presence of a cytosolic N-terminal domain that is anchored to the phospholipid bilayer by a single segment of hydrophobic amino acids close to the C-terminus. The mode of insertion into membranes of these proteins, many of which play key roles in fundamental intracellular processes, is obligatorily post-translational, is highly specific and may be subject to regulatory processes that modulate the protein's function. Recent work has demonstrated that tail-anchored proteins translocate their C-termini across the endoplasmic reticulum membrane by a mechanism different from that used for Sec61-dependent post-translational signal-peptide-driven translocation. Here we summarize recent results on the insertion of tail-anchored proteins and discuss possible mechanisms that could be involved.
Targeting of secretory and membrane proteins by the signal recognition particle (SRP) is evolutionarily conserved, and the multidomain protein SRP54 acts as the key player in SRP-mediated protein transport. Binding of a signal peptide to SRP54 at the ribosome is coordinated with GTP binding and subsequent complex formation with the SRP receptor. Because these functions are localized to distinct domains of SRP54, communication between them is essential. We report the crystal structures of SRP54 from the Archaeon Sulfolobus solfataricus with and without its cognate SRP RNA binding site (helix 8) at 4-A resolution. The two structures show the flexibility of the SRP core and the position of SRP54 relative to the RNA. A long linker helix connects the GTPase (G domain) with the signal peptide binding (M) domain, and a hydrophobic contact between the N and M domains relates the signal peptide binding site to the G domain. Hinge regions are identified in the linker between the G and M domains (292-LGMGD) and in the N-terminal part of the M domain, which allow for structural rearrangements within SRP54 upon signal peptide binding at the ribosome.
C-tail-anchored proteins are defined by an N-terminal cytosolic domain followed by a transmembrane anchor close to the C terminus. Their extreme C-terminal polar residues are translocated across membranes by poorly understood post-translational mechanism(s). Here we have used the yeast system to study translocation of the C terminus of a tagged form of mammalian cytochrome b(5), carrying an N-glycosylation site in its C-terminal domain (b(5)-Nglyc). Utilization of this site was adopted as a rigorous criterion for translocation across the ER membrane of yeast wild-type and mutant cells. The C terminus of b(5)-Nglyc was rapidly glycosylated in mutants where Sec61p was defective and incapable of translocating carboxypeptidase Y, a well known substrate for post-translational translocation. Likewise, inactivation of several other components of the translocon machinery had no effect on b(5)-Nglyc translocation. The kinetics of translocation were faster for b(5)-Nglyc than for a signal peptide-containing reporter. Depletion of the cellular ATP pool to a level that retarded Sec61p-dependent post-translational translocation still allowed translocation of b(5)-Nglyc. Similarly, only low ATP concentrations (below 1 microm), in addition to cytosolic protein(s), were required for in vitro translocation of b(5)-Nglyc into mammalian microsomes. Thus, translocation of tail-anchored b(5)-Nglyc proceeds by a mechanism different from that of signal peptide-driven post-translational translocation.
Snail, a DNA-binding zinc finger protein, functions as a transcriptional repressor for genes including E-cadherin during development and the acquisition of tumor cell invasiveness. Human Snail is a 264-amino acid nuclear protein with an amino-terminal basic amino acid-rich domain (SNAG domain) and a carboxyl-terminal DNA-binding domain (zinc finger domain). A series of fusion proteins composed of green fluorescent protein (GFP) and portions of the Snail protein were generated, and their subcellular localization was examined. Fusion of the four zinc fingers to GFP led to the targeting of GFP to the nucleus, demonstrating that the zinc finger domain is sufficient for nuclear localization. Using an in vitro transport system, the nuclear import of Snail was reconstituted by importin (karyopherin) beta in the presence of Ran and NTF2. We further demonstrated that Snail binds directly to importin beta in a zinc finger domain-dependent manner. These results indicate that zinc finger domain of Snail functions as a nuclear localization signal and Snail can be transported into the nucleus in an importin beta-mediated manner.
BACKGROUND Alternative exons encode different isoforms of the human insulin-like growth factor-I (IGF-I) precursor without altering mature IGF-I. We hypothesized that the various IGF-I precursors may traffic IGF-I differently. Chimeric IGF-I precursors were made with green fluorescent protein (GFP) cloned between the signal and mature IGF-I domains. RESULTS Chimeras containing exons 1 or 2 were located in the cytoplasm, consistent with a secretory pathway, and suggesting that both exons encoded functional signal peptides. Exon 5-containing chimeras localized to the nucleus and strongly to the nucleolus, while chimeras containing exon 6 or the upstream portion of exon 5 did not. Nuclear and nucleolar localization also occurred when the mature IGF-I domain was deleted from the chimeras, or when signal peptides were deleted. CONCLUSIONS We have identified a nucleolar localization for an isoform of the human IGF-I precursor. The findings are consistent with the presence of a nuclear and nucleolar localization signal situated in the C-terminal part of the exon 5-encoded domain with similarities to signals in several other growth factors.
A screening strategy designed to identify cloned cDNAs encoding abundant Macaca fascicularis epididymal transcripts has yielded a clone corresponding to a 1.03-kb transcript present at high levels in the epididymis. Following library rescreening, DNA sequence analysis of several near full-length clones predicts a novel, 151-amino-acid protein, epididymal secretory protein 14.6 (ESP14.6), which contains a strong candidate signal peptide characteristic of secretory proteins.
Mycobacterium tuberculosis uses the ESX-1/Snm system [early secreted antigen 6 kilodaltons (ESAT-6) system 1/secretion in mycobacteria] to deliver virulence factors into host macrophages during infection. Despite its essential role in virulence, the mechanism of ESX-1 secretion is unclear. We found that the unstructured C terminus of the CFP-10 substrate was recognized by Rv3871, a cytosolic component of the ESX-1 system that itself interacts with the membrane protein Rv3870. Point mutations in the signal that abolished binding of CFP-10 to Rv3871 prevented secretion of the CFP-10 (culture filtrate protein, 10 kilodaltons)/ESAT-6 virulence factor complex. Attachment of the signal to yeast ubiquitin was sufficient for secretion from M. tuberculosis cells, demonstrating that this ESX-1 signal is portable.
The thylakoid twin arginine protein translocation (Tat) system operates by a cyclical mechanism in which precursors bind to a cpTatC-Hcf106 receptor complex, which then recruits Tha4 to form the translocase. After translocation, the translocase disassembles. Here, we fine-mapped initial interactions between precursors and the components of the receptor complex. Precursors with (Tmd)Phe substitutions in the signal peptide and early mature domain were bound to thylakoids and photo-cross-linked to components. cpTatC and Hcf106 were found to interact with different regions of the signal peptide. cpTatC cross-linked strongly to residues in the immediate vicinity of the twin arginine motif. Hcf106 cross-linked less strongly to residues in the hydrophobic core and the early mature domain. To determine whether precursors must leave their initial sites of interaction during translocation, cross-linked precursors were subjected to protein transport conditions. tOE17 cross-linked to cpTatC was efficiently translocated, indicating that the mature domain of the precursor can be translocated while the signal peptide remains anchored to the receptor complex.
The gene-poor, hominid-specific Yp11.2/Xq21.3 X-Y homology block encodes two members of the protocadherin group of cell surface molecules, PCDHX and PCDHY. These two genes, mainly expressed in brain, were known to be composed of at least six exons sharing 98.1% DNA identity. The genomic structure of PCDHX/ Y has been reanalyzed in detail, uncovering the existence of at least 11 more exons spanning more than 700 kb. Many of these exons located at the 5' and 3' ends of PCDHX/ Y undergo differential and alternative splicing. Seven of the exons have been found to use alternative splice sites. Most of these variants are expressed within the brain, although some isoforms exhibit a more ubiquitous distribution pattern. PCDHX/ Y transcription appears to be driven from two alternative promoters located usptream of exon 1 and exon 4.1. Assuming that the splicing events at the 5' and 3' ends of these genes are independent of one another, potentially up to 360 different mRNAs could be produced. The main impact on protein function is predicted to be in the efficiency of translation, post-translational processing within the cell, and structure of the cytoplasmic domain that may influence any role the genes have in signaling.
Numerous proteins in pro- and eukaryotes must cross cellular membranes in order to reach their site of function. Many of these proteins carry signal sequences that are removed by specific signal peptidases during, or shortly after, membrane transport. Signal peptidases have been identified in the rough endoplasmic reticulum, the matrix and inner membrane of mitochondria, the stroma and thylakoid membrane of chloroplasts, the bacterial plasma membrane and the thylakoid membrane of cyanobacteria. The composition of these peptidases varies between one and several subunits. No site-specific inhibitors are known for the majority of these enzymes. Accordingly, signal peptidases recognize structural motifs rather than linear amino acid sequences. Such motifs have become evident by employing extensive site-directed mutagenesis to investigate the anatomy of signal sequences. Analysis of the reaction specificities and the primary sequences of several signal peptidases suggests that the enzymes of the endoplasmic reticulum, the inner mitochondrial membrane and the thylakoid membrane of chloroplasts all have evolved from bacterial progenitors.
The targeting mechanism of chloroplast outer envelope membrane proteins remains largely unknown. We investigated the targeting of AtToc64. In protoplasts, the transmembrane domain (TMD) and its C-terminal Iysine-rich flanking region (LFR) were both necessary and sufficient for targeting to the outer envelope membrane. The lysine residues of the flanking region were critical; without the LFR, the TMD was targeted to the ER or the plasma membrane. In addition, the types of amino acid residues of the TMD, but not the amino acid sequence per se, is a signal for targeting to the chloroplast envelope membrane. TMDs containing phenylalanines were not targeted to the chloroplast in vivo. Based on these results, we propose that the chloroplast targeting signal of AtToc64 comprises two different components: 1) the LFR, which is a signal for evading SRP-mediated co-translational translocation and 2) the hydrophobic amino acid side chains of the TMD, whose size functions as a signal for a cytosolic factor that mediates transport to the chloroplast.
Poliovirus receptor (hPVR/CD155) is a cell surface glycoprotein that belongs to the immunoglobulin superfamily but its natural function remains unknown. Two membrane-bound isoforms, hPVRalpha and hPVRdelta, are known to date, and they differ only in the amino acid sequence of their cytoplasmic domains. To gain an insight into the possible function of the cytoplasmic domains, we examined the localization of introduced hPVRalpha and hPVRdelta in polarized epithelial cells deficient of native hPVRs. Basolateral sorting of hPVRalpha was observed in Madine-Darby canine kidney cells expressing mu1B, but not in LLC-PK1 porcine kidney cells deficient in mu1B. Distribution of hPVRdelta, however, occurred both on the apical and basolateral plasma membranes of these two cell lines. Basolateral sorting of hPVRalpha was also seen in LLC-PK1 cells that expressed an intact exogenous mu1B, but not in the cells that expressed a mutant mu1B lacking binding ability to tyrosine-containing signals. These results indicate that mu1B is involved in the distribution of hPVRalpha to the basolateral membrane. Comparative distribution analysis of hPVRalpha using a series of mutants with truncations and substitutions in the cytoplasmic tail demonstrated that determinant for the basolateral sorting resided in the tyrosine-containing motif of the cytoplasmic tail. Furthermore, yeast two hybrid analysis strongly suggested that the tyrosine motif directly interacted with mu1B protein. Thus, basolateral sorting of hPVRalpha appears to involve the interaction with mu1B through a tyrosine motif existing in the cytoplasmic domain.
A newly synthesized secretory protein in cells bears a special sequence, called signal peptide or sequence, which plays the role of "address tag" in guiding the protein to wherever it is needed. Such a unique function of signal sequences has stimulated novel strategies for drug design or reprogramming cells for gene therapy. To realize these new ideas and plans, however, it is important to develop an automated method for fast and accurately identifying the signal sequences or their cleavage sites. In this paper, a new method is developed for predicting the signal sequence of a query secretory protein by fusing the results from a series of global alignments through a voting system. The very high success rates thus obtained suggest that the novel approach is very promising, and that the new method may become a useful vehicle in identifying signal sequence, or at least serve as a complementary tool to the existing algorithms of this field.
The hVPAC1 receptor for vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase activating peptide (PACAP) has an N-terminal signal peptide like all other class II G protein-coupled receptors (GPCRs). We determined the role of the signal peptide in expression of human VPAC1 receptor in transfected CHO cells. Three constructs were transfected: Flag30-hVPAC1, a receptor containing an inserted FLAG sequence between Ala30 and Ala31 and fused in the C-terminal position to GFP; Flag30-[delta1-30]-hVPAC1, the same construct as Flag30-hVPAC1 but lacking the 1-30 putative signal peptide (SP) sequence; Flag0-hVPAC1, a receptor containing an N-terminal FLAG sequence and fused in the C-terminal position to GFP. For each construct, we determined 125I-VIP binding, VIP-induced cAMP production, GFP fluorescence and indirect immunofluorescence on nonpermeabilized cells incubated with mouse monoclonal anti-Flag antibodies. The data were consistent with a crucial role of the signal peptide for expression of functional VPAC1 receptors at the cell surface and suggested that the signal peptide is cleaved during the translocation of the receptor to the plasma membrane, probably in the endoplasmic reticulum.