Secretory protein translocation in a neurospora crassa in vitro system. Hydrolysis of a nucleoside triphosphate is required for posttranslational translocation

An in vitro translocation system has been reconstituted with subcellular fractions from the cell wall-less mutant of Neurospora crassa (fz;sg;os-1). Prepro alpha factor and invertase, secretory proteins from yeast, were faithfully translocated and glycosylated by Neurospora microsomes when presence cotranslationally in the Neurospora translation system. When presence cotranslationally in the Neurospora translation system, microsomes from canine pancreas(cRM) could also translocate and glycosylate the secretory proteins. However, salt-extracted cRM, which is depleted of canine signal recognition particle, could not. Furthermore, prepro alpha factor and a truncated form of invertase, containing the first 262-amino acid residues of the secretory invertase, were glycosylated by Neurospora microsomes posttranslationally, whereas only the truncated form of invertase was glycosylated by cRM when added posttranslationally. The full length invertase was not glycosylated posttranslationally. Posttranslational glycosylation of prepro alpha factor and of the truncated form of invertase is dependent on the hydrolysis of a nucleoside triphosphate. These data suggest that posttranslational glycosylation of prepro alpha factor occurs via a novel type of recognition mechanism which is either absent or ineffective in cRM.     

Secretion of somatostatin by Saccharomyces cerevisiae. Correct processing of an alpha-factor-somatostatin hybrid

Somatostatin is a 14-amino acid peptide hormone that is proteolytically processed from its precursor, prosomatostatin, by a paired-basic-specific protease localized in the Golgi apparatus and secretory vesicles. Yeast (Saccharomyces cerevisiae MAT alpha) synthesize an analogous peptide hormone precursor, pro-alpha-factor, that contains tandem repeats of alpha factor (13 amino acids) flanked by spacers that include paired basic residues. To investigate the role of these two pro regions in mediating intracellular transport and processing, cloned genes specific for preprosomatostatin and prepro-alpha-factor were used to generate recombinants encoding hybrids between the alpha-factor pro region (amino-terminal) and somatostatin (carboxyl-terminal). These recombinants were inserted into yeast expression vectors under control of either the native alpha-factor promoter or the inducible yeast PHO5 (acid phosphatase) promoter. Yeast transformed with these plasmids expressed the hybrid messenger RNAs constitutively (alpha-factor promoter) or when induced in phosphate-deficient medium (PHO5 promoter). Radioimmunoassay of culture media revealed the secretion of up to 200 ng of immunoreactive somatostatin/10(7) cells. Metabolic labeling with [35S]cysteine, followed by immunoprecipitation with anti-somatostatin antibodies revealed two forms of hybrid precursor intracellularly, one of Mr 25,000, containing core carbohydrates, and a second of Mr 11,000, which was unglycosylated. Translation of mRNA extracted from these transformants in the wheat germ cell-free system revealed that the Mr 11,000 form was the primary translation product, whereas the Mr 25,000 species could be generated in vitro by inclusion of mammalian rough microsomes. The secreted immunoreactive material was shown to be authentic somatostatin by high pressure liquid chromatography analysis and protein sequencing. These results demonstrate that the yeast processing enzymes recognize these chimeric precursors, resulting in the secretion of the mature peptide hormone.     

Parathyroid mRNA directs the synthesis of pre-proparathyroid hormone and proparathyroid hormone in the Krebs ascites cell-free system.

Translation in a cell-free extract of Krebs II ascites cells of a mRNA fraction prepared from bovine parathyroid glands results in the synthesis of two radioactive products that appear identical to pre-proparathyroid hormone (Pre-ProPTH) (M.W. ～ 14,000), the suspected earliest biosynthetic precursor of parathyroid hormone (PTH) (M.W. 9,500), and to proparathyroid hormone (ProPTH) (M.W. 10,200), the immediate biosynthetic precursor of PTH. The two products of synthesis in the ascites extract co-electrophoresed on both urea-acetate and urea-SDS acrylamide gels with Pre-ProPTH obtained from cell-free translation of parathyroid RNA in extracts of wheat-germ and with ProPTH isolated from parathyroid slices. Both products were precipitated with an antiserum to PTH. Partial analysis of the amino acid sequence of [³⁵S]methionine-labeled Pre-ProPTH synthesized by the ascites extract indicates that a substantial fraction of the product is lacking the two N-terminal methionines present in the Pre-ProPTH synthesized by the wheat-germ system. The results indicate that, ($\text{i}$), unlike the wheat-germ, ascites extracts contain enzymes that remove the initiator methionine from Pre-ProPTH and convert Pre-ProPTH into ProPTH (no ProPTH was observed in the wheat-germ system) and ($\text{ii}$) the cleavage processes appear to occur in association with synthesis, inasmuch as neither removal of NH₂-terminal methionine nor formation of ProPTH was observed upon incubation of Pre-ProPTH isolated from either the wheat-germ system or from the ascites system when put back into the ascites system.     

Functional analysis of the signal-sequence processing site of yeast acid phosphatase

A systematic study of the signal peptidase cleavage site of the main cell-wall-repressible Saccharomyces cerevisiae acid phosphatase encoded by the PHO5 gene is presented. The last amino acid of the signal sequence, the chromosomally encoded alanine of the wild-type gene, was changed by any of 19 other amino acids in the chromosomal DNA by using in vitro mutagenesis in Escherichia coli and the technique of gene replacement. Processing and secretion are normal when the amino acid at this position is a small neutral amino acid, i.e. alanine, glycine, cysteine, serine or threonine. Processing glycosylation, and secretion of regulated acid phosphatase are distinctly affected with other amino acid substitutions and core-glycosylated protein accumulates in the cell. Surprisingly, PHO5 protein is still secreted to the cell wall and into the growth medium but at a lower rate and without cleavage of the signal sequence. The same features are exhibited by a mutated acid phosphatase with a deletion of four amino acids at the end of the signal peptide (-7 to -4 relative to the processing site) thus preserving the important -3 to -1 region.     

Secretion of human blood coagulation factor XIIIa by the yeast Yarrowia lipolytica.

The industrial yeast, Yarrowia lipolytica, secretes high yields of an alkaline extracellular protease (AEP), which is synthesized as a preproprotein encoded by the XPR2 gene. We investigated the possibility of using this system for the secretion of human coagulation factor XIII subunit a (FXIIIa). This protein is naturally secreted in the plasma by an unknown, signal peptide-independent mechanism and has so far been found to be nonsecretable in yeast. We have designed six hybrid genes encoding fusion proteins between increasing portions of the AEP preprodomain and the precursor or mature forms of FXIIIa. All constructs directed translocation of the FXIIIa precursor into the endoplasmic reticulum. Transport of the translocated and core-glycosylated hybrid precursor to the Golgi apparatus appeared to be strongly rate limiting, and most of the precursors appeared to be partially proteolysed. One of these constructs directed the extracellular secretion of a low amount of hyperglycosylated FXIIIa. These results indicate that fusion to the yeast AEP signal peptide and dipeptide stretch allows FXIIIa to be translocated, albeit inefficiently, through the endoplasmic reticulum and to follow a classical secretory transit.     

Secretion in yeast: in vitro analysis of the sec53 mutant.

Of central importance to studying protein translocation via a combined genetic and biochemical approach is the in vitro analysis of yeast conditionally-lethal secretory mutants. Analysis of sec53 presented an opportunity not only to see if mutants could be examined in recently developed yeast in vitro translocation systems, but also to characterize further the nature of this mutant originally postulated to be defective in protein translocation. Membranes from sec53 were capable of translocating and glycosylating nascent prepro-alpha-factor in vitro in both sec53 and wild-type lysates at temperatures that were non-permissive for growth of the mutant cells. These results suggested that the Sec53 protein does not function directly in the translocation and glycosylation of prepro-alpha-factor. To examine this point further, we isolated membranes from sec53 cells that had been grown at the non-permissive temperature prior to disruption. In such cases, regardless of assay temperature, membranes from sec53 cells efficiently translocated but failed to glycosylate prepro-alpha-factor in vitro. The in vitro phenotype of sec53 could be mimicked by isolating rough microsomes from wild-type cells that had been grown for 1 h in the presence of tunicamycin. Together, these results demonstrate that sec53 is not defective in translocation, rather in assembly of the dolichol-oligosaccharide substrate needed for N-linked glycosylation.     

Regulation of thiamine biosynthesis in Saccharomyces cerevisiae.

A pho6 mutant of Saccharomyces cerevisiae, lacking a regulatory gene for the synthesis of periplasmic thiamine-repressible acid phosphatase activity, was found to be auxotrophic for thiamine. The activities of four enzymes involved in the synthesis of thiamine monophosphate were hardly detectable in the crude extract from the pho6 mutant. On the other hand, the activities of these enzymes and thiamine-repressible acid phosphatase in a wild-type strain of S. cerevisiae, H42, decreased with the increase in the concentration of thiamine in yeast cells. These results suggest that thiamine synthesis in S. cerevisiae is subject to a positive regulatory gene, PHO6, whereas it is controlled negatively by the intracellular thiamine level.     

In vivo translocation of the cell wall acid phosphatase across the yeast endoplasmic reticulum membrane: are there multiple signals for the targeting process?

The repressible Saccharomyces cerevisiae acid phosphatase (APase) coded by the PHO5 gene is a cell wall protein that follows the yeast secretory pathway. We had previously described the in vivo fate of a multicopy plasmid-encoded modified protein, lacking 15 out of 17 signal peptide amino acids. This modified protein accumulates mainly within the cell as an inactive unglycosylated form. However 30% of this precursor is translocated, glycosylated and dispatched to the cell wall. We establish, in the present report, that this phenomenon did not result from an overproduction of the plasmid encoded protein, since it was also observed in a normal single copy situation. The secretion persisted after a deletion including the single hydrophobic segment present in the N-terminus of the mature protein. The entry of both wild type and mutant APase into the ER was inhibited in sec62 mutants suggesting that the SEC62 gene product would not be implicated in signal peptide recognition.     

Signal recognition particle (SRP) stabilizes the translocation-competent conformation of pre-secretory proteins.

When affinity-purified proOmpA was diluted out of 8 M urea into a sample of yeast microsomes, it was translocated and processed in the absence of any cytosolic factors; an intact membrane and ATP were the only requirements. The translocation competence of proOmpA was lost, however, during a 15-h incubation at 0 degrees C. The competence was retained when trigger factor and a yeast cytosolic extract were present during incubations at 0 degrees C. The same reactions were carried out with affinity-purified prepro-alpha-factor, and the same results were obtained with the exception that trigger factor was not required. When the various cytosolic factors were replaced with SRP, the addition of yeast microsomes after 15 h resulted in the translocation and processing (and glycosylation) of both proOmpA and prepro-alpha-factor. Pancreatic microsomes were also used in this type of assay, and it was found that proOmpA (but not prepro-alpha-factor) could be translocated when diluted out of urea. In this case, as with yeast microsomes, translocation competence was maintained by SRP. These results show that in addition to a recognition and targeting function, SRP can stabilize the translocation-competent conformation of pre-secretory proteins in vitro for translocation across eukaryotic membranes.     

Expression of acid phosphatase-beta-galactosidase hybrid proteins prevents translocation by depleting a soluble factor

We have shown that hybrid proteins composed of the yeast repressible acid phosphatase (PHO5) and bacterial beta-galactosidase (lacZ) interfere with secretion of native acid phosphatase (Wolfe, P. B. (1988) J. Biol. Chem. 263, 6908-6915). We now report that PHO5-LacZ hybrid proteins have a more general effect on secretion and prevent translocation of several secreted proteins. Translocation of both the mating pheromone alpha-factor and the vacuolar protease carboxypeptidase Y is partially blocked when PHO5-LacZ hybrids are expressed. Cell fractionation and protease sensitivity indicate that alpha-factor and carboxypeptidase Y accumulate in precursor form on the cytoplasmic surface of the endoplasmic reticulum. Indirect immunofluorescence with antibody directed against beta-galactosidase supports the localization of hybrid proteins to the endoplasmic reticulum. Analysis of the hybrid protein phenotype in vivo and in vitro suggests that the hybrid proteins deplete a soluble factor required for efficient translocation across the endoplasmic reticulum. First, a decrease in the expression of a hybrid protein in vivo decreases its effect on translocation. Second, an in vitro translation/translocation reaction, prepared from a hybrid-bearing strain, is deficient in its ability to translocate prepro-alpha-factor across yeast microsomal membranes. This deficiency is complemented by addition of cytosol prepared from wild type cells. Finally, the hybrid protein phenotype is shown to be independent of the requirement for SSA gene products.     

Expression of human salivary alpha-amylase gene in Saccharomyces cerevisiae and its secretion using the mammalian signal sequence

A cDNA fragment coding for human salivary alpha-amylase precursor was joined to the promoter of the Saccharomyces cerevisiae PHO5 gene, and the recombinant gene was inserted into a vector plasmid capable of autonomous replication in yeast. Yeast cells transformed with this recombinant plasmid synthesized about 5 X 10(5) molecules of the enzyme per cell when synthesis was induced by deprivation of inorganic phosphate and released about half of the synthesized enzyme into the medium. The enzyme is stable, and exhibited the same specific activity as alpha-amylase in human saliva. The amylase-producing yeast grew on starch and produced alcohol.     

Protein transport into mitochondria is conserved between plant and yeast species

Protein targeting into plant mitochondria was investigated by in vitro translocation experiments. The precursor of the mitochondrial F1-ATPase beta subunit from Nicotiana plumbaginifolia was synthesized in vitro, translocated to, processed, and assembled in purified Vicia faba mitochondria. Transport (but not binding) required a membrane potential and external nucleotides and was conserved among plant species. beta subunit precursors from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe were imported and correctly processed in plant mitochondria. This translocation used protease-sensitive components of the outer membrane. Conversely, the N. plumbaginifolia beta subunit precursor was efficiently translocated and cleaved in yeast mitochondria. However, a precursor for a chloroplast protein was not targeted to plant or yeast mitochondria. We conclude that the machinery for protein import into mitochondria is specific and conserved in plant and yeast organisms. These results are discussed in the context of a poly- or monophyletic origin of mitochondria.     

The nucleotide sequence of the yeast PHO5 gene: a putative precursor of repressible acid phosphatase contains a signal peptide.

The nucleotide sequence of the PHO5 gene of the yeast, Saccharomyces cerevisiae, which encodes repressible acid phosphatase (APase) was determined. Comparison of N-terminal amino acid sequence deduced from the nucleotide sequence with that of the purified repressible APase revealed the existence of a putative signal peptide in the precursor protein. The signal peptide was shown to contain 17 amino acid residues and its structural features were quite similar to those of higher eukaryotic and prokaryotic signal peptides. The nucleotide sequence of 5' and 3' noncoding flanking regions of the PHO5 gene are also discussed.     

Regulation of Repressible Acid Phosphatase by Cyclic Amp in SACCHAROMYCES CEREVISIAE

One of the cyr 1 mutants (cyr 1-2) in yeast produced low levels of adenylate cyclase and cyclic AMP at 25 degrees and was unable to derepress acid phosphatase. Addition of cyclic AMP to the cyr1-2 cultures elevated the level of repressible acid phosphatase activity. The bcy1 mutation, which suppresses the cyr1-2 mutation by allowing activity of a cyclic AMP-independent protein kinase, also allows acid phosphatase synthesis without restoring adenylate cyclase activity. The CYR3 mutant had structurally altered cyclic AMP-dependent protein kinase and was unable to derepress acid phosphatase. The cyr1 locus was different from pho2, pho4 and pho81, which were known to regulate acid phosphatase synthesis. Mutants carrying cyr1-2 and pho80, PHO81c, PHO82 or pho85 mutations, which confer constitutive synthesis of repressible acid phosphatase, produced acid phosphatase. The cyr1-2 mutant produced significantly low levels of invertase and alpha-D-glucosidase. These results indicated that cyclic AMP-dependent protein kinase exerts its function in the synthesis of repressible acid phosphatase and other enzymes.     

PHO5-LACZ hybrid proteins block translocation of native acid phosphatase in Saccharomyces cerevisiae

A set of protein hybrids composed of variable portions of the amino-terminal residues of the yeast phosphate-repressible acid phosphatase (product of PHO5) and an active fragment of bacterial beta-galactosidase has been constructed. When these PHO5-LACZ hybrids are expressed in a yeast strain carrying an intact chromosomal PHO5 gene, they show a size-dependent interference with the secretion of native acid phosphatase. Hybrid proteins containing approximately 50 residues of acid phosphatase do not affect secretion of native acid phosphatase. Hybrids containing greater than 200 residues of acid phosphatase reduce the amount of secreted acid phosphatase more than by 50%. The interference with secretion is specific for acid phosphatase. The hybrids do not affect secretion of invertase, and do not confer a growth-deficient phenotype on yeast. Both the hybrid proteins and acid phosphatase accumulate in non-glycosylated, membrane-bound forms which are sensitive to proteolysis from the cytoplasmic side of the membrane. The hybrids and accumulated acid phosphatase co-migrate on Percoll density gradients with markers of the endoplasmic reticulum, but not with markers of the Golgi or secretory vesicles. These results suggest that PHO5-LACZ hybrid proteins specifically block secretion of native acid phosphatase by interfering with enzyme after targeting but before translocation across the endoplasmic reticulum.     

Reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex in yeast: the acceptor Golgi compartment is defective in the sec23 mutant

Using either permeabilized cells or microsomes we have reconstituted the early events of the yeast secretory pathway in vitro. In the first stage of the reaction approximately 50-70% of the prepro-alpha-factor, synthesized in a yeast translation lysate, is translocated into the endoplasmic reticulum (ER) of permeabilized yeast cells or directly into yeast microsomes. In the second stage of the reaction 48-66% of the ER form of alpha-factor (26,000 D) is then converted to the high molecular weight Golgi form in the presence of ATP, soluble factors and an acceptor membrane fraction; GTP gamma S inhibits this transport reaction. Donor, acceptor, and soluble fractions can be separated in this assay. This has enabled us to determine the defective fraction in sec23, a secretory mutant that blocks ER to Golgi transport in vivo. When fractions were prepared from mutant cells grown at the permissive or restrictive temperature and then assayed in vitro, the acceptor Golgi fraction was found to be defective.