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PubMed1991

Correlation of secondary structure with biological activity for a leader peptide: circular dichroism-derived structure and in vitro biological activities of preproparathyroid hormone peptide and its analogs.

Leader or signal sequences are specialized domains within precursor proteins which serve an essential role in interacting with the cellular secretory apparatus to enable intracellular transport and secretion of proteins. Despite many differences in primary amino acid sequences, signal domains interact with a common set of intracellular components, presumably because the signal sequences share an overall conformational similarity. In a few instances, mutant signal peptides from prokaryotes have been studied and their structures correlated with function (export) in vivo. A series of analogs of the precursor-specific region of preproparathyroid hormone have been prepared which contain substitutions of either proline or a charged amino acid within the hydrophobic core. These synthetic "mutants" have previously been evaluated in several in vitro assays to determine their functionality with regard to protein secretion and suitability as substrates for signal peptidase. The secondary structural content of each peptide, as well as the native sequence and sulfur-free analog, was determined in aqueous and nonaqueous conditions by circular dichroism (CD) as a function of time. The structures obtained were correlated with in vitro bioactivities. Unlike the findings or previous CD studies, all the peptides examined here had low to undetectable alpha-helical content in both aqueous and nonaqueous buffers. The unsubstituted and sulfur-free analogs had high (80-85%) beta-structure in aqueous conditions which was reduced to approximately 30% in nonaqueous solvent. The proline- and charged-substituted peptides contained about half the beta-structure content (35-55%) in aqueous buffer; in nonaqueous solvent their structure was similar to the unsubstituted peptides. The structure-activity correlates found were as follows: a high degree of structure (aqueous conditions) correlated with interaction with signal recognition particle and substrate suitability for signal peptidase; a low degree of structure (nonaqueous environment) correlated with activity in the translocation assay.

Amino Acid Sequence↗

PubMed1990

A novel pathway for secretory proteins?

In eukaryotes, most proteins which are transported to the extracellular space, into mitochondria or into chloroplasts are synthesized as precursor polypeptides containing cleavable N-terminal signal or targeting sequences. We have searched the literature for proteins that are exported from the cytosol without being proteolytically processed. Some of these proteins contain uncleaved signal or targeting sequences. However, among secretory proteins there is a class that does not possess hydrophobic signal sequences and appears to leave the cell by a secretory pathway clearly distinct from the classical route through the endoplasmic reticulum and Golgi apparatus.

Acylation↗

PubMed1991

[Evolution of regulatory proteins].

A concept of the evolution of signal molecules and their receptors is proposed. The formation of novel regulatory proteins by way of gene fusion and gene shuffling and the role of these processes in metabolic integration are considered. The different receptor variants may be due to alternative splicing which is regulated in a tissue-specific manner.

Biological Evolution↗

PubMed1985

A former amino terminal signal sequence engineered to an internal location directs translocation of both flanking protein domains.

To determine whether a functional amino terminal signal sequence can be active at an internal position, a hybrid gene was constructed in which the entire coding region of bovine preprolactin cDNA was inserted into chimpanzee alpha-globin cDNA 109 codons downstream from the initiation codon of globin. When RNA synthesized in vitro from this plasmid (pSPGP1) was translated in the rabbit reticulocyte cell-free system, a 32-kD protein was produced that was both prolactin and globin immunoreactive. When microsomal membranes were present during translation (but not when added posttranslationally), a 26-kD and a 14-kD product were also observed. By immunoreactivity and electrophoretic mobility, the 26-kD protein was identical to mature prolactin, and the 14-kD protein appeared to be the globin domain with the prolactin signal sequence attached at its carboxy terminus. From (a) posttranslational proteolysis in the presence and absence of detergent, (b) sedimentation of vesicles in the presence and absence of sodium carbonate pH 11.5, and (c) N-linked glycosylation of the globin-immunoreactive fragment after insertion of an Asn-X-Ser N-linked glycosylation site into the globin coding region of pSPGP1, it appears that all of the 26-kD and some of the 14-kD products, but none of the 32-kD precursor, have been translocated to the lumen of the membrane vesicles. Thus, when engineered to an internal position, the prolactin signal sequence is able to translocate both flanking protein domains. These data have implications for the understanding of translocation of proteins across the membrane of the endoplasmic reticulum.

Amino Acid Sequence↗

PubMed1990

The 15 amino acid residues preceding the amino terminus of the envelope protein in the yellow fever virus polyprotein precursor act as a signal peptide.

The 15 amino acids which precede the sequence of the envelope (E) protein in the yellow fever virus (YFV) polyprotein precursor have been proposed to function as a signal peptide for the E protein (P. Desprès A. Cahour, C. Wychowski, M. Girard and M. Bouloy; Ann. Inst. Pasteur/Virol., 139, 59-67, 1988). To confirm this hypothesis, recombinant SV40 genomes were constructed in which the sequence of the E protein, or that of the poliovirus VP0 capsid polypeptide were placed immediately downstream of and in frame with the sequence of the putative signal peptide, under the control of the late SV40 promoter. The E protein expressed by the hybrid virus SV-E was recognized by two neutralizing monoclonal antibodies directed against the YFV envelope protein. In this construct, the E protein was deleted of its C-terminal transmembrane zone. Therefore, as expected, the protein appeared to be efficiently transported along the exocytic pathway and excreted into the cell culture medium. In addition, when the putative signal peptide was fused in frame with poliovirus polypeptide VP0, the expressed chimeric polypeptide was targeted to the endoplasmic reticulum where it underwent glycosylation.

Amino Acid Sequence↗

PubMed1992

How the degenerate signal is recognized and delivered in the course of protein translocation across biological membranes.

Protein translocation across a membrane is generally directed by a degenerate signal peptide encoded in the nascent polypeptide chains. Effective signal recognition and the subsequent traversal of the polypeptide chain through the membrane are ensured by the mechanism of cotranslational translocation and/or by chaperone molecules which universally bind to nascent polypeptides and keep them under-structured and translocation-competent. In order to decipher the degenerate signal with a high degree of fidelity, the cells seem to have evolved a multistep signal recognition and relay mechanism, by which a weak, less-specific interaction between individual signal peptides and a defined signal recognition factor can take place, while the highly specific signal delivery can be achieved by only allowing the correct, effective signals to pass through all check-points along the relay cascade. In addition, a "double recognition" and energy-dependent "proof-reading" mechanism may be involved in single steps of such weak-bond interactions.

Biological Transport, Active↗

PubMed1991

Analysis of the haemolysin transport process through the secretion from Escherichia coli of PCM, CAT or beta-galactosidase fused to the Hly C-terminal signal domain.

Secretion of haemolysin (HlyA) is secA independent, but depends upon two accessory membrane proteins, HlyB and HlyD, encoded by the hly determinant. A fourth (cytoplasmic) protein, HlyC, is required to activate HlyA post-translationally, but has no role in export. Deletion studies have previously shown that the HlyA molecule contains a targeting signal close to the C-terminus which specifically directs its secretion to the medium. This targeting signal has been variously located within the terminal 27, 53, 60 or 113 amino acids. In this paper, we have sought to confirm the presence of a C-terminal targeting signal and to analyse the specificity of the Hly transport system through fusion of C-terminal fragments of HlyA to heterologous polypeptides. A C-terminal fragment (23 kDa) of HlyA, when fused at the C-terminus, efficiently promoted the secretion of the eukaryotic protein prochymosin (PCM) to the medium via HlyB and HlyD. This result is in contrast to previous findings that prochymosin, preceded by the alkaline phosphatase signal sequence, cannot be translocated across the Escherichia coli inner membrane. The HlyA targeting domain was also used to secrete to the medium varying portions of chloramphenicol acetyltransferase (CAT) and 98 per cent of the beta-galactosidase (LacZ) molecule (both E. coli cytoplasmic proteins). In the case of the PCM and CAT fusions the efficiency of secretion was reduced as the proportion of the PCM and CAT molecule increased. This result is consistent with inhibition of secretion through the irreversible folding of the larger passenger protein fragments, or the occlusion of the HlyA targeting signal by upstream sequences. Analysis of the nature of the C-terminal domain promoting secretion of prochymosin, demonstrated that shortening the signal domain from 218 to 113 amino acids significantly reduced the efficiency of secretion. This result may also reflect the importance of maintaining an independently folded signal motif well separated from a passenger domain.

Amino Acid Sequence↗

PubMed1987

The role of topogenic sequences in the movement of proteins through membranes.

Recent advances have led to considerable convergence in ideas of the way topogenic sequences act to translocate proteins across various intracellular membranes (Table 2). Whereas co-translational translocation and processing were previously considered the norm at the endoplasmic reticulum membrane, several instances of post-translational translocation into endoplasmic reticulum microsomes in vitro have now been described. However, it must be noted that post-translational translocation in vitro is much less efficient than when endoplasmic reticulum membranes are present during translation, and it is possible that in the intact cell translocation occurs during translation. Movement of proteins into chloroplasts and mitochondria occurs after translation. When translocation is post-translational, proteins may perhaps traverse the membrane as folded domains, and the conformational effects of topogenic sequences on these domains may be as envisaged in Wickner's 'membrane-trigger hypothesis'. Both signal and transit sequences possess amphipathic structures which are capable of interacting with phospholipid bilayers, and these interactions may disturb the bilayer sufficiently to allow entry of the following domains of protein. There is increasing evidence that GTP is required to bind ribosomes and their associated nascent chains to the endoplasmic reticulum membrane. Precisely how the cell's energy is applied to achieve translocation is not clear, but one possibility at the endoplasmic reticulum is that a GTP-hydrolysing transducing mechanism may exist to couple signal sequence receptor binding to movement of the nascent chain across the membrane. Electrochemical gradients are required for protein movement to the mitochondrial inner membrane and across the bacterial inner membrane. Cytoplasmic factors such as SRP, the secA gene product or a 40 kDa protein (for mitochondrial precursors) may act by binding to topogenic sequences and preventing precursor proteins as they are translated from folding into forms which cannot be translocated. Specificity in the cell may be achieved both by targetting interactions between these cytoplasmic factors and their receptors located in target membranes, and also by specific binding of the topogenic sequences to specific proteins integrated into the target membranes. Possible candidates for the latter are the protein of microsomal membranes that reacts with a photoreactive signal peptide to give a 45 kDa complex (Fig. 1), the secY gene product of the bacterial inner membrane, and receptors on the outer membranes of chloroplasts and mitochondria. Whether these aid translocation as well as recognition is not clear.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

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.

Amino Acid Sequence↗

PubMed1991

Protein export in prokaryotes and eukaryotes. Theme with variations.

Protein export in prokaryotes as well as in eukaryotes can be defined as protein transport across the plasma membrane. In both types of organisms there are various apparently ATP-dependent transport mechanisms which can be distinguished from one another and which show similarities when the prokaryotic mechanism is compared with the respective eukaryotic mechanism. First, one can distinguish between transport mechanisms which involve so-called signal or leader peptides and those which do not. The latter mechanisms seem to employ ATP-dependent transport systems which belong to the family of oligopeptide permeases and multiple drug resistance proteins. Second, in signal or leader peptide-dependent transport one can distinguish between transport mechanisms which involve ribonucleoparticles and those which employ molecular chaperones. Both mechanisms appear to converge at the level of ATP-dependent translocases.

Animals↗

PubMed1992

Antifolding activity of hsp60 couples protein import into the mitochondrial matrix with export to the intermembrane space.

Cytochrome b2 reaches the intermembrane space of mitochondria by transport into the matrix followed by export across the inner membrane. While in the matrix, the protein interacts with hsp60, which arrests its folding prior to export. The bacterial-type export sequence in pre-cytochrome b2 functions by inhibiting the ATP-dependent release of the protein from hsp60. Release for export apparently requires, in addition to ATP, the interaction of the signal sequence with a component of the export machinery in the inner membrane. Export can occur before import is complete provided that a critical length of the polypeptide chain has been translocated into the matrix. Thus, hsp60 combines two activities: catalysis of folding of proteins destined for the matrix, and maintaining proteins in an unfolded state to facilitate their channeling between the machineries for import and export across the inner membrane. Anti-folding signals such as the hydrophobic export sequence in cytochrome b2 may act as switches between these two activities.

Base Sequence↗

PubMed1990

Targeting of proteins into the eukaryotic secretory pathway: signal peptide structure/function relationships.

Much progress has been made in recent years regarding the mechanisms of targeting of secretory proteins to, and across, the endoplasmic reticulum (ER) membrane. Many of the cellular components involved in mediating translocation across this bilayer have been identified and characterized. Polypeptide domains of secretory proteins, termed signal peptides, have been shown to be necessary, and in most cases sufficient, for entry of preproteins into the lumen of the ER. These NH2-terminal segments appear to serve multiple roles in targeting and translocation. The structural features which mediate their multiple functions are currently the subject of intense study.

Animals↗

PubMed1992

A novel calcium-binding protein from Euglena gracilis. Characterisation of a cDNA encoding a 74-kDa acidic-repeat protein targeted across the endoplasmic reticulum.

We have isolated a novel cDNA from Euglena gracilis that encodes a protein composed of 24.9% aspartate with an estimated pI of 3.56, and a deduced molecular mass of 73,542 Da. The first 20 or so amino acids are hydrophobic and resemble a signal sequence. The rest of the polypeptide is composed of a 23-amino-acid repeat. There are 30 repeats, of which 23 are full length. Part of the consensus sequence derived from the repeats has some similarity to the loop of the EF-hand type calcium-binding motif. Evidence is presented that a fusion protein of this novel protein with beta-galactosidase can bind calcium. Northern blotting indicates a single transcript of 2.3 kb (the same size as the cDNA). In-vitro translation of the cDNA gives a protein that migrates on SDS/PAGE with an apparent molecular mass of 120-125 kDa. The protein is processed into a smaller, protease-protected form (110-120 kDa) when translated in the presence of canine pancreatic microsomal vesicles. This suggests that the protein is targeted across the endoplasmic reticulum membrane in vivo, and is the first report of a signal sequence from E. gracilis. We propose that the cDNA obtained encodes a novel calcium-binding protein that is either secreted or resident in the endomembrane system of E. gracilis, and call it the acidic-repeat protein.

Amino Acid Sequence↗

PubMed1985

Ribosome-SRP-signal sequence interactions. The relay helix hypothesis.

The role of the signal sequence in protein export is reviewed, and some difficulties inherent in the conventional picture of how it interacts with other components of the export machinery are pointed out. An alternative model is suggested, which seems to account better for some of the critical experimental findings made so far.

Amino Acid Sequence↗

PubMed1989

Peptide signals for protein degradation within lysosomes.

In this article we summarize our findings concerning a pathway by which cytosolic proteins can be selectively taken up and degraded within lysosomes. Serum deprivation of cells in culture activates this pathway, and only proteins that contain peptide sequences related to KFERQ are degraded at an enhanced rate. Approximately 30% of intracellular proteins contain such peptide sequences, and we speculate about the physiological relevance of the selective degradation of these proteins in response to serum withdrawal. Several rat tissues also contain proteins with peptide sequences related to KFERQ, and the amount of these proteins is reduced in response to starvation. Finally, we present recent results suggesting that this selective uptake of cytosolic proteins by lysosomes is not through classical macroautophagy. Instead, the selective uptake appears to be similar to other protein sorting pathways such as protein translocation through the endoplasmic reticulum or protein import into mitochondria.

Amino Acid Sequence↗

PubMed1992

Characterization by cDNA cloning of the mRNA of a new growth factor from bovine seminal plasma: acidic seminal fluid protein.

A cDNA expression library in lambda gt11 prepared from cDNA derived of seminal vesicle tissue was screened by means of monospecific rabbit anti-aSFP IgG. The sequence of clone pTF21, containing an insert of 668 bp comprised an open reading frame from position 7 to 411 terminated by two stop codons. From this sequence a protein of 134 amino acid residues can be deduced. The mature aSFP was preceded by a signal peptide of 20 amino acids length. The protein sequence contains no signal for N-glycosylation. The molecular weight calculated from the amino acid sequence is 12922 Da. The start codon ATG is part of the sequence AAGATGA which fulfills the criteria of an initiation consensus sequence. The coding region was followed by 257bp of the complete 3'-untranslated region (3'UTR). A putative polyadenylation signal AATAAT, although not of the standard type, is observed at position 650. According to Northern analysis, aSFP mRNA is expressed in seminal vesicle tissue, ampulla and weakly in tissue of epididymis, but not in testis or other bovine tissue. aSFP is specified by a single copy gene. Attempts to detect homologies to known protein sequences were not successful.

Amino Acid Sequence↗

PubMed1986

Protein translocation across and integration into membranes.

This review concentrates mainly on the translocation of proteins across the endoplasmic reticulum membrane and cytoplasmic membrane in bacteria. It will start with a short historical review and will pinpoint the crucial questions in the field. Special emphasis will be given to the present knowledge on the molecular details of the first steps, i.e., on the function of the signal recognition particle and its receptor. The knowledge on the signal peptidase and the ribosome receptor(s) will also be summarized. The various models for the translocation of proteins across and the integration of proteins into membranes will be critically discussed. In particular, the function of signal, stop-transfer, and insertion sequences will be dealt with and molecular differences discussed. The cotranslational mode of membrane transfer will be compared with the post-translational transport found for mitochondria and chloroplasts. This review will conclude with open questions and an outlook.

Amino Acid Sequence↗

PubMed1987

Product of SEC53 is required for folding and glycosylation of secretory proteins in the lumen of the yeast endoplasmic reticulum.

Yeast secretory mutant sec53 cells accumulate inactive secretory glycoprotein precursors that remain associated with the endoplasmic reticulum (ER) at the restrictive temperature (37 degrees C). The possibility that precursor polypeptides fail to penetrate completely into the ER lumen was tested by examining the protease accessibility of accumulated invertase, mating pheromone precursor prepro-alpha-factor and the vacuolar protein precursor procarboxypeptidase Y in cell lysates. In all three cases, the secretory protein precursors are protected from the action of exogenous protease unless the membrane is permeabilized by including Triton X-100 or saponin in the incubation. These results suggest that the sec53 defect allows complete polypeptide translocation. Consistent with this interpretation, the precursor of invertase accumulates in a signal peptide-processed form. In addition, invertase and prepro-alpha-factor precursors contain a small amount of possibly aberrant carbohydrate. In mutant cells or in wild type cells treated with tunicamycin, a 10-kDa fragment of the N terminus of mature invertase assumes a conformation that is resistant to trypsin with or without detergent. This domain may be associated with an ER protein or may simply assume an unusual conformation as a consequence of deficient glycosyl modification.

Carboxypeptidases↗