Active site mutations in yeast protein disulfide isomerase cause dithiothreitol sensitivity and a reduced rate of protein folding in the endoplasmic reticulum.

Aspects of protein disulfide isomerase (PDI) function have been studied in yeast in vivo. PDI contains two thioredoxin-like domains, a and a', each of which contains an active-site CXXC motif. The relative importance of the two domains was analyzed by rendering each one inactive by mutation to SGAS. Such mutations had no significant effect on growth. The domains however, were not equivalent since the rate of folding of carboxypeptidase Y (CPY) in vivo was reduced by inactivation of the a domain but not the a' domain. To investigate the relevance of PDI redox potential, the G and H positions of each CGHC active site were randomly mutagenized. The resulting mutant PDIs were ranked by their growth phenotype on medium containing increasing concentrations of DTT. The rate of CPY folding in the mutants showed the same ranking as the DTT sensitivity, suggesting that the oxidative power of PDI is an important factor in folding in vivo. Mutants with a PDI that cannot perform oxidation reactions on its own (CGHS) had a strongly reduced growth rate. The growth rates, however, did not correlate with CPY folding, suggesting that the protein(s) required for optimal growth are dependent on PDI for oxidation. pdi1-deleted strains overexpressing the yeast PDI homologue EUG1 are viable. Exchanging the wild-type Eug1p C(L/I)HS active site sequences for C(L/I)HC increased the growth rate significantly, however, further highlighting the importance of the oxidizing function for optimal growth.

Yeast carboxypeptidase Y vacuolar targeting signal is defined by four propeptide amino acids.

The amino-terminal propeptide of carboxypeptidase Y (CPY) is necessary and sufficient for targeting this glycoprotein to the vacuole of Saccharomyces cerevisiae. A 16 amino acid stretch of the propeptide was subjected to region-directed mutagenesis using randomized oligonucleotides. Mutations altering any of four contiguous amino acids, Gln-Arg-Pro-Leu, resulted in secretion of the encoded CPY precursor (proCPY), demonstrating that these residues form the core of the vacuolar targeting signal. Cells that simultaneously synthesize both wild-type and sorting-defective forms of proCPY efficiently sort and deliver only the wild-type molecule to the vacuole. These results indicate that the PRC1 missorting mutations are cis-dominant, implying that the mutant forms of proCPY are secreted as a consequence of failing to interact with the sorting apparatus, rather than a general poisoning of the vacuolar protein targeting system.

Functional differences in yeast protein disulfide isomerases.

PDI1 is the essential gene encoding protein disulfide isomerase in yeast. The Saccharomyces cerevisiae genome, however, contains four other nonessential genes with homology to PDI1: MPD1, MPD2, EUG1, and EPS1. We have investigated the effects of simultaneous deletions of these genes. In several cases, we found that the ability of the PDI1 homologues to restore viability to a pdi1-deleted strain when overexpressed was dependent on the presence of low endogenous levels of one or more of the other homologues. This shows that the homologues are not functionally interchangeable. In fact, Mpd1p was the only homologue capable of carrying out all the essential functions of Pdi1p. Furthermore, the presence of endogenous homologues with a CXXC motif in the thioredoxin-like domain is required for suppression of a pdi1 deletion by EUG1 (which contains two CXXS active site motifs). This underlines the essentiality of protein disulfide isomerase-catalyzed oxidation. Most mutant combinations show defects in carboxypeptidase Y folding as well as in glycan modification. There are, however, no significant effects on ER-associated protein degradation in the various protein disulfide isomerase-deleted strains.

Active-site residues of procarboxypeptidase Y are accessible to chemical modification.

The accessibility of the active-site cleft of procarboxypeptidase Y from Saccharomyces cerevisiae has been studied by chemical modifications of two specific amino-acid residues. Previous studies have shown that these residues, Cys-341 and Met-398 in the mature enzyme, are located in the S1 and S'1 substrate binding sites, respectively, of carboxypeptidase Y. We have found that these residues also in proCPY are accessible to modification with fairly bulky reagents and in the case of Met-398 the rate of modification is even faster than in carboxypeptidase Y. While the catalytic serine in the mature enzyme reacts with diisopropylfluorophosphate, this is not the case for procarboxypeptidase Y.

Functional properties of the two redox-active sites in yeast protein disulphide isomerase in vitro and in vivo.

Protein folding catalysed by protein disulphide isomerase (PDI) has been studied both in vivo and in vitro using different assays. PDI contains a CGHC active site in each of its two catalytic domains (a and a'). The relative importance of each active site in PDI from Saccharomyces cerevisiae (yPDI) has been analysed by exchanging the active-site cysteine residues for serine residues. The activity of the mutant forms of yPDI was determined quantitatively by following the refolding of bovine pancreatic trypsin inhibitor in vitro. In this assay the activity of the wild-type yPDI is quite similar to that of human PDI, both in rearrangement and oxidation reactions. However, while the a domain active site of the human enzyme is more active than the a'-site, the reverse is the case for yPDI. This prompted us to set up an assay to investigate whether the situation would be different with a native yeast substrate, procarboxypeptidase Y. In this assay, however, the a' domain active site also appeared to be much more potent than the a-site. These results were unexpected, not only because of the difference with human PDI, but also because analysis of folding of procarboxypeptidase Y in vivo had shown the a-site to be most important. We furthermore show that the apparent difference between in vivo and in vitro activities is not due to catalytic contributions from the other PDI homologues found in yeast.

Surprisingly high stability of barley lipid transfer protein, LTP1, towards denaturant, heat and proteases.

Barley LTP1 belongs to a large family of plant proteins termed non-specific lipid transfer proteins. The in vivo function of these proteins is unknown, but it has been suggested that they are involved in responses towards stresses such as pathogens, drought, heat, cold and salt. Also, the proteins have been suggested as transporters of monomers for cutin synthesis. We have analysed the stability of LTP1 towards denaturant, heat and proteases and found it to be a highly stable protein, which apparently does not denature at temperatures up to 100 degrees C. This high stability may be important for the biological function of LTP1.

Mutation of yeast Eug1p CXXS active sites to CXXC results in a dramatic increase in protein disulphide isomerase activity.

Protein disulphide isomerase (PDI) is an essential protein which is localized to the endoplasmic reticulum of eukaryotic cells. It catalyses the formation and isomerization of disulphide bonds during the folding of secretory proteins. PDI is composed of domains with structural homology to thioredoxin and with CXXC catalytic motifs. EUG1 encodes a yeast protein, Eug1p, that is highly homologous to PDI. However, Eug1p contains CXXS motifs instead of CXXC. In the current model for PDI function both cysteines in this motif are required for PDI-catalysed oxidase activity. To gain more insight into the biochemical properties of this unusual variant of PDI we have purified and characterized the protein. We have furthermore generated a number of mutant forms of Eug1p in which either or both of the active sites have been mutated to a CXXC sequence. To determine the catalytic capacity of the wild-type and mutant forms we assayed activity in oxidative refolding of reduced and denatured procarboxypeptidase Y as well as refolding of bovine pancreatic trypsin inhibitor. The wild-type protein showed very little activity, not only in oxidative refolding but also in assays where only isomerase activity was required. This was surprising, in particular since mutant forms of Eug1p containing a CXXC motif displayed activity close to that of genuine PDI. These results lead us to propose that general disulphide isomerization is not the main function of Eug1p in vivo.

Exchange of regions of the carboxypeptidase Y propeptide. Sequence specificity and function in folding in vivo.

The propeptide of carboxypeptidase Y from Saccharomyces cerevisiae is important for folding of the enzyme. Previous work [Ramos, C., Winther, J.R. & Kielland-Brandt, M. C. (1994) J. Biol. Chem. 269, 7006-7012] suggested that the sequences essential for in vivo folding were situated in the COOH-proximal third of the propeptide. Concentrating on this region we have investigated the functionality of propeptide variants. Using a random mutagenesis approach we found that two segments can be defined: one in which there is a fairly high tolerance for substitution with unrelated sequences and another that has a more strict requirement for sequence conservation. Nevertheless, an overall lack of requirement for propeptide sequence conservation was found by substitution of the carboxypeptidase Y propeptide with that of a highly divergent propeptide sequence from an otherwise similar carboxypeptidase from Candida albicans. This propeptide was partially functional when combined with carboxypeptidase Y. Analysis of the biosynthesis of the mutant forms of the zymogen showed that a fraction of the molecules proceeded from the endoplasmic reticulum with fairly rapid kinetics, while the rest was degraded.

Propeptide of carboxypeptidase Y provides a chaperone-like function as well as inhibition of the enzymatic activity.

The zymogen of the vacuolar carboxypeptidase Y from Saccharomyces cerevisiae was purified and characterized with respect to activation as well as refolding in vitro. The purified procarboxypeptidase Y has no detectable activity but can be efficiently activated by proteinase K from Tritirachium album. We used this method of activation as a tool for the investigation of refolding procarboxypeptidase Y in vitro. The proenzyme, denatured in 6 M guanidinium chloride, is renatured efficiently after dilution of the denaturant, whereas the mature enzyme regains little activity in the same procedure. Changes in intrinsic fluorescence reveal the mature enzyme to be considerably more stable than the proenzyme toward denaturation with guanidinium chloride. This suggests that the propeptide induces a metastable structure important for overcoming energy barriers that might otherwise obstruct a productive folding pathway. The relatively large number of charged amino acid residues and a high theoretical potential for alpha-helix formation in the carboxypeptidase Y propeptide suggest a structural similarity to a number of other propeptides and heat shock proteins.

Refolding of a carboxypeptidase Y folding intermediate in vitro by low-affinity binding of the proregion.

Efficient folding of carboxypeptidase Y is dependent on the presence of the proregion. Thus, denatured procarboxypeptidase Y, in contrast to the mature enzyme, refolds efficiently in vitro in low ionic strength buffers. Under these conditions denatured mature carboxypeptidase Y forms an inactive, soluble folding intermediate, which has been characterized in the present study. The inactive intermediate can be folded into the active enzyme at a low efficiency (5-10%) by the addition of 0.9 M ammonium sulfate. The refolding is accompanied by pronounced structural changes. As seen for other protease zymogens the isolated proregion from carboxypeptidase Y was found to stimulate refolding without covalent linkage to the mature part. However, the added proregion does not form a stable complex with the native enzyme and requires the presence of 0.9 M ammonium sulfate to exhibit its function. The proregion increases the yield of correctly folded enzyme, and kinetic analysis suggests that this is due to a reduction of the rate of nonproductive folding or aggregation. In addition, the proregion stabilizes carboxypeptidase Y toward thermoinactivation.

Requirement of the propeptide for in vivo formation of active yeast carboxypeptidase Y.

Deletions have been constructed in the region encoding the 91-amino acid propeptide of the vacuolar enzyme carboxypeptidase Y of Saccharomyces cerevisiae, and in vivo effects of these mutations on the intracellular transport of the mutant proenzymes have been examined. Deletions did not include the vacuolar targeting signal, and none of the mutated forms of procarboxypeptidase Y was found to be secreted. All deletions, however, resulted in a decreased rate of transport of the truncated proenzymes from the endoplasmic reticulum to the Golgi apparatus. Up to 29 residues close to the N terminus can be removed without completely eliminating transport of the mutated proenzymes to the vacuole. However, the C-terminal part of the propeptide contains elements which are essential, since two small deletions, of 9 and 15 residues, respectively, within this area resulted in loss of carboxypeptidase Y activity. This region is, however, not sufficient for efficient formation of active carboxypeptidase Y, since truncated precursors in which the vacuolar targeting signal was fused to the C-terminal part of the proregion did not give rise to active enzyme. Based on the results, we propose that the carboxypeptidase Y propeptide plays an essential role in guiding the proper folding of the protein in vivo and that many parts of the propeptide contribute, in an additive way, to this function.

Ligand recognition and domain structure of Vps10p, a vacuolar protein sorting receptor in Saccharomyces cerevisiae.

Vp10p is a receptor that sorts several different vacuolar proteins by cycling between a late Golgi compartment and the endosome. The cytoplasmic tail of Vps10p is necessary for the recycling, whereas the lumenal domain is predicted to interact with the soluble ligands. We have studied ligand binding to Vps10p by introducing deletions in the lumenal region. This region contains two domains with homology to each other. Domain 2 binds carboxypeptidase Y (CPY), proteinase A (PrA) and hybrids of these proteases with invertase. Moreover, we show that aminopeptidase Y (APY) is a ligand of Vps10p. The native proteases compete for binding to domain 2. Binding of CPY(156)-invertase or PrA(137)-invertase, on the other hand, do not interfere with binding of CPY to Vps10p. Furthermore, the Q24RPL27 sequence known to be important for vacuolar sorting of CPY, is of little importance in the Vps10p-dependent sorting of CPY-invertase. Apparently, domain 2 contains two different binding sites; one for APY, CPY and PrA, and one for CPY-invertase and PrA-invertase. The latter interaction seems not to be sequence specific, and we suggest that an unfolded structure in these ligands is recognized by Vps10p.

The pro region required for folding of carboxypeptidase Y is a partially folded domain with little regular structural core.

The pro region of carboxypeptidase Y (CPY) from yeast is necessary for the correct folding of the enzyme [Winther, J. R., & Sørensen P. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 9330-9334]. Using fluorescence, circular dichroism, and heteronuclear NMR analyses, it is demonstrated that the isolated pro region is a partially folded protein domain under the conditions where it is functional. It is characterized by a relatively high content of secondary structural elements but a very low content of defined tertiary structure. Although these characteristics are reminiscent of the compact denatured states that have been identified as intermediates in protein folding ("molten globules"), the pro region exhibits only very weak binding of the hydrophobic probe 1-anilino-8-naphthalenesulfonate, and it is resistant toward complete thermal unfolding. Altogether the data indicate an extremely flexible structure that has little regular structural core. It is proposed that the feature of a partially folded domain per se is important for the function of the pro region of CPY as a "co-translational chaperone".

Barley lipid transfer protein, LTP1, contains a new type of lipid-like post-translational modification.

In plants a group of proteins termed nonspecific lipid transfer proteins are found. These proteins bind and catalyze transfer of lipids in vitro, but their in vivo function is unknown. They have been suggested to be involved in different aspects of plant physiology and cell biology, including the formation of cutin and involvement in stress and pathogen responses, but there is yet no direct demonstration of an in vivo function. We have found and characterized a novel post-translational modification of the barley nonspecific lipid transfer protein, LTP1. The protein-modification bond is of a new type in which an aspartic acid in LTP1 is bound to the modification through what most likely is an ester bond. The chemical structure of the modification has been characterized by means of two-dimensional homo- and heteronuclear nuclear magnetic resonance spectroscopy as well as mass spectrometry and is found to be lipid-like in nature. The modification does not resemble any standard lipid post-translational modification but is similar to a compound with known antimicrobial activity.

Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein.

To visualize the formation of disulfide bonds in living cells, a pair of redox-active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2-fold decrease in the intrinsic fluorescence. Inter conversion between the two redox states could thus be followed in vitro as well as in vivo by non-invasive fluorimetric measurements. The 1.5 A crystal structure of the oxidized protein revealed a disulfide bond-induced distortion of the beta-barrel, as well as a structural reorganization of residues in the immediate chromophore environment. By combining this information with spectroscopic data, we propose a detailed mechanism accounting for the observed redox state-dependent fluorescence. The redox potential of the cysteine couple was found to be within the physiological range for redox-active cysteines. In the cytoplasm of Escherichia coli, the protein was a sensitive probe for the redox changes that occur upon disruption of the thioredoxin reductive pathway.

Production of a heterologous proteinase A by Saccharomyces kluyveri.

In order to evaluate the potential of Saccharomyces kluyveri for heterologous protein production, S. kluyveri Y159 was transformed with a S. cerevisiae-based multi-copy plasmid containing the S. cerevisiae PEP4 gene, which encodes proteinase A, under the control of its native promoter. As a reference, S. cerevisiae CEN.PK 113-5D was transformed with the same plasmid and the two strains were characterised in batch cultivations on glucose. The glucose metabolism was found to be less fermentative in S. kluyveri than in S. cerevisiae. The yield of ethanol on glucose was 0.11 g/g in S. kluyveri, compared to a yield of 0.40 g/g in S. cerevisiae. Overexpression of PEP4 led to the secretion of active proteinase A in both S. kluyveri and S. cerevisiae. The yield of active proteinase A during growth on glucose was found to be 3.6-fold higher in S. kluyveri than in the S. cerevisiae reference strain.

Autoactivation of proteinase A initiates activation of yeast vacuolar zymogens.

The Saccharomyces cerevisiae PEP4 gene encodes proteinase A, an aspartyl protease. pep4 mutants are defective in the activation of many vacuolar hydrolases, including proteinase B. We have expressed a pep4 mutation which directs the accumulation of pro-proteinase A with a defective active site. Co-expression with PEP4 leads to normal processing, i.e. the mutant zymogen is functional as a substrate for the maturation reaction in trans. We conclude that wild-type pro-proteinase A has the ability to mediate its own activation. Elimination of the co-expressed PEP4 gene did not effectively stop the processing of the mutant zymogen, owing to a strong, proteinase-B-dependent, phenotypic lag. In a proteinase-B-negative strain, processing of pro-proteinase A led to an active form of a higher molecular mass than the normal mature form.

Yeast carboxypeptidase Y requires glycosylation for efficient intracellular transport, but not for vacuolar sorting, in vivo stability, or activity.

Functions of the carbohydrate side chains of the yeast vacuolar enzyme carboxypeptidase Y (CPY) were investigated by removal, through site-directed mutagenesis, of the sequences which act as signals for N-linked glycosylation. The mutant forms of the enzyme were analysed with respect to activity and intracellular sorting, and the stabilities in vivo and in vitro were studied. It was found that carbohydrate was not important for accurate vacuolar targeting of CPY, but that the rate of transport of the unglycosylated CPY through the secretory pathway to the vacuole was reduced. Tunicamycin, which inhibits the formation of asparagine-linked glycosylation, had a similar effect on the transport of CPY at 23 degrees C. However, the absence of N-linked carbohydrate in general had the more dramatic result of blocking the transport of CPY altogether at an increased temperature (37 degrees C). The unglycosylated mutant CPY was not temperature sensitive for transport in the absence of tunicamycin. Analysis of mutant enzymes containing a single glycosyl residue at each of the four positions showed that the residue at position 87 was particularly important for transport. There was no decrease in the intracellular stability of the completely unglycosylated enzyme, and in vitro the rate of heat inactivation of this species was not increased.

Mutational analysis of the vacuolar sorting signal of procarboxypeptidase Y in yeast shows a low requirement for sequence conservation.

The core of the vacuolar targeting signal of yeast carboxypeptidase Y (CPY) is recognized by the receptor Vps10p and consists of four contiguous amino acid residues, Gln24-Arg-Pro-Leu27, near the amino terminus of the propeptide (Valls, L.A., Winther, J. R., and Stevens, T. H. (1990) J. Cell Biol. 111, 361-368; Marcusson, E. G., Horazdovsky, B. F., Cereghino, J. L., Gharakhanian, E., and Emr, S. D. (1994) Cell 77, 579-586). In order to determine the sequence specificity of the interaction with the sorting receptor, substitutions were introduced into this part of the propeptide by semirandom site-directed mutagenesis. The efficiency of vacuolar sorting by the mutants was determined by immunoprecipitation of CPY from pulse-labeled cells. It was found that amino acid residues Gln24 and Leu27 were the most important ones. While it appears that Gln24 is essential for proper function, Leu27 can be exchanged with the other hydrophobic amino acid residues, isoleucine, valine, and phenylalanine. Tolerance toward various substitutions for Arg25 is fairly high, while substitution of Pro26 for uncharged amino acid residues also resulted in only weak missorting. In addition to the low requirement for sequence conservation, the position of the targeting element relative to the amino terminus of the propeptide was analyzed and found not to be critical.