. Surprisingly, SCF and the Cdc53/Hrt1 subcomplex activate autoubiquitination of Cdc34 E2 enzyme by a mechanism that does not appear to require a reactive thiol. The highly conserved human HRT1 complements the lethality of hrt1⌬, and human HRT2 binds CUL-1. We conclude that Cdc53/Hrt1 comprise a highly conserved module that serves as the functional core of a broad variety of heteromeric ubiquitin ligases.

Over 190 independent insertions into target plasmids of the retro virus-like element Ty3 were recovered and mapped. Ty3 was shown to insert upstream of tRNA, 5S, and U6 genes, all of which are transcribed by RNA polymerase III. Integration sites were within 1-4 nucleotides of the position of transcription initiation, even for one mutant gene where the polymerase III initiation site was shifted to a completely new context. Mutagenesis of a SUP2 tRNA gene target showed that integration required functional promoter elements but that it did not correlate in a simple way with target transcription. This is the first report directly linking a discrete genomic function with preferential insertion of a retrotransposon.

Ty3, a retrotransposon of Saccharomyces cerevisiae, is found within 20 base pairs (bp) of the 5' ends of different tRNA genes. Determination of the complete nucleotide sequence of one Ty3 retrotransposon shows that the element is composed of an internal domain 4,748 bp long flanked by long terminal repeats of the 340-bp sigma element. Three open reading frames (ORFs) longer than 100 codons are present in the sense strand. The first ORF, TYA3, encodes a protein with a motif found in the nucleic acid-binding protein of retroviruses. The second ORF, TYB3, has homology to retroviral pol genes. The deduced amino acid sequence of the reverse transcriptase domain shows the greatest similarity to Drosophila retrotransposon 17.6, with 43% identical residues. The inferred order of functional domains within TYB3-protease, reverse transcriptase, and endonuclease-resembles the order in Drosophila element 17.6 and in animal retroviruses but is different from that found in yeast elements Tyl and Ty2. A second Ty3 element (Ty3-1) from a standard laboratory strain was overexpressed and shown to transpose.The genome of Saccharomyces cerevisiae contains at least three families of retrotransposons: Tyl, Ty2 (reviewed in references 24 and 54), and Ty3 (9). Retrotransposons are mobile genetic elements that transpose through an RNA intermediate and resemble retroviruses except for the apparent absence of an extracellular phase. The first retrotransposon identified in S. cerevisiae was named Ty, for transposable element in yeast (5). Two closely related forms of that element have been characterized and designated Tyl and Ty2 (8,35,47,88). Tyl and Ty2 are similar in nucleotide and amino acid sequences but have two large regions of heterogeneity which were first demonstrated by heteroduplex analysis (47). Tyl and Ty2 have an internal domain called epsilon which is 5.3 kilobase pairs (kbp) long. Epsilon is flanked by direct repeats of delta elements, which are 332 to 338 base pairs (bp) long (22,29). Transcription initiates in the upstream delta long terminal repeat (LTR), and the signal for polyadenylation occurs in the second LTR, downstream of the initiation site sequence (19). Thus, an almost full-length transcript with redundant termini is generated. Tyl and Ty2 insertions can influence the transcription of neighboring genes positively (21, 89) or negatively (6, 18, 67). The epsilon region contains two overlapping open reading frames (ORFs), which have similarity to the retroviral gag and pol genes. Boeke and co-workers (2) showed that Tyl transposition is dependent on Tyl transcription and that the transposition occurs through an RNA intermediate. These results demonstrated the functional similarity of retrotransposons to retroviruses.Ty3 is a more recently discovered element (9). It consists of a 4.7-kbp internal domain flanked by direct repeats of sigma elements, each 340 bp long. Characterization of one Ty3 element, now designated Ty3-1, showed that it has the following retroviruslike features: (i) flanking direct repeats of th...

The yeast retroviruslike element Ty3 inserts at the transcription initiation sites of genes transcribed by RNA polymerase III (Pol III). An in vitro integration assay was developed with the use of Ty3 viruslike particles and a modified SUP2 tyrosine transfer RNA (tRNA(Tyr)) gene target. Integration was position-specific and required Ty3 integrase, Pol III transcription factor (TF) IIIB-, TFIIIC-, and Pol III-containing fractions showed that TFIIIB and TFIIIC, together, were sufficient for position-specific Ty3 integration, but not for transcription. This report demonstrates that in vitro integration of a retroelement can be targeted by cellular proteins.

Retroviruses and retrotransposons assemble intracellular immature core particles around a RNA genome, and nascent particles collect in association with membranes or as intracellular clusters. How and where genomic RNA are identified for retrovirus and retrotransposon assembly, and how translation and assembly processes are coordinated is poorly understood. To understand this process, the subcellular localization of Ty3 RNA and capsid proteins and virus-like particles was investigated. We demonstrate that mRNAs, proteins, and virus-like particles of the yeast Ty3 retrotransposon accumulate in association with cytoplasmic P-bodies, which are sites of mRNA translation repression, storage, and degradation. Deletions of genes encoding P-body proteins decreased Ty3 transposition and caused changes in the pattern of Ty3 foci, underscoring the biological significance of the association of Ty3 virus-like protein components and P-bodies. These results suggest the hypothesis that P-bodies may serve to segregate translation and assembly functions of the Ty3 genomic RNA to promote assembly of virus-like particles. Because Ty3 has features of a simple retrovirus and P-body functions are conserved between yeast and metazoan organisms, these findings may provide insights into host factors that facilitate retrovirus assembly.

A collection of 4457 Saccharomyces cerevisiae mutants deleted for nonessential genes was screened for mutants with increased or decreased mobilization of the gypsylike retroelement Ty3. Of these, 64 exhibited increased and 66 decreased Ty3 transposition compared with the parental strain. Genes identified in this screen were grouped according to function by using GOnet software developed as part of this study. Gene clusters were related to chromatin and transcript elongation, translation and cytoplasmic RNA processing, vesicular trafficking, nuclear transport, and DNA maintenance. Sixty-six of the mutants were tested for Ty3 proteins and cDNA. Ty3 cDNA and transposition were increased in mutants affected in nuclear pore biogenesis and in a subset of mutants lacking proteins that interact physically or genetically with a replication clamp loader. Our results suggest that nuclear entry is linked mechanistically to Ty3 cDNA synthesis but that host replication factors antagonize Ty3 replication. Some of the factors we identified have been previously shown to affect Ty1 transposition and others to affect retroviral budding. Host factors, such as these, shared by distantly related Ty retroelements and retroviruses are novel candidates for antiviral targets.

In order to compete with petroleum-based fuel and chemicals, engineering a robust biocatalyst that can convert renewable feedstocks into biorenewable chemicals, such as carboxylic acids, is increasingly important. However, product toxicity is often problematic. In this study, the toxicity of the carboxylic acids hexanoic, octanoic, and decanoic acid on Saccharomyces cerevisiae was investigated, with a focus on octanoic acid. These compounds are completely inhibitory at concentrations of magnitude 1 mM, and the toxicity increases as chain length increases and as media pH decreases. Transciptome analysis, reconstruction of gene regulatory network, and network component analysis suggested decreased membrane integrity during challenge with octanoic acid. This was confirmed by quantification of dose-dependent and chain length-dependent induction of membrane leakage, though membrane fluidity was not affected. This induction of membrane leakage could be significantly decreased by a period of pre-adaptation, and this preadaptation was accompanied by increased oleic acid content in the membrane, significantly increased production of saturated lipids relative to unsaturated lipids, and a significant increase in the average lipid chain length in the membrane. However, during adaptation cell surface hydrophobicity was not altered. The supplementation of oleic acid to the medium not only elevated the tolerance of yeast cells to octanoic acid but also attenuated the membrane leakiness. However, while attempts to mimic the oleic acid supplementation effects through expression of the Trichoplusia ni acyl-CoA Δ9 desaturase OLE1(TniNPVE desaturase) were able to increase the oleic acid content, the magnitude of the increase was not sufficient to reproduce the supplementation effect and increase octanoic acid tolerance. Similarly, introduction of cyclopropanated fatty acids through expression of the Escherichia coli cfa gene was not helpful for tolerance. Thus, we have provided quantitative evidence that carboxylic acids damage the yeast membrane and that manipulation of the lipid content of the membrane can increase tolerance, and possibly production, of these valuable products.

A set of shuttle vectors was constructed to facilitate expression of genes for metabolic engineering in Saccharomyces cerevisiae. Selectable markers include the URA3, TRP1, MET15, LEU2-d8, HIS3 and CAN1 genes. Differential expression of genes can be achieved as each marker is available on both CEN/ARS-and 2 µ-containing plasmids. Unique restriction sites downstream of TEF1, PGK1 or HXT7-391 promoters and upstream of the CYC1 terminator allow insertion of open-reading frame cassettes for expression. Furthermore, a fragment appropriate for integration into the genome via homologous recombination can be readily generated in a polymerase chain reaction. Vector marker genes are flanked by loxP recognition sites for the CreA recombinase to allow efficient site-specific marker deletion and recycling. Expression and copy number were characterized for representative high-and low-copy vectors carrying the different marker and promoter sequences. Metabolic engineering typically requires the stable introduction of multiple genes and genomic integration is often preferred. This requires an expanded number of stable expression sites relative to standard gene expression studies. This study demonstrated the practicality of polymerase chain reaction amplification of an expression cassette and genetic marker, and subsequent replacement of endogenous retrotransposons by homologous recombination with flanking sequences. Such reporters were expressed comparably to those inserted at standard integration loci. This expands the number of available characterized integration sites and demonstrates that such sites provide a virtually inexhaustible pool of integration targets for stable expression of multiple genes. Together these vectors and expression loci will facilitate combinatorial gene expression for metabolic engineering.