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We have shown that the unfolded protein response (UPR) in Pichia pastoris requires splicing of a non-conventional intron in the HAC1u mRNA in common with other eukaryotes. P. pastoris is a favoured yeast expression host for secreted production of heterologous proteins and the regulation of the UPR in P. pastoris may hold the key to its effective folding and secretion of proteins. We have also shown that the C-terminal region of the Hac1p from P. pastoris is required for functionality. Although the C-terminal regions of Hac1p from both S. cerevisiae and P. pastoris are rich in phenylalanine residues, the P. pastoris Hac1p lacks a C-terminal serine that is known to be important in the efficient functionality of Hac1p from S. cerevisiae.

The Ess/Type VII protein secretion system, essential for virulence of pathogenic Staphylococcus aureus, is dependent upon the four core membrane proteins EssA, EssB, EssC and EsaA. Here, we use crosslinking and blue native PAGE analysis to show that the EssB, EssC and EsaA proteins individually form homomeric complexes. Surprisingly, these components appear unable to interact with each other, or with the EssA protein. We further show that two high molecular weight multimers of EssC detected in whole cells are not dependent upon the presence of EsxA, EsxB or any other Ess component for their assembly.

SecDFyajC of Escherichia coli is required for efficient export of proteins in vivo. However, the functional role of SecDFyajC in protein translocation is unclear. We evaluated the postulated function of SecDFyajC in the maintenance of the proton motive force. As previously reported, inner membrane vesicles (IMVs) lacking SecDFyajC are defective in the generation of a stable proton motive force when energized with succinate. This phenomenon is, however, not observed when NADH is used as an electron donor. Moreover, the proton motive force generated in SecDFyajC-depleted vesicles stimulated translocation to the same extent as seen with IMVs containing SecDFyajC. Further analysis demonstrates that the reduced proton motive force with succinate in IMVs lacking SecDFyajC is due to a lower amount of the enzyme succinate dehydrogenase. The expression of this enzyme complex is repressed by growth on glucose media, the condition used to deplete SecDFyajC. These results demonstrate that SecDFyajC is not required for proton motive force-driven protein translocation.

A cell-free protein translocation system derived exclusively from a Gram-positive bacterium is described here for the first time. Highly efficient in vitro synthesis of plasmid encoded preprolipase of Staphylococcus hyicus is accomplished by coupled transcription/translation using either a cytosolic extract of S. carnosus alone or in combination with T7-RNA-polymerase. Addition of inside-out cytoplasmic membrane vesicles of S. carnosus leads to the partial conversion (processing) of preprolipase to prolipase. In addition, as shown in a protease protection assay, a significant part of preprolipase plus prolipase is translocated in vitro into the lumen of the vesicles. Translocation of preprolipase into the membrane vesicles requires the proton-motive force and the S. carnosus SecA protein.

Translocation of proteins through the inner membrane of E. coli is normally catalyzed by the so-called sec-machinery. Yet, many integral inner membrane proteins appear not to require a fully functional sec-machinery for proper insertion, in spite of the fact that sometimes quite sizable domains have to be translocated to the periplasmic side. This review will focus on recent studies of sec-independent translocation events in an attempt to pin-point the main differences between sec-dependent and sec-independent translocation.

We describe a secretory E. coli protein with a novel phenotype: signal peptide cleavage is largely unaffected whereas chain translocation is efficiently blocked under conditions where SecA, a central component of the secretory machinery, is rendered non-functional, and we have traced this phenotype to the presence of a mildly hydrophobic segment located approximately 30 residues downstream of the signal peptide. When this segment is deleted, normal SecA-dependent signal peptide cleavage and chain translocation is observed; when its hydrophobicity is increased, it becomes a permanent membrane anchor with cleavage of the signal peptide and membrane insertion both being SecA-independent. These findings suggest that the initial insertion of the signal peptide across the membrane can be uncoupled from the translocation process proper.

Proline residues are rarely found in the three most C-terminal positions of bacterial signal peptides, and have never been found in position +1 immediately following the cleavage site. It was recently shown that a Pro+1 mutation in the E. coli maltose binding protein precursor not only prevents cleavage of the signal peptide but also inhibits the leader peptidase enzyme, resulting in cessation of cell growth (Barkocy-Gallagher, G.A. and Bassford, P.J. (1992) J. Biol. Chem. (in press)). Since maltose binding protein is dependent on the sec machinery for translocation across the inner membrane, it was not clear if this ‘Pro+1’ effect was restricted to sec-dependent proteins, or whether it applies also to proteins that do not require the sec functions for translocation. We now present data suggesting that the striking phenotypic effects of Pro+1 mutations can be elicited also by sec-independent proteins.

Human A431 cells express a 100 kDa EGF-receptor homolog that contains only the external domain. The kinetics of its maturation and export are slow and comparable to those of the transmembrane receptor. Here we demonstrate that exogenously added EGF stimulates post-translational processing and export of this receptor through a pathway that is insensitive to inhibitors of protein synthesis. The results suggest that EGF may influence one or more of the rate determining steps that control receptor export from endoplasmic reticulum. This may constitute one of the path-ways by which EGP regulates the expression of its receptor.

We have developed a defined medium which can maintain efficient growth of HepG2 cells sustaining the synthesis of a variety of plasma proteins including apolipoprotein B. This defined system was used to investigate long-term effects of insulin, estrogen, triiodothyronine, cholesterol, and oleate on the growth pattern of HepG2 cells and secretion rate of apolipoprotein B. Oleate and triiodothyronine caused significant increases in secretion of apolipoprotein B. The stimulatory effect of triiodothyronine was only observed after long (6 days) exposure of cells to the hormone. In contrast, insulin caused up to a 4-fold decrease in the secretion rate of apolipoprotein B during the early growth periods. This inhibitory effect appeared to be partially abolished after 6 days. Our data suggest that some important questions on regulation of apolipoprotein B expression can be addressed by the long-term culture of HepG2 cells in defined medium.

Terminal residues in SecA, the dimeric ATPase motor of bacterial preprotein translocase, were proposed to be required for function and dimerization. To test this, we generated truncation mutants of the 901aa long SecA of Escherichia coli. We now show that deletions of carboxy-terminal domain (CTD), the extreme CTD of 70 residues, or of the N-terminal nonapeptide or of both, do not compromise protein translocation or viability. Deletion of additional C-terminal residues upstream of CTD compromised function. Functional truncation mutants like SecA9-861 are dimeric, conformationally similar to SecA, fully competent for nucleotide and SecYEG binding and for ATP catalysis. Our data demonstrate that extreme terminal SecA residues are not essential for SecA catalysis and dimerization.

A protein translocation system was reconstituted from inverted membrane vesicles prepared from Na+ pump-possessing Vibrio alginolyticus and purified Escherichia coli SecA. The translocation required ATP and was stimulated by the functioning of the Na+ pump, suggesting that the electrochemical potential of Na+ but not that of H+, is important for protein translocation in Vibrio.

A derivative of the α-amylase from Bacillus licheniformis (AmyL) engineered to give an active enzyme with increased net positive charge is secreted by Bacillus subtilis with a yield that is significantly lower than that of the native enzyme. This reduction in yield is the result of increased proteolysis during or shortly after translocation through the cytoplasmic membrane. When we compared the overall rate of folding of the engineered derivative (AmyLQS50.5) with that of AmyL it exhibited a greater dependency on Ca2+ ions for in vitro folding. When the concentration of Ca2+ in the growth medium was increased, so too did the relative yield of AmyLQS50.5. We discuss the importance of secretory protein folding at the membrane/cell wall interface with respect to the yield of native and heterologous proteins from B. subtilis.

Leader peptidase is an integral membrane protein of E. coli and it catalyses the removal of most signal peptides from translocated precursor proteins. In this study it is shown that when the transmembrane anchors are removed in vivo, the remaining catalytic domain can bind to inner and outer membranes of E. coli. Furthermore, the purified catalytic domain binds to inner membrane vesicles and vesicles composed of purified inner membrane lipids with comparable efficiency. It is shown that the interaction is caused by penetration of a part of the catalytic domain between the lipids. Penetration is mediated by phosphatidylethanolamine, the most abundant lipid in E. coli, and does not seem to depend on electrostatic interactions. A hydrophobic segment around the catalytically important residue serine 90 is required for the interaction with membranes.

The export of colicin A and of colicin E1 is not equally affected in both secA and secY mutants of E. Echerichia coli: release of colicin A occurs slowly while that of colicin E1 is blocked. Processing and functioning of Cal, the colicin A lysis protein, seem to be slightly or not at all modified in these mutants, whereas synthesis and assembly of CelA, the colicin E1 lysis protein, are highly inhibited. These variations observed in the dependence of the two lysis proteins on secA and secY gene products are interpreted as being either the cause or the consequence of the differences observed in their rate of biogenesis.

Brefeldin A (BFA) causes rapid redistribution of Golgi proteins into the endoplasmic reticulum (ER), leaving no definable Golgi-apparatus, and blocks transport of proteins from the ER to distal secretory compartments of the cell. Using pulse-chase experiments the present study shows that BFA (1 microgram/ml) inhibits basal and CCK-stimulated protein secretion in isolated pancreatic acinar cells by 65 +/- 6% and 84 +/- 5%, respectively. In isolated permeabilized cells higher concentrations of BFA (30 micrograms/ml) were necessary to obtain inhibition of protein secretion. In parallel experiments protein secretion was stimulated by GTP (1 mM). BFA had no inhibitory effect on protein secretion in the presence of GTP, indicating that BFA might act on a GTP-binding protein. Investigating the effect of BFA on small molecular weight GTP-binding proteins we observed that [alpha-32P]GTP binding to a 21 kDa protein in a subcellular fraction enriched in ER was increased in the presence of BFA. We conclude that this 21 kDa and possibly also other GTP-binding proteins may be the molecular target of Brefeldin A in pancreatic acinar cells.

SecY is an integral membrane protein which participates in the translocation of proteins through the bacterial cell membrane. We have cloned the secY gene of Lactococcus lactis, and found its deduced protein sequence, 439 amino acids long, to be similar in length to the previously determined SecY proteins of Escherichia coli, Bacillus subtilis and Mycoplasma capricolum. Comparison of the L. lactis SecY to the 3 other SecY proteins revealed 90 conserved amino acid residues (21%). Nearly half of the conserved residues are clustered in 2 of the 10 transmembrane segments, and in 2 of the 6 cytoplasmic regions. Some of the conserved regions are apparently responsible for the interactions of SecY with signal sequences, and the proteins SecE and SecA.

The chemical cross-linking between the two cysteine residues at positions + 290 and + 302 of proOmpA was performed with N,N'-bis(3-maleimidopropionyl)-2-hydroxy-1,3-propanediamine. In the absence of the proton motive force (delta muH+), the cross-linked proOmpA was only partially translocated into everted membrane vesicles, leading to accumulation of translocation intermediates. In the presence of delta mu H+, the cross-linked proOmpA was completely translocated. The translocated OmpA still possessed the cross-linked loop composed of 13 amino acid residues and the cross-linker. It is concluded that polypeptide chains need not be necessarily linear and fully expanded to be translocated.

The lspA gene coding for lipoprotein signal peptidase is located very close to the ileS gene coding for isoleucyl-tRNA synthetase on the Escherichia coli chromosome. Deletions were generated in vitro from both ends of the 4.3 kb fragment that carries the lspA gene and the ileS gene, and the expression of the two genes was examined before and after insertion of the trp promoter-operator at one end. The results indicate that the lspA and ileS genes constitute a cotranscriptional unit in the order of promoter- ileS - lspA . The gene order of dnaJ - rpsT - ileS - lspA - dapB around 0.5 min on the E. coli chromosome map was also determined.

A decapeptide highly homologous to the STa Escherichia coli heat-stable enterotoxin and to several other heat-stable enterotoxins was fused genetically to the amino-end of the B-subunit of cholera toxin (CTB) and the hybrid protein gene expressed from a tacP overexpression system. The STa-related decapeptide used, which was encoded by a synthetic oligodeoxynucleotide, contained a single mutation which substituted a disulfide-linked cysteine by alanine. After its fusion to CTB the decapeptide was able to both react with and to give rise to anti-STa antibodies. Expression of the decapeptide-CTB hybrid by non-toxigenic Vibrio cholerae resulted in its full secretion into the extracellular milieu from where it could then be readily purified by single-step affinity chromatography using immobilized GM1 ganglioside. Bacteria producing this non-toxic, immunogenic decapeptide-CTB toxoid might be useful for the development of oral vaccines against diarrhea caused by E.coli and other bacteria producing immunologically related heat-stable enterotoxins, and as a source of immunoreagents for methods used to diagnose disease caused by these bacteria.

A high-expression plasmid for the secA gene was constructed. The SecA protein was then overproduced in E. coli and purified. The purified SecA stimulated the in vitro translocation of a model secretory protein into inverted membrane vesicles pretreated with 4 M urea. Membrane vesicles from a secAts mutant exhibited lower translocation activity, which was enhanced by SecA. These results indicate that SecA is directly involved in protein secretion across the cytoplasmic membrane.

In rat lacrimal gland, cholinergic, α- or β-adrenergic or methylxanthine stimulations of protein secretion are extracellular calcium dependent. 10 μM trifluoperazine (TFP) inhibited only cholinergic and α-adrenergic stimulations. Half maximal effect was observed at 30 μM, with all inducers except norepinephrine (3 μM). 10 or 30 μM TFP also suppressed the decrease of L-[3H]leucine incorporation into protein due to carbamylcholine. 100 μM TFP inhibited protein secretion and L-[3H]leucine incorporation. 500 μM TFP promoted cell lysis. It is suggested that: (a) at 100 μM TFP, inhibition is not specific for protein secretion; (b) at 30 μM TFP, inhibition could be related to a role of calmodulin in the secretory regulation process.

The nucleotide sequence of the lspA gene coding for lipoprotein signal peptidase of Escherichia coli was determined and the amino acid sequence of the peptidase was deduced from it. The molecular mass and amino acid composition of the predicted lipoprotein signal peptidase were consistent with those of the signal peptidase purified from cells harboring the lspA gene-carrying plasmid. The peptidase most probably has no cleavable signal peptide. The lspA gene was preceded by the ileS gene coding for isoleucylt RNA synthetase and the tandem termination codons of the ileS gene overlapped with the initiation codon of the lspA gene.

The function of the SRH1 product, the yeast homologue of the 54 kDa subunit of the mammalian signal recognition particle, has been analyzed using a galactose dependent mutant of the gene. SRH1 has been placed under control of the GALI promoter and introduced into a haploid cell that had its chromosomal SRH1 copy disrupted. This mutant grows normally on galactose medium but slows down the growth about 10 h after transfer to glucose medium. At the same time, precursor forms of secretory proteins, α-mating factor and invertase, accumulate in the cells. This result indicates that the SRH1 product is involved in translocation of precursors of secretory proteins across the endoplasmic reticulum membrane in yeast cells.

Protein sulfation was studied in germ-free rats by prolonged in vivo labeling with [35S]sulfate. Specific sets of sulfated proteins were observed in all tissues examined, in leucocytes, and in blood plasma. No protein sulfation was detected in erythrocytes. Analysis of the type of sulfate linkage showed that sulfated proteins secreted into the plasma contained predominantly tyrosine sulfate, whereas sulfated proteins found in tissues contained largely carbohydrate sulfate. This implies some kind of selection concerning the intracellular processing, secretion, turnover or re-uptake of sulfated proteins which is responsible for the enrichment of tyrosine-sulfated proteins in the plasma.

The Ddi1 protein of the yeast Saccharomyces cerevisiae is involved in numerous interactions with the ubiquitin system, which may be mediated by its N-terminal ubiquitin like domain and its C-terminal ubiquitin associated domain. Ddi1 also contains a central region with all the features of a retroviral aspartic proteinase, which was shown to be important in cell-cycle control. Here we demonstrate an additional role for this domain, along with the N-terminal region, in protein secretion. These results further substantiate the hypothesis that Ddi1 functions in vivo as a catalytically-active aspartic proteinase.

An intact C-terminus was previously shown to be required for stability and secretion of the polygalacturonase (PehA) in Erwinia carotovora ssp. carotovora. Here we have analyzed the effects of amino acid (aa) substitutions generated to five C-terminal positions of PehA. Conservation of two hydrophobic and one non-hydrophobic residue (V372, V374 and N371, respectively) was found to be essential for maintenance of the protein stability. As an exception, one of the mutants (V372G) did not show major effects on protein stability, as determined by immunoblots and enzyme activity assay, yet it prevented the secretion completely. We conclude that the C-terminus of PehA is directly involved in the formation or stabilization of a conformation-sensitive structure needed for recognition of the protein as secreted.

Application of a differential hybridization technique led to the isolation of a human pituitary cDNA clone encoding the complete structure of the polypeptide 7B2. This protein of unknown function, which is sorted to secretory granules, appears to be present selectively in neurons and endocrine cells. The polypeptide chain of human 7B2, preceded by a cleaved signal peptide, comprises 185 amino acids (a calculated Mr of 20 793). Interesting features of the highly-conserved 7B2 structure include (i) a serine phosphorylation consensus sequence, (ii) the occurrence of three pairs of dibasic amino acids representing potential proteolytic cleavage sites and, in particular, (iii) the presence of three regions homologous to GTP-binding domains giving 7B2 structural characteristics of a GTP-binding protein.

The secY and secE genes were individually cloned and placed under the control of the tac promoter on plasmids. Induction with isopropyl-β-D-thiogalactopyranoside resulted in the overproduction of SecE, but not that of SecY. The simultaneous induced expression of both genes in the same cells resulted in the overproduction of SecY together with that of SecE. SecY and SecE thus overproduced were localized in the cytoplasmic membrane as those expressed at the normal levels were. It is suggested that SecY and SecE interact with each other in the cytoplasmic membrane. The numbers of the SecY and SecE molecules per cell were estimated.

The equilibrium transition curves for thermal unfolding of levansucrase were established at several pH values. At pH 7 and within the temperature range of bacterial growth, the unfolded from is predominant. However, under such conditions, refolding is promoted by the only addition of Ca2+ or Fe3+. We propose that the tertiary structure flexibility of levansucrase plays a key role in its secretion process.

In order to test whether SecY and SecE proteins constitute the SecA receptor inside out membrane vesicles where prepared from strains producing greatly different levels of these two proteins, and their SecA binding activity was quantitated. Substantial overproduction of SecE or SecY and SecE proteins resulted in no increase or only 50% increase, respectively, in the number of high affinity SecA binding sites. These results suggest that SecY and SecE proteins appear insufficient to constitute the primary SecA receptor. The existence of a cycle of SecA association with the inner membrane and its modulation by particular integral membrane proteins is discussed.

The function of the domains of Trichoderma reesei endoglucanase I (EGI) has been studied. Truncated EGI proteins were expressed from the 3′-end deleted cDNAs in the yeast Saccharomyces cerevisiae under the control of the ADCI expression cassette. EGI protein was detected by monoclonal antibody EI-2 and EGI activity as cleared zones around growing colonies on agar plates containing hydroxyethylcellulose (HEC) covalently stained with Ostazin brilliant red (OBR). The results showed that the Thr—Ser-rich hinge region and the conserved ‘tail’ are not necessary for the efficient synthesis and secretion of EGI in yeast, but the intact core region is necessary for the enzymatic activity.

Organelles of the early protein secretion pathway (ER, Golgi) are involved in biosynthesis and intracellular migration of the yeast sphingolipids, inositolphosphorylceramide (IPC), mannosylinositolphosphorylceramide (MIPC), and mannosyldiinositolphosphorylceramide (M(IP)2C). Cycloheximide and nocodazole neither block biosynthesis of sphingolipids, nor ER to Golgi transport of IPC. In contrast, treatment of yeast cells with brefeldin A, which affects integrity of the Golgi, decreases formation of IPC and MIPC. Interruption of late steps of protein secretion (Golgi to plasma membrane transport) in temperature-sensitive secretory mutants prevents sphingolipids from being transported to the cell periphery.

The sequence of EspB, a secreted protein required for virulence of enteropathogenic Escherichia coli (EPEC), reveals a motif common to enzymes that bind pyridoxal phosphate. Pyridoxal phosphate was not found by fluorometry in concentrated supernatants of EPEC cultures that contain EspB. Plasmids containing cloned espB, in which the lysine residue conserved in the motif was substituted with either an arginine or methionine residue, remained capable of complementing an espB deletion mutant to restore EspB function. The results of these studies do not support a role for pyridoxal phosphate in EspB function.

The twin-arginine translocation (Tat) machinery is able to transport fully folded proteins across bacterial and thylakoidal membranes. Previous in vivo and in vitro studies indicated that the model Tat substrate TorA–PhoA acquires Tat-competence only if its four cysteines form disulfide bonds. We now show that removal of the last 33 amino acids of PhoA, although not affecting the formation of disulfide bonds, converts TorA–PhoA into a poor Tat substrate. This finding suggests that even incomplete folding of a substrate can interfere with transport by the Tat translocase of Escherichia coli.

The minimal glycosylation requirement for acid phosphatase secretion and activity was investigated using tunicamycin, an inhibitor of protein glycosylation, and a yeast mutant defective in the synthesis of oligosaccharide outer chains. The results obtained show that outer chain addition is not essential for secretion of active enzyme and that only 4 core chains, out of 8 normally attached to a protein subunit, are sufficient for enzyme transport to the periplasmic space. Enzyme forms with less than 4 chains were retained in membranes of endoplasmic reticulum. Secreted underglycosylated enzyme forms are partially or completely inactive.

Microinjection of human liver mRNA from a patient homozygous for α 1 -antitrypsin deficiency (PiZZ) into Xenopus oocytes led to a 2–10-fold increase in lysosomal activity. Stimulation of lysosomal activity was not observed when mRNA from a normal human liver (α 1 -antitrypsin PiMM), or water was injected into the oocyte. This lysosomal activity was oocyte derived and was not due to translation products of the human liver mRNA. Thus a protein that accumulates intracellularly in the secretory pathway is capable of stimulating lysosomal activity.

Our study shows that the secretion of a major non-glycosylated, phosphoprotein of 69 kDa (pp69) is a specific marker for non-transformed NRK-49F cells. Treatment of NRK-49F cells with EGF alone or with different combinations of EGF plus TGF-β modulates the secretion of pp69, suggesting its relationship with cellular proliferation. Antibody raised against pp69 recognizes, in addition to pp69, another major phosphoprotein of 62 kDa (pp62) secreted by RR1022 and spontaneously transformed NRK-49F cells. Immunoprecipitation of total cell lysates from both NRK-49F and RR1022 cells with anti-pp69 antibody detected only pp69. These observations suggest a precursor-product relationship between pp69 secreted by non-transformed NRK-49F cells and pp62 secreted by transformed cells.

Secretory signal peptides from individual prokaryotic and eukaryotic species have been analyzed, and the lengths and amino acid compositions of the positively charged amino-terminal region, the central hydrophobic region, and the carboxy-terminal cleavage-region have been compared. We find distinct differences between species in all three regions. Implications for protein secretion in foreign hosts are discussed.

In rat lacrimal glands, Forskolin induces a dose-dependent [ 3 H]protein release. This effect can be potentiated by papaverine. As for the other inducers whose effects on protein secretion are assumed to be cAMP-mediated, Forskolin secretion time course shows a latency. Isoproterenol decreases the Forskolin EC 5- at least 60--times. On the other hand, Forskolin enhances the efficacy of isoproterenol without affecting its potency. As a whole, the data collected show that isoproterenol-induced [ 3 H]protein secretion in rat lacrimal glands involved adenylate cyclase activation by coupling with β-adrenergic receptors.