Synthesis of glia-derived nexin in yeast.

Glia-derived nexin (GDN) is a 43-kDa glycoprotein isolated from rat glioma cell cultures. It promotes neurite extension in cultures of neuroblastoma cells and chick sympathetic neurons. Moreover, GDN is a potent serine protease inhibitor (serpin), belonging to the family of protease nexins. We report here the expression of rat GDN in the Saccharomyces cerevisiae strain GRF18 under the control of the PHO5 promoter. We describe the purification of more than 6 mg total GDN from the cellular extract of 1 liter of yeast culture. The amino acid composition and the sequence of CNBr-fragments of the recombinant protein correlate with the values deduced from the rat GDN cDNA. We provide evidence that the recombinant GDN has exactly the same properties as the glioma-derived protein with respect to its protease-inhibitory activity and its ability to promote the extension of neurites from neuroblastoma cells. The large amounts of recombinant protein obtained from this expression system will allow further biochemical and physiological analysis of GDN and of the serpins in general.

Novel BNIP1 variants and their interaction with BCL2 family members.

By PCR and EST database searches we have identified three novel BNIP1 splice variants, and found that one of them, BNIP1-b, contains a highly conserved BH3 domain. The BNIP1 gene has been assigned to chromosome 5q33-34. Using in vitro protein-protein interaction assays, all BNIP1 variants were shown to interact with BCL2 and also with BCL2L1 (previously Bcl-xL). These interactions are BH3-independent. Furthermore, the BNIP1 variants cannot interact with BAX. The results suggest that the BNIP1 variants are novel members of the BCL2 family but function through a mechanism different from other BH3-only members.

Production of disulfide-linked hirudin dimer by in vitro folding.

A simple process of in vitro folding has been developed for the preparation of hirudin dimer. A variant of recombinant hirudin with Asp33 replaced by Cys was expressed in yeast and isolated by HPLC. Crude Cys33-hirudin contains heterogeneous products that are made of one species of primary sequence. They were together reduced/denatured, and allowed to re-fold in the sodium bicarbonate buffer (pH 8.3) alone. Active, homogeneous Cys33-hirudin monomer folded spontaneously with a first order rate constant of 0.05 +/- 0.01 min-1, followed by the oxidation of two Cys33 to produce the pure dimer. The folding yield was 90%. On an equal weight basis, both Cys33-hirudin monomer and the dimer exhibit thrombin inhibitory activity comparable to that of wild-type hirudin. Due to the presence of an extra cysteine, the folding of active hirudin monomer (formation of three native disulfides) was accelerated by at least 12-fold.

Mutations in FL5.12 cells conferring resistance to apoptosis induced by interleukin-3 deprivation.

In an attempt to dissect the signal pathway in which Bax increases cellular responses to apoptotic stimuli and leads to the activation of the caspase cascade, we mutated FL5.12 Bax CL16 cells with a chemical mutagen. In this report we characterize two mutant clones, FL5.12 ms1 and m3. Both clones are resistant to IL-3 deprivation exhibiting no changes in mitochondrial membrane potential, annexin V and propidium iodide binding. FL5.12 ms1 is also resistant to staurosporine and anti-Fas antibody. In cell fusion experiments m3 behaves genetically dominant and ms1 is recessive. The results suggest that m3 has a mutation in a specific function upstream of Bax, while ms1 has a mutational block in the general pathway downstream of the 'Bcl-2 checkpoint'.

Expression of rat 5 alpha-reductase in Saccharomyces cerevisiae.

Dihydrotestosterone (DHT) is the principle androgen in certain tissues such as the prostate. DHT is formed from testosterone by the NADPH-dependent enzyme 5 alpha-reductase (5AR). In this paper we report the expression of catalytically active steroid 5AR from the rat in Saccharomyces cerevisiae. A full length cDNA coding for 5AR was isolated from a rat liver cDNA library and fixed in frame to the signal sequence of yeast acid phosphatase. A constitutive short promoter fragment of the acid phosphatase gene (PHO5) and the PHO5 transcriptional terminator were added and the expression cassette ligated into the yeast 2 mu vector pDP34. S. cerevisiae transformed with the 5AR expression plasmid pDP34/PHO5AR exhibited about 100-fold more activity per gram wet weight than rat prostate.

Refolding, isolation and characterization of crystallizable human interferon-alpha 8 expression in Saccharomyces cerevisiae.

Human interferon-alpha 8 was expressed in Saccharomyces cerevisiae and found to accumulate intracellularly in an insoluble form. The protein could be solubilized and converted to a biologically active form with high yield by a denaturation-refolding procedure. The interferon-alpha 8 was further purified to apparent homogeneity by copper-chelate affinity chromatography and anion-exchange chromatography and fully characterized by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequence analysis, mass spectrometry, circular-dichroism (CD) spectroscopy and specific activity. Secondary-structure predictions from CD spectroscopy indicate that the molecule is correctly folded. Peptide mapping supported the correct sequence and the expected disulfide-bridge connectivity. The purified protein elutes on reversed-phase high-pressure liquid chromatography (RP-HPLC) as two peaks. Electrospray mass spectrometry and N-terminal sequence analysis of the minor component indicated the existence of an N-terminal acetyl group for the later eluting HPLC-component. In anti-viral assays, the two IFN forms were equally active. Hexagonal crystals of this interferon preparation could be obtained. On the basis of the electrophoretic mobility, HPLC profile, and biological activity assay, the crystalline material was judged to be identical to the uncrystallized interferon. Interferon in crystallized form was found to be stable for up to 24 months and, therefore, could be used for long-term storage, particularly for material intended for clinical use.

Colicin E3: a unique endoribonuclease.

The question of whether or not a cellular ribonuclease is involved in the cleavage of 16S ribosomal RNA by colicin E3 was investigated. For this purpose ribosomes from strains devoid of some ribonucleases or ribosomes in which ribonucleases had been inactivated by heat or removed by extensive washings were used for the colicin reaction. Since the 16S RNA of all these different ribosomes, and even of the most extensively washed ribosomes, was cleaved by colicin E3, it is suggested that cellular ribonucleases are not involved in colicin E3 action. Thus, colicin E3 seems to be a unique endoribonuclease.

Redistribution of Bax from cytosol to membranes is induced by apoptotic stimuli and is an early step in the apoptotic pathway.

It is known that overexpression of Bax accelerates apoptosis, but the biochemical mechanism of the signal transduction from Bax to downstream targets has still not been fully determined. In the present study, we demonstrate that upon apoptotic stimuli, Bax moves from the cytosolic to the membrane fraction. The redistribution of Bax is not inhibited by a caspase inhibitor, zVAD-fmk, which blocks caspase-3 activity and prevents apoptosis in vivo. A FL5.12 Bax CL16 mutant cell, ms3, which is resistant to apoptosis induced by staurosporine, retains the activity of Bax redistribution but shows no caspase-3 activity. Our results revealed that Bax accumulation on membranes precedes caspase-3 activation, indicating that redistribution of Bax is an early event in apoptosis. These results suggest that Bax may be functionally significant in the regulation of caspase-3 activation.

Transcriptional studies on yeast SEC genes provide no evidence for regulation at the transcriptional level.

A number of proteins have been identified as components of the secretory pathway of Saccharomyces cerevisiae (SEC gene products). However, very little is known about the expression of these components and their regulation at the transcriptional level. In this study yeast cells were exposed to conditions that changed the secretory activity of the cells. The conditions analysed include the different stages of the cell cycle, overexpression of secretory proteins, and block of secretion and endocytosis. The effect of these conditions on the transcriptional expression levels of a number of SEC genes (SAR1, SEC1, SEC14, SEC17, SEC18, SEC23, SEC62, YPT1) was analysed. In summary, no major changes in transcriptional expression levels could be detected. From these results we conclude that the components of the secretory pathway are expressed constitutively and that no general regulation of transcription exists, that could adjust the expression level of the SEC genes to the secretory activity of the cells.

Involvement of the mature domain in the in vitro maturation of Bacillus subtilis precursor 5S ribosomal RNA.

A precursor of 5S ribosomal RNA from Bacillus subtilis (p5A rRNA, 179 nucleotides in length) is cleaved by RNase M5, a specific maturation endonuclease which releases the mature 5S rRNA (m5, 116 nucleotides) and precursor fragments derived from the 5' (21 nucleotides) and 3' (42 nucleotides) termini of p5A rRNA. Previous results (Meyhack, B., et al. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3045) led to the conclusion that recognition elements in potential RNase M5 substrates mainly reside in the mature moiety of the precursor. Limited digestion of p5A rRNA with RNase T1 permitted the isolation of a number of test substrates which contained both precursor-specific segments and were unaltered in the immediate vicinity of the cleavage sites, but which differed in that more or less extensive regions of the mature moiety of the p5A rRNA were deleted. Tests of the capacity of these partial molecules to serve as substrates for RNase M5 indicate clearly that the enzyme recognizes the overall conformation of potential substrates, neglecting only the double-helical "prokaryotic loop" (Fox, G.E., & Woese, C.R. (1975) Nature (London) 256, 505).

Recognition of local nucleotide conformation in contrast to sequence by a rRNA processing endonuclease.

RNase M5 of Bacillus subtilis cleaves twice in a double-helical region of a 179-nucleotide precursor of 5S rRNA to yield mature 5S rRNA (116 nucleotides) plus fragments (21 and 42 nucleotides) derived from both termini. Previous experiments had shown that the major recognition elements for the highly specific RNase M5 are in the mature domain of the precursor. However, one precursor residue, a G adjacent to the 5' cleavage site, significantly enhances the rate of its own cleavage as well as that of the 3' precursor fragment, so it must be an important component of the features recognized by the enzyme. This G residue is opposed in the helical substrate region to a C residue, which is at the 3' terminus of the mature domain, presenting the question of whether RNase M5 specifically contacts the cleavage site on the basis of nucleotide sequence (the G residue per se) or on the basis of more general aspects of helical conformation. We tested these alternatives by fabricating partially synthetic test substrates for RNase M5. Experiments were performed on 5' and 3' half-molecules derived from mature 5S rRNA. The 3'-terminal C was removed by periodate oxidation and beta elimination and replaced in a T4 RNA ligase condensation with each of the four mononucleoside bisphosphates. Artificial "precursor" segments containing each of the four nucleotides adjacent to the 5' cleavage site were added to the 5' terminus of the 5S rRNA half-molecule. We then annealed the modified half-molecules to yield test substrates containing all permutations of complementary in contrast to noncomplementary nucleotides at the cleavage site. The susceptibilities of these test substrates show that conformation, not sequence, is the important feature in the locale of the cleaved bonds.

The influence of GAP promoter variants on hirudin production, average plasmid copy number and cell growth in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has been engineered to synthesize and secrete desulfato-hirudin (hirudin), a thrombin inhibitor from the leech Hirudo medicinalis. The synthetic gene coding for hirudin was expressed constitutively under the control of four size-variants of the yeast glyceraldehyde-3-phosphate dehydrogenase promoter (GAP) and cloned into a 2 mu based multicopy yeast vector. The constitutive action of the four promoter variants was confirmed by demonstrating that the expression and secretion of hirudin is growth-related. The different efficiencies of the promoter variants not only affected hirudin expression but also led to changes in several cellular parameters, such as cell growth, average plasmid copy number and plasmid stability. The observed changes show that yeast cells establish a specific equilibrium for each promoter variant. We conclude, that the adjustment of cellular parameters in response to the expression levels of a heterologous protein is regulated by two counteracting selective forces: (1) the need for complementation of the auxotrophic host marker by the plasmid-encoded selection gene which, in the case of dLEU2, requires several plasmid copies; and (2) a selective advantage of cells with a lower copy number enabling them to escape the burden of heterologous protein production.

Involvement of precursor-specific segments in the in vitro maturation of Bacillus subtilis precursor 5S ribosomal RNA.

In vitro maturation of precursor 5S ribosomal RNA (p5A) from Bacillus subtilis effected by RNase M5 yields mature 5S RNA (m5, 116 nucleotides), and 3' precursor-specific segment (42 nucleotides), and a 5' precursor-specific segment (21 nucleotides) (Sogin, M.L., Pace, B., and Pace, N.R. (1977), J. Biol. Chem. 252, 1350). Limited digestion of p5A with RNase T2 introduces a single scission at position 60 of the molecule; m5 is cleaved at the corresponding nucleotide residue. The complementary "halves" of the molecules could be isolated from denaturing polyacrylamide gels. The isolated fragments of p5A are not substrates for RNase M5, suggesting that some recognition elements can be utilized by RNase M5 only when presented in double-helical form. In exploring the involvement of the precursor-specific segments in the RNase M5-p5A interaction, substrate molecules lacking the 3' or 5' precursor-specific segment were constructed by reannealing complementary "halves" from p5A and m5 RNA. The artificial substrate lacking the 5'-terminal precursor segment was cleaved very much more slowly than the lacking t' segment; the 5' precursor-specific segment therefore contains one or more components recognized by RNase M5 during its interaction with the p5A substrate.

Two yeast acid phosphatase structural genes are the result of a tandem duplication and show different degrees of homology in their promoter and coding sequences.

We have cloned the structural genes for a regulated ( PHO5 ) and a constitutive ( PHO3 ) acid phosphatase from yeast by transformation and complementation of a yeast pho3 , pho5 double mutant. Both genes are located on a 5.1-kb BamHI fragment. The cloned genes were identified on the basis of genetic evidence and by hybrid selection of mRNA coupled with in vitro translation and immunoprecipitation. Subcloning of partial Sau3A digests and functional in vivo analysis by transformation together with DNA sequence analysis showed that the two genes are oriented in the order (5') PHO5 , PHO3 (3'). While the nucleotide sequences of the two coding regions are quite similar, the putative promoter regions show a lower degree of sequence homology. Partly divergent promoter sequences may explain the different regulation of the two genes.

Precursor-specific nucleotide sequences can govern RNA folding.

An immediate precursor of 5S ribosomal RNA (rRNA) from Bacillus subtilis has 21 and 42 nucleotide precursor-specific segments associated with its 5' and 3' termini, respectively. On the basis of its nucleotide sequence, predicted secondary structure and location in the rRNA transcriptional unit, the 3' precursor element apparently functions during the termination of transcription. A portion of the 5' precursor element is shown to facilitate the native folding of the mature domain of the precursor. Precursor 5S rRNA molecules which lack the 5' terminal 8-9 nucleotides of the 5' precursor elements were fabricated. These abbreviated constructs assume a non-native conformation, as revealed by their behavior during polyacrylamide gel electrophoresis. The aberrant conformation is evidently forced upon the abbreviated constructs by the residual 5' precursor sequence, since its removal by the maturation endonuclease RNAase M5 precipitates the reordering of the mature domain into its native conformation. Inspection of the nucleotide sequence of the 5S precursor suggested the nature of the conformational aberration, and gel electrophoresis analyses of limited nuclease digests of end-labeled precursors in the native and aberrant conformations are consistent with the derived model. We conclude taht the 5' terminal six nucleotides in the intact 5S precursor assist in the folding of the mature domain by forming a base-paired duplex with neighboring nucleotides, thereby preventing that adjacent sequence from engendering the abnormal conformation. The involvement of precursor-specific sequences and conformational dynamics in RNA function are discussed.

C-terminal proteolytic degradation of recombinant desulfato-hirudin and its mutants in the yeast Saccharomyces cerevisiae.

The potent thrombin inhibitor hirudin variant 1, originally isolated from the leech Hirudo medicinalis, was expressed in Saccharomyces cerevisiae under the control of a truncated glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter fragment. Fusion of the yeast acid phosphatase (PHO5) signal sequence to the hirudin gene led to quantitative secretion of recombinant desulfato-hirudin variant 1 (r-hirudin) into the extracellular medium in a growth-dependent manner. In comparison to the genuine molecule, r-hirudin lacks the sulfate group at the Tyr in position 63. Besides the full-length protein of 65 amino acids (hir65), chemical analysis revealed the presence mainly of two derivatives lacking the last amino acid Gln (hir64) or the penultimate Leu (hir63) in addition. When expressing r-hirudin in mutant strains defective in all but one of the three major known carboxypeptidases, it turned out that the vacuolar carboxypeptidase yscY as well as the alpha-factor precursor-processing carboxypeptidase, ysc alpha, participate in the C-terminal degradation of r-hirudin. Direct involvement of yscY and ysc alpha was confirmed by sequential disruption of their structural genes PRC1 and KEX1, respectively. Disruption of PRA1, coding for the yscY-processing proteinase yscA, also abolished yscY-mediated C-terminal r-hirudin degradation, but clearly reduced the overall expression yield. Since ysc alpha is described to be highly specific for basic amino acids which are not present at the C-terminus of r-hirudin, a series of r-hirudin mutants with changes in the C-terminal amino acids were constructed and analysed for ysc alpha-mediated and yscY-mediated degradation. Chromatographic analysis of the expression products confirmed the preference of ysc alpha for basic amino acids, although Tyr, Leu and Gln were also hydrolysed. It could further be concluded that ysc alpha might also be responsible for the C-terminal degradation of recombinant atrial natriuretic factor and epidermal growth factor expressed in yeast.

Structural basis of BFL-1 for its interaction with BAX and its anti-apoptotic action in mammalian and yeast cells.

BFL-1 is the smallest member of the BCL-2 family and has been shown to retard apoptosis in various cell lines. However, the structural basis for its function remains unclear. Molecular modeling showed that BFL-1 could have a similar core structure as BCL-xL, consisting of seven alpha helices, although both proteins share only the conserved BCL-2 homology domains (BH1 and BH2 domains), but otherwise have very limited sequence homology, particularly in the N-terminal region. We demonstrated in the yeast two-hybrid system that BFL-1 interacts strongly with human BAX but is not able to form homodimers nor to interact with human BCL-2 or BCL-xL. Overexpression experiments in REF52 rat fibroblasts showed that BFL-1 conferred increased resistance to apoptosis induced by serum deprivation. BFL-1 had also the ability to neutralize BAX lethality in yeast. BAX requires the BH3 domain for interaction with BFL-1. However, the minimal region of BFL-1 for the interaction with BAX in coimmunoprecipitation experiments was not sufficient to protect cells from apoptosis. Further examination of BFL-1 and several other anti-apoptotic proteins suggests a more general type of structure based on structural motifs, i.e. a hydrophobic pocket for the binding of proapoptotic proteins, rather than extended sequence homologies.