Cloning and functional expression of a cDNA encoding a pheromone gland-specific acyl-CoA Δ11-desaturase of the cabbage looper moth, Trichoplusia ni

Desaturation of coenzyme-A esters of saturated fatty acids is a common feature of sex pheromone biosynthetic pathways in the Lepidoptera. The enzymes that catalyze this step share several biochemical properties with the ubiquitous acyl-CoA Δ9-desaturases of animals and fungi, suggesting a common ancestral origin. Unlike metabolic acyl-CoA Δ9-desaturases, pheromone desaturases have evolved unusual regio- and stereoselective activities that contribute to the remarkable diversity of chemical structures used as pheromones in this large taxonomic group. In this report, we describe the isolation of a cDNA encoding a pheromone gland desaturase from the cabbage looper moth, Trichoplusia ni, a species in which all unsaturated pheromone products are produced via a Δ11Z-desaturation mechanism. The largest ORF of the ≈1,250-bp cDNA encodes a 349-aa apoprotein (PDesat-Tn Δ11Z) with a predicted molecular mass of 40,240 Da. Its hydrophobicity profile is similar overall to those of rat and yeast Δ9-desaturases, suggesting conserved transmembrane topology. A 182-aa core domain delimited by conserved histidine-rich motifs implicated in iron-binding and catalysis has 72 and 58% similarity (including conservative substitutions) to acyl-CoA Δ9Z-desaturases of rat and yeast, respectively. Northern blot analysis revealed an ≈1,250-nt PDesat-Tn Δ11Z mRNA that is consistent with the spatial and temporal distribution of Δ11-desaturase enzyme activity. Genetic transformation of a desaturase-deficient strain of the yeast Saccharomyces cerevisiae with an expression plasmid encoding PDesat-Tn Δ11Z resulted in complementation of the strain’s fatty acid auxotrophy and the production of Δ11Z-unsaturated fatty acids.

A yeast genetic system for selecting small molecule inhibitors of protein–protein interactions in nanodroplets

Cellular processes are mediated by complex networks of molecular interactions. Dissection of their role most commonly is achieved by using genetic mutations that alter, for example, protein–protein interactions. Small molecules that accomplish the same result would provide a powerful complement to the genetic approach, but it generally is believed that such molecules are rare. There are several natural products, however, that illustrate the feasibility of this approach. Split-pool synthesis now provides a simple mechanical means to prepare vast numbers of complex, even natural product-like, molecules individually attached to cell-sized polymer beads. Here, we describe a genetic system compatible with split-pool synthesis that allows the detection of cell-permeable, small molecule inhibitors of protein–protein interactions in 100- to 200-nl cell culture droplets, prepared by a recently described technique that arrays large numbers of such droplets. These “nanodroplets” contain defined media, cells, and one or more beads containing ≈100 pmol of a photoreleasable small molecule and a controlled number of cells. The engineered Saccharomyces cerevisiae cells used in this study express two interacting proteins after induction with galactose whose interaction results in cell death in the presence of 5-fluoroorotic acid (inducible reverse two-hybrid assay). Disruption of the interaction by a small molecule allows growth, and the small molecule can be introduced into the system hours before induction of the toxic interaction. We demonstrate that the interaction between the activin receptor R1 and the immunophilin protein FKBP12 can be disrupted by the small molecule FK506 at nanomolar concentrations in nanodroplets. This system should provide a general method for selecting cell-permeable ligands that can be used to study the relevance of protein–protein interactions in living cells or organisms.

Histones H3 and H4 are components of upstream activation factor required for the high-level transcription of yeast rDNA by RNA polymerase I

RNA polymerase I (Pol I) transcription in the yeast Saccharomyces cerevisiae is greatly stimulated in vivo and in vitro by the multiprotein complex, upstream activation factor (UAF). UAF binds tightly to the upstream element of the rDNA promoter, such that once bound (in vitro), UAF does not readily exchange onto a competing template. Of the polypeptides previously identified in purified UAF, three are encoded by genes required for Pol I transcription in vivo: RRN5, RRN9, and RRN10. Two others, p30 and p18, have remained uncharacterized. We report here that the N-terminal amino acid sequence, its mobility in gel electrophoresis, and the immunoreactivity of p18 shows that it is histone H3. In addition, histone H4 was found in UAF, and myc-tagged histone H4 could be used to affinity-purify UAF. Histones H2A and H2B were not detectable in UAF. These results suggest that histones H3 and H4 probably account for the strong binding of UAF to DNA and may offer a means by which general nuclear regulatory signals could be transmitted to Pol I.

Interactions of the chaperone Hsp104 with yeast Sup35 and mammalian PrP

[PSI+] is a genetic element in yeast for which a heritable change in phenotype appears to be caused by a heritable change in the conformational state of the Sup35 protein. The inheritance of [PSI+] and the physical state of Sup35 in vivo depend on the protein chaperone Hsp104 (heat shock protein 104). Although these observations provide a strong genetic argument in support of the “protein-only” or “prion” hypothesis for [PSI+], there is, as yet, no direct evidence of an interaction between the two proteins. We report that when purified Sup35 and Hsp104 are mixed, the circular dichroism (CD) spectrum differs from that predicted by the addition of the proteins’ individual spectra, and the ATPase activity of Hsp104 is inhibited. Similar results are obtained with two other amyloidogenic substrates, mammalian PrP and β-amyloid 1-42 peptide, but not with several control proteins. With a group of peptides that span the PrP protein sequence, those that produced the largest changes in CD spectra also caused the strongest inhibition of ATPase activity in Hsp104. Our observations suggest that (i) previously described genetic interactions between Hsp104 and [PSI+] are caused by direct interaction between Hsp104 and Sup35; (ii) Sup35 and PrP, the determinants of the yeast and mammalian prions, respectively, share structural features that lead to a specific interaction with Hsp104; and (iii) these interactions couple a change in structure to the ATPase activity of Hsp104.

Selection of peptides that functionally replace a zinc finger in the Sp1 transcription factor by using a yeast combinatorial library

We have developed a strategy for the identification of peptides able to functionally replace a zinc finger domain in a transcription factor. This strategy could have important ramifications for basic research on gene regulation and for the development of therapeutic agents. In this study in yeast, we expressed chimeric proteins that included a random peptide combinatorial library in association with two zinc finger domains and a transactivating domain. The library was screened for chimeric proteins capable of activating transcription from a target sequence in the upstream regulatory regions of selectable or reporter genes. In a screen of approximately 1.5 × 107 transformants we identified 30 chimeric proteins that exhibited transcriptional activation, some of which were able to discriminate between wild-type and mutant DNA targets. Chimeric library proteins expressed as glutathione S-transferase fusions bound to double-stranded oligonucleotides containing the target sequence, suggesting that the chimeras bind directly to DNA. Surprisingly, none of the peptides identified resembled a zinc finger or other well-known transcription factor DNA binding domain.

The crystal structure of the nitrogen regulation fragment of the yeast prion protein Ure2p

The yeast nonchromosomal gene [URE3] is due to a prion form of the nitrogen regulatory protein Ure2p. It is a negative regulator of nitrogen catabolism and acts by inhibiting the transcription factor Gln3p. Ure2p residues 1–80 are necessary for prion generation and propagation. The C-terminal fragment retains nitrogen regulatory activity, albeit somewhat less efficiently than the full-length protein, and it also lowers the frequency of prion generation. The crystal structure of this C-terminal fragment, Ure2p(97–354), at 2.3 Å resolution is described here. It adopts the same fold as the glutathione S-transferase superfamily, consistent with their sequence similarity. However, Ure2p(97–354) lacks a properly positioned catalytic residue that is required for S-transferase activity. Residues within this regulatory fragment that have been indicated by mutational studies to influence prion generation have been mapped onto the three-dimensional structure, and possible implications for prion activity are discussed.

Molecular cloning of a cytochrome P450 taxane 10β-hydroxylase cDNA from Taxus and functional expression in yeast

The early steps in the biosynthesis of Taxol involve the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene followed by cytochrome P450-mediated hydroxylation at C5, acetylation of this intermediate, and a second cytochrome P450-dependent hydroxylation at C10 to yield taxadien-5α-acetoxy-10β-ol. Subsequent steps of the pathway involve additional cytochrome P450 catalyzed oxygenations and CoA-dependent acylations. The limited feasibility of reverse genetic cloning of cytochrome P450 oxygenases led to the use of Taxus cell cultures induced for Taxol production and the development of an approach based on differential display of mRNA-reverse transcription-PCR, which ultimately provided full-length forms of 13 unique but closely related cytochrome P450 sequences. Functional expression of these enzymes in yeast was monitored by in situ spectrophotometry coupled to in vivo screening of oxygenase activity by feeding taxoid substrates. This strategy yielded a family of taxoid-metabolizing enzymes and revealed the taxane 10β-hydroxylase as a 1494-bp cDNA that encodes a 498-residue cytochrome P450 capable of transforming taxadienyl acetate to the 10β-hydroxy derivative; the identity of this latter pathway intermediate was confirmed by chromatographic and spectrometric means. The 10β-hydroxylase represents the initial cytochrome P450 gene of Taxol biosynthesis to be isolated by an approach that should provide access to the remaining oxygenases of the pathway.

Characterization and mutational analysis of yeast Dbp8p, a putative RNA helicase involved in ribosome biogenesis

RNA helicases of the DEAD box family are involved in almost all cellular processes involving RNA molecules. Here we describe functional characterization of the yeast RNA helicase Dbp8p (YHR169w). Our results show that Dbp8p is an essential nucleolar protein required for biogenesis of the small ribosomal subunit. In vivo depletion of Dbp8p resulted in a ribosomal subunit imbalance due to a deficit in 40S ribosomal subunits. Subsequent analyses of pre-rRNA processing by pulse–chase labeling, northern hybridization and primer extension revealed that the early steps of cleavage of the 35S precursor at sites A1 and A2 are inhibited and delayed at site A0. Synthesis of 18S rRNA, the RNA moiety of the 40S subunit, is thereby blocked in the absence of Dbp8p. The involvement of Dbp8p as a bona fide RNA helicase in ribosome biogenesis is strongly supported by the loss of Dbp8p in vivo function obtained by site-directed mutagenesis of some conserved motifs carrying the enzymatic properties of the protein family.

Excision of 8-oxoguanine within clustered damage by the yeast OGG1 protein

Clustered damages are formed in DNA by ionising radiation and radiomimetic anticancer agents and are thought to be biologically severe. 7,8-dihydro-8-oxoguanine (8-oxoG), a major DNA damage resulting from oxidative attack, is highly mutagenic leading to a high level of G·C→T·A transversions if not previously excised by OGG1 DNA glycosylase/AP lyase proteins in eukaryotes. However, 8-oxoG within clustered DNA damage may present a challenge to the repair machinery of the cell. The ability of yeast OGG1 to excise 8-oxoG was determined when another type of damage [dihydrothymine, uracil, 8-oxoG, abasic (AP) site or various types of single-strand breaks (SSBs)] is present on the complementary strand 1, 3 or 5 bases 5′ or 3′ opposite to 8-oxoG. Base damages have little or no influence on the excision of 8-oxoG by yeast OGG1 (yOGG1) whereas an AP site has a strong inhibitory effect. Various types of SSBs, obtained using either oligonucleotides with 3′- and 5′-phosphate termini around a gap or through conversion of an AP site with either endonuclease III or human AP endonuclease 1, strongly inhibit excision of 8-oxoG by yOGG1. Therefore, this large inhibitory effect of an AP site or a SSB may minimise the probability of formation of a double-strand break in the processing of 8-oxoG within clustered damages.

Translational control of human p53 expression in yeast mediated by 5′-UTR–ORF structural interaction

We have expressed human p53 cDNA in the yeast Saccharomyces cerevisiae and shown that the level of production and the length of the p53 protein depends on the presence of untranslated mRNA regions (UTRs). The expression of the ORF alone leads to a p53 protein of correct size (53 kDa) that accumulates to high levels, concomitantly with the presence of a small amount of a p40 protein (40 kDa). However, when either the entire 5′-UTR and a part of the 3′- or 5′-UTR alone is used, this leads to the production of small amounts of the 40 kDa truncated form only. The p40 protein corresponds to a truncated form of p53 at the C-terminal extremity since it reacts only with a monoclonal antibody recognising the N-terminal epitope. This effect on the amount and length of p53 protein had no correlation at the mRNA level, suggesting that translational control probably occurs through the 5′-UTR. We propose a model of structural interaction between this UTR and a part of the ORF mRNA for the regulation of p53 expression in this heterologous context.

Identification of partial loss of function p53 gene mutations utilizing a yeast-based functional assay

Missense mutations within the central DNA binding region of p53 are the most prevalent mutations found in human cancer. Numerous studies indicate that ‘hot-spot’ p53 mutants (which comprise ∼30% of human p53 gene mutations) are largely devoid of transcriptional activity. However, a growing body of evidence indicates that some non-hot-spot p53 mutants retain some degree of transcriptional activity in vivo, particularly against strong p53 binding sites. We have modified a previously described yeast-based p53 functional assay to readily identify such partial loss of function p53 mutants. We demonstrate the utility of this modified p53 functional assay using a diverse panel of p53 mutants.

The dhp1+ gene, encoding a putative nuclear 5′→3′ exoribonuclease, is required for proper chromosome segregation in fission yeast

The Schizosaccharomyces pombe dhp1+ gene is an ortholog of the Saccharomyces cerevisiae RAT1 gene, which encodes a nuclear 5′→3′ exoribonuclease, and is essential for cell viability. To clarify the cellular functions of the nuclear 5′→3′ exoribonuclease, we isolated and characterized a temperature-sensitive mutant of dhp1 (dhp1-1 mutant). The dhp1-1 mutant showed nuclear accumulation of poly(A)+ RNA at the restrictive temperature, as was already reported for the rat1 mutant. Interestingly, the dhp1-1 mutant exhibited aberrant chromosome segregation at the restrictive temperature. The dhp1-1 cells frequently contained condensed chromosomes, most of whose sister chromatids failed to separate during mitosis despite normal mitotic spindle elongation. Finally, chromosomes were displaced or unequally segregated. As similar mitotic defects were also observed in Dhp1p-depleted cells, we concluded that dhp1+ is required for proper chromosome segregation as well as for poly(A)+ RNA metabolism in fission yeast. Furthermore, we isolated a multicopy suppressor of the dhp1-1 mutant, referred to as din1+. We found that the gene product of dhp1-1 was unstable at high temperatures, but that reduced levels of Dhp1-1p could be suppressed by overexpressing Din1p at the restrictive temperature. Thus, Din1p may physically interact with Dhp1p and stabilize Dhp1p and/or restore its activity.

An amyloid-forming peptide from the yeast prion Sup35 reveals a dehydrated β-sheet structure for amyloid

X-ray diffraction and other biophysical tools reveal features of the atomic structure of an amyloid-like crystal. Sup35, a prion-like protein in yeast, forms fibrillar amyloid assemblies intrinsic to its prion function. We have identified a polar peptide from the N-terminal prion-determining domain of Sup35 that exhibits the amyloid properties of full-length Sup35, including cooperative kinetics of aggregation, fibril formation, binding of the dye Congo red, and the characteristic cross-β x-ray diffraction pattern. Microcrystals of this peptide also share the principal properties of the fibrillar amyloid, including a highly stable, β-sheet-rich structure and the binding of Congo red. The x-ray powder pattern of the microcrystals, extending to 0.9-Å resolution, yields the unit cell dimensions of the well-ordered structure. These dimensions restrict possible atomic models of this amyloid-like structure and demonstrate that it forms packed, parallel-stranded β-sheets. The unusually high density of the crystals shows that the packed β-sheets are dehydrated, despite the polar character of the side chains. These results suggest that amyloid is a highly intermolecularly bonded, dehydrated array of densely packed β-sheets. This dry β-sheet could form as Sup35 partially unfolds to expose the peptide, permitting it to hydrogen-bond to the same peptide of other Sup35 molecules. The implication is that amyloid-forming units may be short segments of proteins, exposed for interactions by partial unfolding.

Stable expression in yeast of the mature form of human telomerase RNA depends on its association with the box H/ACA small nucleolar RNP proteins Cbf5p, Nhp2p and Nop10p

Telomerase is a ribonucleoprotein (RNP) particle required for the replication of telomeres. The RNA component, termed hTR, of human telomerase contains a domain structurally and functionally related to box H/ACA small nucleolar RNAs (snoRNAs). Furthermore, hTR is known to be associated with two core components of H/ACA snoRNPs, hGar1p and Dyskerin (the human counterpart of yeast Cbf5p). To assess the functional importance of the association of hTR with H/ACA snoRNP core proteins, we have attempted to express hTR in a genetically tractable system, Saccharomyces cerevisiae. Both mature non-polyadenylated and polyadenylated forms of hTR accumulate in yeast. The former is associated with all yeast H/ACA snoRNP core proteins, unlike TLC1 RNA, the endogenous RNA component of yeast telomerase. We show that the presence of the H/ACA snoRNP proteins Cbf5p, Nhp2p and Nop10p, but not Gar1p, is required for the accumulation of mature non-polyadenylated hTR in yeast, while accumulation of TLC1 RNA is not affected by the absence of any of these proteins. Our results demonstrate that yeast telomerase is unrelated to H/ACA snoRNPs. In addition, they show that the accumulation in yeast of the mature RNA component of human telomerase depends on its association with three of the four core H/ACA snoRNP proteins. It is likely that this is the case in human cells as well.