Identification of signal sequence binding proteins integrated into the rough endoplasmic reticulum membrane.

An azidophenacyl derivative of a chemically synthesized consensus signal peptide has been prepared. The peptide, when photoactivated in the presence of rough or high-salt-stripped microsomes from pancreas, leads to inhibition of their activity in cotranslational processing of secretory pre-proteins translated from their mRNA in vitro. The peptide binds specifically with high affinity to components in the microsomal membranes from pancreas and liver, and photoreaction of a radioactive form of the azidophenacyl derivative leads to covalent linkage to yield two closely related radiolabelled proteins of Mr about 45,000. These proteins are integrated into the membrane, with large 30,000-Mr domains embedded into the phospholipid bilayer to which the signal peptide binds. A smaller, endopeptidase-sensitive, domain is exposed on the cytoplasmic surface of the microsomal vesicles. The specificity and selectivity of the binding of azidophenacyl-derivatized consensus signal peptide was demonstrated by concentration-dependent inhibition of photolabelling by the 'cold' synthetic consensus signal peptide and by a natural internal signal sequence cleaved and isolated from ovalbumin. The properties of the labelled 45,000-Mr protein-signal peptide complexes, i.e. mass, pI, ease of dissociation from the membrane by detergent or salts and immunological properties, distinguish them from other proteins, e.g. subunits of signal recognition particle, docking protein and signal peptidase, already known to be involved in targetting and processing of nascent secretory proteins at the rough endoplasmic reticulum membrane. Although the 45,000-Mr signal peptide binding protein displays properties similar to those of the signal peptidase, a component of the endoplasmic reticulum, the azido-derivatized consensus signal peptide does not interact with it. It is proposed that the endoplasmic reticulum proteins with which the azidophenacyl-derivatized consensus signal peptide interacts to yield the 45,000-Mr adducts may act as receptors for signals in nascent secretory pre-proteins in transduction of changes in the endoplasmic reticulum which bring about translocation of secretory protein across the membrane.     

The hydrophobic core of twin-arginine signal sequences orchestrates specific binding to Tat-pathway related chaperones

Redox enzyme maturation proteins (REMPs) bind pre-proteins destined for translocation across the bacterial cytoplasmic membrane via the twin-arginine translocation system and enable the enzymatic incorporation of complex cofactors. Most REMPs recognize one specific pre-protein. The recognition site usually resides in the N-terminal signal sequence. REMP binding protects signal peptides against degradation by proteases. REMPs are also believed to prevent binding of immature pre-proteins to the translocon. The main aim of this work was to better understand the interaction between REMPs and substrate signal sequences. Two REMPs were investigated: DmsD (specific for dimethylsulfoxide reductase, DmsA) and TorD (specific for trimethylamine N-oxide reductase, TorA). Green fluorescent protein (GFP) was genetically fused behind the signal sequences of TorA and DmsA. This ensures native behavior of the respective signal sequence and excludes any effects mediated by the mature domain of the pre-protein. Surface plasmon resonance analysis revealed that these chimeric pre-proteins specifically bind to the cognate REMP. Furthermore, the region of the signal sequence that is responsible for specific binding to the corresponding REMP was identified by creating region-swapped chimeric signal sequences, containing parts of both the TorA and DmsA signal sequences. Surprisingly, specificity is not encoded in the highly variable positively charged N-terminal region of the signal sequence, but in the more similar hydrophobic C-terminal parts. Interestingly, binding of DmsD to its model substrate reduced membrane binding of the pre-protein. This property could link REMP-signal peptide binding to its reported proofreading function.     

The physical and functional borders of transit peptide-like sequences in secondary endosymbionts

Background  

Plastids rely on protein supply by their host cells. In plastids surrounded by two membranes (primary plastids) targeting of these proteins is facilitated by an N-terminal targeting signal, the transit peptide. In secondary plastids (surrounded by three or four membranes), transit peptide-like regions are an essential part of a bipartite topogenic signal sequence (BTS), and generally found adjacent to a N-terminally located signal peptide of the plastid pre-proteins. As in primary plastids, for which no wealth of functional information about transit peptide features exists, the transit peptide-like regions used for import into secondary ones show some common features only, which are also poorly characterized.     

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.     

On the use of the antibiotic chloramphenicol to target polypeptide chain mimics to the ribosomal exit tunnel

The ribosomal exit tunnel had recently become the centre of many functional and structural studies. Accumulated evidence indicates that the tunnel is not simply a passive conduit for the nascent chain, but a rather functionally important compartment where nascent peptide sequences can interact with the ribosome to signal translation to slow down or even stop. To explore further this interaction, we have synthesized short peptides attached to the amino group of a chloramphenicol (CAM) base, such that when bound to the ribosome these compounds mimic a nascent peptidyl-tRNA chain bound to the A-site of the peptidyltransferase center (PTC). Here we show that these CAM-peptides interact with the PTC of the ribosome while their effectiveness can be modulated by the sequence of the peptide, suggesting a direct interaction of the peptide with the ribosomal tunnel. Indeed, chemical footprinting in the presence of CAM-P2, one of the tested CAM-peptides, reveals protection of 23S rRNA nucleotides located deep within the tunnel, indicating a potential interaction with specific components of the ribosomal tunnel. Collectively, our findings suggest that the CAM-based peptide derivatives will be useful tools for targeting polypeptide chain mimics to the ribosomal tunnel, allowing their conformation and interaction with the ribosomal tunnel to be explored using further biochemical and structural methods.     

Signal recognition particle binds to ribosome-bound signal sequences with fluorescence-detected subnanomolar affinity that does not diminish as the nascent chain lengthens

The binding of signal recognition particle (SRP) to ribosome-bound signal sequences has been characterized directly and quantitatively using fluorescence spectroscopy. A fluorescent probe was incorporated cotranslationally into the signal sequence of a ribosome.nascent chain complex (RNC), and upon titration with SRP, a large and saturable increase in fluorescence intensity was observed. Spectral analyses of SRP and RNC association as a function of concentration allowed us to measure, at equilibrium, K(d) values of 0.05-0.38 nm for SRP.RNC complexes with different signal sequences. Competitive binding experiments with nonfluorescent RNC species revealed that the nascent chain probe did not alter SRP affinity and that SRP has significant affinity for both nontranslating ribosomes (K(d) = 71 nm) and RNCs that lack an exposed signal sequence (K(d) = 8 nm). SRP can therefore distinguish between translating and nontranslating ribosomes. The very high signal sequence-dependent SRP.RNC affinity did not decrease as the nascent chain lengthened. Thus, the inhibition of SRP-dependent targeting of RNCs to the endoplasmic reticulum membrane observed with long nascent chains does not result from reduced SRP binding to the signal sequence, as widely thought, but rather from a subsequent step, presumably nascent chain interference of SRP.RNC association with the SRP receptor and/or translocon.     

Signal peptide fragments of preprolactin and HIV-1 p-gp160 interact with calmodulin.

Secretory proteins and most membrane proteins are synthesized with a signal sequence that is usually cleaved from the nascent polypeptide during transport into the lumen of the endoplasmic reticulum. Using site-specific photo-crosslinking we have followed the fate of the signal sequence of preprolactin in a cell-free system. This signal sequence has an unusually long hydrophilic n-region containing several positively charged amino acid residues. We found that after cleavage by signal peptidase the signal sequence is in contact with lipids and subunits of the signal peptidase complex. The cleaved signal sequence is processed further and an N-terminal fragment is released into the cytosol. This signal peptide fragment was found to interact efficiently with calmodulin. Similar to preprolactin, the signal sequence of the HIV-1 envelope protein p-gp160 has the characteristic feature for calmodulin binding in its n-region. We found that a signal peptide fragment of p-gp160 was released into the cytosol and interacts with calmodulin. Our results suggest that signal peptide fragments of some cellular and viral proteins can interact with cytosolic target molecules. The functional consequences of such interactions remain to be established. However, our data suggest that signal sequences may be functionally more versatile than anticipated up to now.     

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.     

The complete nucleotide sequence of the Pseudomonas gene coding for carboxypeptidase G2

The complete nucleotide sequence of the Pseudomonas chromosomal gene coding for the enzyme carboxypeptidase G2 (CPG2) has been determined. The nucleotide sequence obtained has been confirmed by comparing the predicted amino acid sequence with that of randomly derived peptide fragments and by N-terminal sequencing of the purified protein. The gene has been shown to code for a 22 amino acid signal peptide at its N-terminus which closely resembles the signal peptides of other secreted proteins. An alternative 36 amino acid signal peptide which may function in Pseudomonas has also been identified. The codon utilisation of the gene is influenced by the high G + C (67.2%) content of the DNA and exhibits a 92.8% preference for codons ending in G or C. This unusual codon preference may contribute to the generally observed weak expression of Pseudomonas genes in Escherichia coli. A region of DNA upstream of the structural gene has also been sequenced and a ribosome binding site and two putative promoter sequences identified.     

Trafficking of lysosomal enzymes

The targeting of lysosomal enzymes from their site of synthesis in the rough endoplasmic reticulum (RER) to their final destination in lysosomes is directed by a series of protein and carbohydrate recognition signals on the enzymes. Lysosomal enzymes, along with secretory and plasma membrane proteins, contain amino-terminal signal sequences that direct the vectorial discharge of the nascent proteins into the lumen of the RER. The three classes of proteins also share a common peptide signal for asparagine glycosylation. The next signal is unique to lysosomal enzymes and permits their high-affinity binding to a specific phosphotransferase that catalyzes the formation of the mannose 6-phosphate recognition marker. This carbohydrate determinant allows binding to specific receptors that translocate the lysosomal enzymes from the Golgi complex to an acidified prelysosomal compartment. There the lysosomal enzymes are discharged for final packaging into lysosomes. Two distinct mannose 6-phosphate receptors have been identified, and cDNAs encoding their entire sequences have been cloned. An analysis of the deduced amino acid sequences of the receptors shows that each is composed of four structural domains: a signal sequence, an extracytoplasmic amino-terminal domain, a hydrophobic membrane-spanning region, and a cytoplasmic domain. The entire extracytoplasmic region of the small receptor is homologous to the 15 repeating domains that constitute the extracytoplasmic portion of the large receptor.     

Signal peptides open protein-conducting channels in E. coli.

Plasma membrane vesicles and protoplasts of Escherichia coli were fused to planar lipid bilayers and studied with electrophysiological techniques. Large transmembrane aqueous channels were opened when 0.2 nM LamB signal peptide was added to the cytoplasmic side of the membrane. These aqueous pores are similar in conductance to those previously observed in mammalian endoplasmic reticulum when puromycin is used to release and thus unplug nascent translocating chains. Signal sequences have been previously shown to be necessary and sufficient for targeting proteins to cellular membranes. These results demonstrate that signal peptides are sufficient for opening the protein-conducting channels. We suggest that they are the physiological ligands that open protein-conducting channels at the initiation of protein translocation across prokaryotic plasma membrane and mammalian endoplasmic reticulum.     

Cloning and sequence analysis of cDNA encoding a precursor for rat brain natriuretic peptide

Brain natriuretic peptide (BNP) is a new type of natriuretic peptide, which has so far been identified only in porcine brain and atrium. Immunological observations suggest that rat and porcine BNP may have structural difference according to species. To identify rat BNP, we constructed a rat atrial cDNA library, and screened for clones encoding rat BNP-precursor by using part of porcine BNP cDNA as a probe. By sequencing a cloned cDNA, the amino acid sequence of rat BNP-precursor comprising 121 residues was deduced as carrying a 26-residue putative signal peptide at the N-terminus and a region homologous to porcine BNP-32 at the C-terminus. In addition, remarkably high homology between rat and porcine BNP-precursors was observed in the 3'-untranslated AT-rich region. Comparing sequences of precursors of ANP and BNP thus far identified, structural and processing features characteristic of the BNP family were discussed.     

Nucleotide and derived amino acid sequences of the major porin of Comamonas acidovorans and comparison of porin primary structures.

The DNA sequence of the gene which codes for the major outer membrane porin (Omp32) of Comamonas acidovorans has been determined. The structural gene encodes a precursor consisting of 351 amino acid residues with a signal peptide of 19 amino acid residues. Comparisons with amino acid sequences of outer membrane proteins and porins from several other members of the class Proteobacteria and of the Chlamydia trachomatis porin and the Neurospora crassa mitochondrial porin revealed a motif of eight regions of local homology. The results of this analysis are discussed with regard to common structural features of porins.     

Intragenic suppressor mutations that restore export of maltose binding protein with a truncated signal peptide

A deletion mutation, malE delta 12-18, removes seven residues from the hydrophobic core of the maltose binding protein (MBP) signal peptide and thus prevents secretion of this protein to the periplasm of E. coli. Intragenic suppressor mutations of malE delta 12-18 have been obtained, some highly efficient in their ability to restore proper MBP export. Twelve independently isolated suppressors represent six unique mutational events. Five result in alterations within the MBP signal peptide; one changes the amino acid at residue 19 of the mature MBP. Analysis of these suppressors indicates that the length of the hydrophobic core is a major determinant of signal peptide function. The experiments further suggest that the hydrophobic core region serves primarily a structural role in mediating protein secretion, and that other sequences outside of this region may be responsible for providing the initial recognition of the MBP nascent chain as a secreted protein.     

A signal sequence for the insertion of a transmembrane glycoprotein. Similarities to the signals of secretory proteins in primary structure and function.

The biosynthesis of a secretory protein and a transmembrane viral glycoprotein are compared by two different experimental approaches. (a) NH2-terminal sequence analysis has been performed on various forms of the transmembrane glycoprotein of vesicular stomatitis virus synthesized in cell-free systems. The sequence data presented demonstrate that the nascent precursor of the glycoprotein contains a "signal sequence" of 16 amino acids at the NH2 terminus, whose sequence is Met-Lys-Cys-Leu-Leu-Tyr-Leu-Ala-Phe-Leu-Phe-Ile-(His-Val-Asn)-Cys. This signal sequence is proteolytically cleaved during the process of insertion into microsomal membranes prior to chain completion. The new NH2 terminus of the inserted, cleaved, and glycosylated membrane protein is located within the lumen of the microsomal vesicles and is identical to that of the authentic glycoprotein from virions. (b) Nascent chain competition experiments were performed between this glycoprotein, bovine pituitary prolactin (a secretory protein), and rabbit globin (a cytosolic protein). It was found that the nascent membrane glycoprotein, but not nascent globin, competed with nascent prolactin for membrane sites involved in the early biosynthetic event of transfer across membranes. These data suggest that an initially common pathway is involved in the biogenesis of secretory proteins and at least one class of integral membrane proteins.     

Alternate recruitment of signal recognition particle and trigger factor to the signal sequence of a growing nascent polypeptide

Different from cytoplasmic membrane proteins, presecretory proteins of bacteria usually do not require the signal recognition particle for targeting to the Sec translocon. Nevertheless signal sequences of presecretory proteins have been found in close proximity to signal recognition particle immediately after they have emerged from the ribosome. We show here that at the ribosome, the molecular environment of a signal sequence depends on the nature of downstream sequence elements that can cause an alternate recruitment of signal recognition particle and the ribosome-associated chaperone Trigger factor to a growing nascent chain. While signal recognition particle and Trigger factor might remain bound to the same ribosome, both ligands are clearly able to displace each other from a nascent chain. The data also imply that a signal sequence owes its molecular environment to the fact that it remains closely apposed to the ribosomal exit site during growth of a nascent secretory protein.