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Changing an existing lipid or appending a lipid to a cytosolic protein has emerged as an important technique for targeting proteins to membranes and for constitutively activating the membrane-bound protein. The potential for more precise or regulated interactions of lipidated proteins in membrane subdomains suggests that this method for membrane targeting will be of increasing usefulness.
Streptokinase (SK), an extracellular protein from Streptococcus equisimilis, is secreted post-translationally by Escherichia coli using both its native and E. coli-derived transport signals. In this communication we report that cleavage specificity of signal peptidase I, and thus efficiency of secretion, varies in E. coli when SK export is directed by different transport signals. The native (+1) N-terminus of mature SK was retained when it was transported under the control of its own, PelB or LamB signal peptide. However, when translocation of SK was controlled by the OmpA or MalE signal peptide, Ala2 of mature SK was preferred as a cleavage site for the pre-SK processing. Our results indicate that compatibility of the leader peptide with the mature sequences of SK, which fulfills the requirement for a given secondary structure within the cleavage region, is essential for maintaining the correct processing of pre-SK. An OmpA-SK fusion, which results in the deletion of two N-terminal amino acid residues of mature SK, was further studied with respect to the recognition of alternative cleavage site in E. coli. The alanine at +2 in mature SK was changed to glycine or its relative position was changed to +3 by introducing a methionine residue at the +1 position. Both alterations resulted in the correct cleavage of pre-SK at the original OmpA fusion site. In contrast, introduction of an additional alanine at +4, creating three probable cleavage sites (Ala-x-Ala-x-Ala-x-Ala), resulted in the recognition of all three target sites for cleavage, with varying efficiency. The results indicate that the nature of the secondary structure generated at the cleavage junction of pre-SK, resulting from the fusion of different signal peptides, modulates the cleavage specificity of signal peptidase I during extracellular processing of SK. Based on these findings it is proposed that flexibility in the interaction of the active site of signal peptidase I with the cleavage sites of signal peptides may occur when it encounters two or more juxtaposed cleavage sites. Preference for one cleavage site over another, then, may depend on fulfillment of secondary structure requirements in the vicinity of the pre-protein cleavage junction.
Little is known about the identity of endoplasmic reticulum (ER) export signals and how they are used to regulate the number of proteins on the cell surface. Here, we describe two ER export signals that profoundly altered the steady-state distribution of potassium channels and were required for channel localization to the plasma membrane. When transferred to other potassium channels or a G protein-coupled receptor, these ER export signals increased the number of functional proteins on the cell surface. Thus, ER export of membrane proteins is not necessarily limited by folding or assembly, but may be under the control of specific export signals.
Lipid modification of the N-terminal Cys residue (N-acyl-S-diacylglyceryl-Cys) has been found to be an essential, ubiquitous, and unique bacterial posttranslational modification. Such a modification allows anchoring of even highly hydrophilic proteins to the membrane which carry out a variety of functions important for bacteria, including pathogenesis. Hence, being able to identify such proteins is of great value. To this end, we have created a comprehensive database of bacterial lipoproteins, called DOLOP, which contains information and links to molecular details for about 278 distinct lipoproteins and predicted lipoproteins from 234 completely sequenced bacterial genomes. The website also features a tool that applies a predictive algorithm to identify the presence or absence of the lipoprotein signal sequence in a user-given sequence. The experimentally verified lipoproteins have been classified into different functional classes and more importantly functional domain assignments using hidden Markov models from the SUPERFAMILY database that have been provided for the predicted lipoproteins. Other features include the following: primary sequence analysis, signal sequence analysis, and search facility and information exchange facility to allow researchers to exchange results on newly characterized lipoproteins. The website, along with additional information on the biosynthetic pathway, statistics on predicted lipoproteins, and related figures, is available at http://www.mrc-lmb.cam.ac.uk/genomes/dolop/.
Novel open reading frames coding for cytochrome c peroxidase (CcP) belonging to the superfamily of bacterial, fungal, and plant heme peroxidases were analyzed in the available fungal genomes. Multiple sequence alignment of 71 selected peroxidase genes revealed the presence of three conserved regions essential for their function: one on the distal and two on the proximal side of the prosthetic heme group. Conserved sequence motifs on the proximal heme side are peculiar for CcPs and are responsible for their reactivity. Phylogenetic analysis performed with the distance method as well as with the maximum likelihood method revealed the existence of three distinct subfamilies of fungal CcP and their relationship to other members of the peroxidase superfamily. These divergent CcP evolutionary lines apparently evolved from a single primordial heme peroxidase gene in parallel with the evolution of ascorbate peroxidase genes. Analyzed CcPs differ significantly in their N-terminal sequences. Only subfamily I did not exhibit a presence of any signal sequence. Subfamily II members possess a well defined signal sequence allowing processing and release into mitochondrion and also in subfamily III a signal sequence was detected. Several here analyzed peroxidase genes mainly from Candida albicans and from Rhizopus oryzae can be considered interesting for the investigation of the structure-function relationship of novel CcPs revealing differences to the well documented properties of cytochrome c peroxidase from Saccharomyces cerevisiae.
The crystal structure of the signal sequence binding subunit of the signal recognition particle (SRP) from Thermus aquaticus reveals a deep groove bounded by a flexible loop and lined with side chains of conserved hydrophobic residues. The groove defines a flexible, hydrophobic environment that is likely to contribute to the structural plasticity necessary for SRP to bind signal sequences of different lengths and amino acid sequence. The structure also reveals a helix-turn-helix motif containing an arginine-rich alpha helix that is required for binding to SRP RNA and is implicated in forming the core of an extended RNA binding surface.
Newly synthesized proteins have an intrinsic signal sequence, functioning as "address tags" or "zip codes", that is essential for guiding them wherever they are needed. Owing to such a unique function, protein signals have become a crucial tool in finding new drugs or reprogramming cells for gene therapy. However, to effectively use protein signals as a desirable vehicle in the field of proteomics, the first important thing is to find a fast and powerful method to identify the "address tag" or "zip code" entity. Although all signal sequences contain a hydrophobic core region, they show great variation in both overall length and amino acid sequence. It is this variation that makes it possible to deliver thousands of proteins to many different cellular locations by varieties of modes. It is also this variation that makes it very difficult to formulate a general algorithm to predict signal sequences. Nevertheless, various prediction models and algorithms have been developed during the past 17 years. This Review summarizes the development in this area, from the pioneering methods to neural network approaches, and to the sub-site coupling approaches. Meanwhile, the future challenges in this area, as well as some promising avenues for further improving the prediction quality, have been briefly addressed as well.
The CASR, a cell surface glycoprotein expressed in parathyroid gland and kidney, is critical for maintaining extracellular calcium homeostasis. The inherited disorders, familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT), are caused by inactivating mutations in the CASR gene. The CASR has an N-terminal, 19 amino acid signal peptide that is predicted to direct the nascent polypeptide chain, as it emerges from the ribosome, into the endoplasmic reticulum (ER). Here, we report the functional characterization of three CASR mutations identified in hypercalcemic/hyperparathyroid patients. The mutations, L11S, L13P and T14A, lie within the signal peptide hydrophobic core. When transiently transfected into kidney cells, L11S and L13P mutants demonstrated reduced intracellular and plasma membrane expression and signaling to the mitogen-activated protein kinase pathway in response to extracellular calcium relative to wild-type CASR and the T14A mutant. All mutant CASR RNAs translated into protein normally. In cotranslational processing assays, which test the functionality of the signal peptide in the early secretory pathway, the wild-type CASR and mutant T14A nascent polypeptides were targeted to microsomal vesicles, representing the ER, translocated into the vesicular lumen and underwent core N-glycosylation. In contrast, the L11S and L13P mutants failed to be inserted in the microsomes and undergo glycosylation. This is the first study examining the function of the CASR signal sequence and reveals that both L11S and L13P mutants are markedly impaired with respect to cotranslational processing, accounting for the observed parathyroid dysfunction.
A group of bacterial exported proteins are synthesized with N-terminal signal peptides containing a SRRxFLK 'twin-arginine' amino acid motif. Proteins bearing twin-arginine signal peptides are targeted post-translationally to the twin-arginine translocation (Tat) system which transports folded substrates across the inner membrane. In Escherichia coli, most integral inner membrane proteins are assembled by a co-translational process directed by SRP/FtsY, the SecYEG translocase, and YidC. In this work we define a novel class of integral membrane proteins assembled by a Tat-dependent mechanism. We show that at least five E. coli Tat substrate proteins contain hydrophobic C-terminal transmembrane helices (or 'C-tails'). Fusions between the identified transmembrane C-tails and the exclusively Tat-dependent reporter proteins TorA and SufI render the resultant chimeras membrane-bound. Export-linked signal peptide processing and membrane integration of the chimeras is shown to be both Tat-dependent and YidC-independent. It is proposed that the mechanism of membrane integration of proteins by the Tat system is fundamentally distinct from that employed for other bacterial inner membrane proteins.
While it has been demonstrated that GFP-tagged proteins were transported to their correct cellular compartments in most cells, attempts to secrete GFP/GFP-fusion through the default secretory pathway have not been as successful. In an attempt to induce secretion of GFP and Hexokinase (HXK)-GFP fusion in Saccharomycescerevisiae, we have cloned constructs that employed four different yeast secretion signal sequences, i.e., INU1, SUC2, PHO5, and MEL1. The expression is under the control of the galactose-inducible GAL1 promoter. Our results showed that all eight constructs entered the secretory pathway successfully, and the signal peptides were all cleaved off. However, none of the eight constructs were able to lead to secretion into the culture media or the periplasmic space. The expression levels of the eight constructs differ dramatically, depending on both the signal peptide and whether GFP was fused with HXK. Confocal microscopy studies revealed that the eight constructs also led to very different localization patterns. Among them, two constructs targeted GFP to the vacuole partially or exclusively, whereas others were mostly retained in the ER/Golgi compartments. Our efforts, together with those of others, seem to suggest that the signal peptide itself is not enough to lead to secretion of GFP from S. cerevisiae, although it has been successful in some other organisms. Nonetheless, the advantage of GFP's in vivo detection makes it a powerful tool for investigating protein localization events.
Dentine dysplasia type II is an autosomal dominant disorder in which mineralization of the dentine of the primary teeth is abnormal. On the basis of the phenotypic overlap between, and shared chromosomal location with, dentinogenesis imperfecta type II, a second disorder of dentine mineralization, it has been proposed that the two conditions are allelic. As recent studies have shown that dentinogenesis imperfecta type II results from mutation of the bicistronic dentine sialophosphoprotein gene (DSPP ), we have tested this hypothesis by sequencing DSPP in a family with a history of dentine dysplasia type II. Our results have shown that a missense change, which causes the substitution of a tyrosine for an aspartic acid in the hydrophobic signal peptide domain of the protein, underlies the phenotype in this family. Biochemical analysis has further demonstrated that this mutation causes a failure of translocation of the encoded proteins into the endoplasmic reticulum, and is therefore likely to lead to a loss of function of both dentine sialoprotein and dentine phosphoprotein.
BACKGROUND AND OBJECTIVES: Most secreted proteins, including coagulation factor X (FX), are synthesized with a signal peptide, which is necessary for targeting the nascent polypeptide into the endoplasmic reticulum. Characterization of naturally occurring mutations may provide insights into the functional roles of the amino acids in the signal peptide. DESIGN AND METHODS: A 52-year old male patient with type I FX deficiency was studied. Mutations were searched for by FX gene (F10) sequencing. The wild-type and the mutant FX proteins were expressed in transfected cells and then immunological assays were performed. Pulse-chase experiments and cell-free expression studies were conducted to determine the cellular fate of the mutant FX molecules. RESULTS: The patient we studied was homozygous for a substitution of arginine for serine at codon -30 in the signal sequence of F10. Immunoassays detected low FX antigen levels in both the conditioned media and lysates of the cells expressing the mutant protein. Pulse-chase analysis showed that only trace amounts of the mutant FX protein were detectable in the conditioned media, and that the mutant molecules did not accumulate inside the cells either. The results of cell-free expression studies showed that although the transcription and translation of the mutant construct were normal, no post-translational processing, such as N-linked glycosylation, occurred in the presence of microsomes. INTERPRETATION AND CONCLUSIONS: These findings suggest that substitution of a neutral polar amino acid, serine by arginine, in the hydrophobic core of FX signal peptide severely impairs the ability of the protein to enter the endoplasmic reticulum and results in FX deficiency.
We investigated the role of the stop transfer sequence of human UGT1A6 in ER assembly and enzyme activity. We found that this sequence was able to address and translocate the upstream lumenal domain into microsomal membranes in vitro co- and posttranslationally. The signal activity of this sequence was further demonstrated in HeLa cells by its ability to target and maintain the CD4 protein deleted from both the N-terminal signal peptide and C-terminal transmembrane domain into the ER. We showed that total or partial deletion of the stop transfer sequence of UGT1A6 severely impaired enzyme activity highlighting its importance in both membrane assembly and function.
Variegate porphyria is an autosomal dominant disorder of heme metabolism resulting from a deficiency in protoporphyrinogen oxidase, an enzyme located on the inner mitochondrial membrane. This study examined the effect of three South African VP-causing mutations (H20P, R59W, R168C) on mitochondrial targeting. Only H20P did not target, and of eight protoporphyrinogen oxidase-GFP chimeric fusion proteins created, N-terminal residues 1-17 were found to be the minimal protoporphyrinogen oxidase sequence required for efficient mitochondrial targeting. Removal of this N-terminal sequence displayed mitochondrial localization, suggesting internal mitochondrial targeting signals. In addition, six constructs were engineered to assess the effect of charge and helicity on mitochondrial targeting of the protein. Of those engineered, only the PPOX20/H20P-GFP construct abolished mitochondrial targeting, presumably through disruption of the protoporphyrinogen oxidase alpha-helix. Based on our results we propose a mechanism for protoporphyrinogen oxidase targeting to the mitochondrion.
Protein folding and quality control in the early secretory pathway function as posttranslational checkpoints in eukaryote gene expression. Herein, an aberrant form of the hepatic secretory protein alpha1-antitrypsin was stably expressed in a human embryonic kidney cell line to elucidate the mechanisms by which glycoprotein endoplasmic reticulum-associated degradation (GERAD) is administered in cells from higher eukaryotes. After biosynthesis, genetic variant PI Z underwent alternative phases of secretion and degradation, the latter of which was mediated by the proteasome. Degradation required release from calnexin- and asparagine-linked oligosaccharide modification by endoplasmic reticulum mannosidase I, the latter of which occurred as PI Z was bound to the molecular chaperone grp78/BiP. That a distinct GERAD program operates in human embryonic kidney cells was supported by the extent of PI Z secretion, apparent lack of polymerization, inability of calnexin to participate in the degradation process, and sequestration of the glycoprotein folding sensor UDP-glucose:glycoprotein glucosyltransferase in the Golgi complex. Because UDP-glucose:glycoprotein glucosyltransferase sustains calnexin binding, its altered distribution is consistent with a GERAD program that hinders the reentry of substrates into the calnexin cycle, allowing grp78/BiP to partner with a lectin, other than calnexin, in the recognition of a two-component GERAD signal to facilitate substrate recruitment. How the processing of a mutant protein, rather than the mutation itself, can contribute to disease pathogenesis, is discussed.
Pore-forming colicins are plasmid-encoded bacteriocins that kill Escherichia coli and closely related bacteria. They bind to receptors in the outer membrane and are translocated across the cell envelope to the inner membrane where they form voltage-dependent ion-channels. Colicins are composed of three domains, with the C-terminal domain responsible for pore-formation. Isolated C-terminal pore-forming domains produced in the cytoplasm of E. coli are inactive due to the polarity of the transmembrane electrochemical potential, which is the opposite of that required. However, the pore-forming domain of colicin A (pfColA) fused to a prokaryotic signal peptide (sp-pfColA) is transported across and inserts into the inner membrane of E. coli from the periplasmic side, forming a functional channel. Sp-pfColA is specifically inhibited by the colicin A immunity protein (Cai). This construct has been used to investigate colicin A channel formation in vivo and to characterise the interaction of pfColA with Cai within the inner membrane. These points will be developed further in this review.
Cellular pathways relay information through dynamic protein interactions. We have assessed the kinetic properties of the murine double minute protein (MDM2) and von Hippel-Lindau (VHL) ubiquitin ligases in living cells under physiological conditions that alter the stability of their respective p53 and hypoxia-inducible factor substrates. Photobleaching experiments reveal that MDM2 and VHL are highly mobile proteins in settings where their substrates are efficiently degraded. The nucleolar architecture converts MDM2 and VHL to a static state in response to regulatory cues that are associated with substrate stability. After signal termination, the nucleolus is able to rapidly release these proteins from static detention, thereby restoring their high mobility profiles. A protein surface region of VHL's beta-sheet domain was identified as a discrete [H+]-responsive nucleolar detention signal that targets the VHL/Cullin-2 ubiquitin ligase complex to nucleoli in response to physiological fluctuations in environmental pH. Data shown here provide the first evidence that cells have evolved a mechanism to regulate molecular networks by reversibly switching proteins between a mobile and static state.
The AGA gene is mutated in patients with aspartylglucosaminuria (AGU), a lysosomal storage disease enriched in the Finnish population. The disease mechanism of AGU and the biochemistry and cell biology of the lysosomal aspartylglucosaminidase (AGA) enzyme are well characterized. Here, we have investigated a novel AGU mutation found in a Finnish patient. The mutation was detected as a compound heterozygote with the Finnish major mutation in the other allele. The novel point mutation, c.44T>G, causes the L15R amino acid substitution in the signal sequence of the AGA enzyme. The mutated AGA enzyme was here analyzed by over expression in BHK and COS-1 cells. The L15R AGA protein was only faintly detectable by immunofluorescence analysis and observed in the endoplasmic reticulum. Metabolic labeling and immunoprecipitation revealed only a small amount of AGA polypeptides but the specific activity of the mutant enzyme was surprisingly high, 37% of the wild type. The amino acid substitution probably affects translocation of AGA polypeptides by altering a critical hydrophobic core structure of the signal sequence. It appears that the small amounts of active enzyme are not able to reach the lysosomes thus explaining the development of AGU disease in the patient.