Ras Regulates the Polarity of the Yeast Actin Cytoskeleton through the Stress Response Pathway

Polarized growth in yeast requires cooperation between the polarized actin cytoskeleton and delivery of post-Golgi secretory vesicles. We have previously reported that loss of the major tropomyosin isoform, Tpm1p, results in cells sensitive to perturbations in cell polarity. To identify components that bridge these processes, we sought mutations with both a conditional defect in secretion and a partial defect in polarity. Thus, we set up a genetic screen for mutations that conferred a conditional growth defect, showed synthetic lethality with tpm1Δ, and simultaneously became denser at the restrictive temperature, a hallmark of secretion-defective cells. Of the 10 complementation groups recovered, the group with the largest number of independent isolates was functionally null alleles of RAS2. Consistent with this, ras2Δ and tpm1Δ are synthetically lethal at 35°C. We show that ras2Δ confers temperature-sensitive growth and temperature-dependent depolarization of the actin cytoskeleton. Furthermore, we show that at elevated temperatures ras2Δ cells are partially defective in endocytosis and show a delocalization of two key polarity markers, Myo2p and Cdc42p. However, the conditional enhanced density phenotype of ras2Δ cells is not a defect in secretion. All the phenotypes of ras2Δ cells can be fully suppressed by expression of yeast RAS1 or RAS2 genes, human Ha-ras, or the double disruption of the stress response genes msn2Δmsn4Δ. Although the best characterized pathway of Ras function in yeast involves activation of the cAMP-dependent protein kinase A pathway, activation of the protein kinase A pathway does not fully suppress the actin polarity defects, suggesting that there is an additional pathway from Ras2p to Msn2/4p. Thus, Ras2p regulates cytoskeletal polarity in yeast under conditions of mild temperature stress through the stress response pathway.

Yeast Gga Coat Proteins Function with Clathrin in Golgi to Endosome Transport

Gga proteins represent a newly recognized, evolutionarily conserved protein family with homology to the “ear” domain of the clathrin adaptor AP-1 γ subunit. Yeast cells contain two Gga proteins, Gga1p and Gga2p, that have been proposed to act in transport between the trans-Golgi network and endosomes. Here we provide genetic and physical evidence that yeast Gga proteins function in trans-Golgi network clathrin coats. Deletion of Gga2p (gga2Δ), the major Gga protein, accentuates growth and α-factor maturation defects in cells carrying a temperature-sensitive allele of the clathrin heavy chain gene. Cells carrying either gga2Δ or a deletion of the AP-1 β subunit gene (apl2Δ) alone are phenotypically normal, but cells carrying both gga2Δ and apl2Δ are defective in growth, α-factor maturation, and transport of carboxypeptidase S to the vacuole. Disruption of both GGA genes and APL2 results in cells so severely compromised in growth that they form only microcolonies. Gga proteins can bind clathrin in vitro and cofractionate with clathrin-coated vesicles. Our results indicate that yeast Gga proteins play an important role in cargo-selective clathrin-mediated protein traffic from the trans-Golgi network to endosomes.

The yeast GAL11 protein binds to the transcription factor IIE through GAL11 regions essential for its in vivo function.

The GAL11 gene encodes an auxiliary transcription factor required for full expression of many genes in yeast. The GAL11-encoded protein (Gal11p) has recently been shown to copurify with the holoenzyme of RNA polymerase II. Here we report that Gal11p stimulates basal transcription in a reconstituted transcription system composed of recombinant or highly purified transcription factors, TFIIB, TFIIE, TFIIF, TFIIH, and TATA box-binding protein and core RNA polymerase II. We further demonstrate that each of the two domains of Gal11p essential for in vivo function respectively participates in the binding to the small and large subunits of TFIIE. The largest subunit of RNA polymerase II was coprecipitated by anti-hemagglutinin epitope antibody from crude extract of GAL11 wild type yeast expressing hemagglutinintagged small subunit of TFIIE. Such a coprecipitation of the RNA polymerase subunit was seen but in a greatly reduced amount, if extract was prepared from gal11 null yeast. In light of these findings, we suggest that Gal11p stimulates promoter activity by enhancing an association of TFIIE with the preinitiation complex in the cell.

The translational function of nucleotide C1054 in the small subunit rRNA is conserved throughout evolution: genetic evidence in yeast.

Mutations at position C1054 of 16S rRNA have previously been shown to cause translational suppression in Escherichia coli. To examine the effects of similar mutations in a eukaryote, all three possible base substitutions and a base deletion were generated at the position of Saccharomyces cerevisiae 18S rRNA corresponding to E. coli C1054. In yeast, as in E. coli, both C1054A (rdn-1A) and C1054G (rdn-1G) caused dominant nonsense suppression. Yeast C1054U (rdn-1T) was a recessive antisuppressor, while yeast C1054-delta (rdn-1delta) led to recessive lethality. Both C1054U and two previously described yeast 18S rRNA antisuppressor mutations, G517A (rdn-2) and U912C (rdn-4), inhibited codon-nonspecific suppression caused by mutations in eukaryotic release factors, sup45 and sup35. However, among these only C1054U inhibited UAA-specific suppressions caused by a UAA-decoding mutant tRNA-Gln (SLT3). Our data implicate eukaryotic C1054 in translational termination, thus suggesting that its function is conserved throughout evolution despite the divergence of nearby nucleotide sequences.

Characterization and functional ordering of Slu7p and Prp17p during the second step of pre-mRNA splicing in yeast.

Temperature-sensitive alleles in four genes (slu7-1, prp16-2, prp17-1, and prp18-1) are known to confer a specific block to the second chemical step of pre-mRNA splicing in vivo in the yeast Saccharomyces cerevisiae. Previous studies showed that Prp16p and Prp18p are required solely for the second step in vitro. The RNA-dependent ATPase, Prp16p, functions at a stage in splicing when ATP is required, whereas Prp18p functions at an ATP-independent stage. Here we use immunodepletion to show that the roles of Slu7p and Prp17p are also confined to the second step of splicing. We find that extracts depleted of Prp17p require both Prp17p and ATP for slicing complementation, whereas extracts depleted of Slu7p require only the addition of Slu7p. These different ATP requirements suggest that Prp16p and Prp17p function before Prp18p and Slu7p. Although SLU7 encodes an essential gene product, we find that a null allele of prp17 is temperature-sensitive for growth and has a partial splicing defect in vitro. Finally, high-copy suppression experiments indicate functional interactions between PRP16 and PRP17, PRP16 and SLU7, and SLU7 and PRP18. Taken together, the results suggest that these four factors may function within a multi-component complex that has both an ATP-dependent and an ATP-independent role in the second step of pre-mRNA splicing.

The only essential function of TFIIIA in yeast is the transcription of 5S rRNA genes.

We have developed a system to transcribe the yeast 5S rRNA gene in the absence of the transcription factor TFIIIA. A long transcript was synthesized both in vitro and in vivo from a hybrid gene in which the tRNA-like promoter sequence of the RPR1 gene was fused to the yeast 5S RNA gene. No internal initiation directed by the endogenous 5S rDNA promoter or any processing of the hybrid transcript was observed in vitro. Yeast cells devoid of transcription factor TFIIIA, which, therefore, could not synthesize any 5S rRNA from the endogenous chromosomal copies of 5S rDNA, could survive if they carried the hybrid RPR1-5S construct on a multicopy plasmid. In this case, the only source of 5S rRNA was the precursor RPR1-5S transcript that gave rise to two RNA species slightly larger than wild-type 5S rRNA. This establishes that the only essential function of TFIIIA is to promote the synthesis of 5S rRNA. However, cells devoid of TFIIIA and surviving with these two RNAs grew more slowly at 30 degrees C compared with wild-type cells and were thermosensitive at 37 degrees C.

Functional complementation of the ste6 gene of Saccharomyces cerevisiae with the pfmdr1 gene of Plasmodium falciparum.

The pfmdr1 gene has been associated with a drug-resistant phenotype in Plasmodium falciparum, and overexpression of pfmdr1 has been associated with mefloquine- and halofantrine-resistant parasites, but little is known about the functional role of pfmdr1 in this process. Here, we demonstrate that the pfmdr1 gene expressed in a heterologous yeast system functions as a transport molecule and complements a mutation in ste6, a gene which encodes a mating pheromone a-factor export molecule. In addition, the pfmdr1 gene containing two mutations which are associated with naturally occurring chloroquine resistance abolishes this mating phenotype, suggesting that these genetic polymorphisms alter this transport function. Our results support the functional role of pfmdr1 as a transport molecule in the mediation of drug resistance and provide an assay system to address the nature of this transport function.

Structure and function of a human transcription factor TFIIIB subunit that is evolutionarily conserved and contains both TFIIB- and high-mobility-group protein 2-related domains.

Transcription factor TFIIIB plays a central role in transcription initiation by RNA polymerase III on genes encoding tRNA, 5S rRNA, and other small structural RNAs. We report the purification of a human TFIIIB-derived complex containing only the TATA-binding polypeptide (TBP) and a 90-kDa subunit (TFIIIB90) and the isolation of a cDNA clone encoding the 90-kDa subunit. The N-terminal half of TFIIIB90 exhibits sequence similarity to the yeast TFIIIB70 (BRF) and the class II transcription factor TFIIB and interacts weakly with TBP. The C-terminal half of TFIIIB90 contains a high-mobility-group protein 2 (HMG2)-related domain and interacts strongly with TBP. Recombinant TFIIIB90 plus recombinant human TBP substitute for human TFIIIB in a complementation assay for transcription of 5S, tRNA, and VA1 RNA genes, and both the TFIIB-related domain and the HMG2-related domain are required for this activity. TFIIIB90 is also required for transcription of human 7SK and U6 RNA genes by RNA polymerase III, but apparently within a complex distinct from the TBP/TFIIIB90 complex.

Yeast histone H3 and H4 N termini function through different GAL1 regulatory elements to repress and activate transcription.

Previous work has shown that N-terminal deletions of yeast histone H3 cause a 2- to 4-fold increase in the induction of GAL1 and a number of other genes involved in galactose metabolism. In contrast, deletions at the H4 N terminus cause a 10- to 20-fold decrease in the induction of these same GAL genes. However, H3 and H4 N-terminal deletions each decrease PHO5 induction only 2- to 4-fold. To define the GAL1 gene regulatory elements through which the histone N termini activate or repress transcription, fusions were made between GAL1 and PHO5 promoter elements attached to a beta-galactosidase reporter gene. We show here that GAL1 hyperactivation caused by the H3 N-terminal deletion delta 4-15 is linked to the upstream activation sequence. Conversely, the relative decrease in GAL1 induction caused by the H4N-terminal deletion delta 4-28 is linked to the downstream promoter which contains the TATA element. These data indicate that the H3 N terminus is required for the repression of the GAL1 upstream element, whereas the H4N terminus is required for the activation of the GAL1 downstream promoter element.

Development of a binary positive and negative protein fragment complementation assay using yeast cytosine deaminase

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Development of a binary positive and negative protein fragment complementation assay using yeast cytosine deaminase

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Protein complementation assay as a display system for screening protein libraries in the intracellular environment

A wide range of screening strategies have been employed to isolate antibodies and other proteins with specific attributes, including binding affinity, specificity, stability and improved expression. However, there remains no high-throughput system to screen for target-binding proteins in a mammalian, intracellular environment. Such a system would allow binding reagents to be isolated against intracellular clinical targets such as cell signalling proteins associated with tumour formation (p53, ras, cyclin E), proteins associated with neurodegenerative disorders (huntingtin, betaamyloid precursor protein), and various proteins crucial to viral replication (e.g. HIV-1 proteins such as Tat, Rev and Vif-1), which are difficult to screen by phage, ribosome or cell-surface display. This study used the β-lactamase protein complementation assay (PCA) as the display and selection component of a system for screening a protein library in the cytoplasm of HEK 293T cells. The colicin E7 (ColE7) and Immunity protein 7 (Imm7) *Escherichia coli* proteins were used as model interaction partners for developing the system. These proteins drove effective β-lactamase complementation, resulting in a signal-to-noise ratio (9:1 – 13:1) comparable to that of other β-lactamase PCAs described in the literature. The model Imm7-ColE7 interaction was then used to validate protocols for library screening. Single positive cells that harboured the Imm7 and ColE7 binding partners were identified and isolated using flow cytometric cell sorting in combination with the fluorescent β-lactamase substrate, CCF2/AM. A single-cell PCR was then used to amplify the Imm7 coding sequence directly from each sorted cell. With the screening system validated, it was then used to screen a protein library based the Imm7 scaffold against a proof-of-principle target. The wild-type Imm7 sequence, as well as mutants with wild-type residues in the ColE7- binding loop were enriched from the library after a single round of selection, which is consistent with other eukaryotic screening systems such as yeast and mammalian cell-surface display. In summary, this thesis describes a new technology for screening protein libraries in a mammalian, intracellular environment. This system has the potential to complement existing screening technologies by allowing access to intracellular proteins and expanding the range of targets available to the pharmaceutical industry.

A fission yeast homolog of Int-6, the mammalian oncoprotein and eIF3 subunit, induces drug resistance when overexpressed

Through a screen to identify genes that induce multi-drug resistance when overexpressed, we have identified a fission yeast homolog of Int-6, a component of the human translation initiation factor eIF3. Disruption of the murine Int-6 gene by mouse mammary tumor virus (MMTV) has been implicated previously in tumorigenesis, although the underlying mechanism is not yet understood. Fission yeast Int6 was shown to interact with other presumptive components of eIF3 in vivo, and was present in size fractions consistent with its incorporation into a 43S translation preinitiation complex. Drug resistance induced by Int6 overexpression was dependent on the AP-1 transcription factor Pap1, and was associated with increased abundance of Pap1-responsive mRNAs, but not with Pap1 relocalization. Fission yeast cells lacking the int6 gene grew slowly. This growth retardation could be corrected by the expression of full length Int6 of fission yeast or human origin, or by a C-terminal fragment of the fission yeast protein that also conferred drug resistance, but not by truncated human Int-6 proteins corresponding to the predicted products of MMTV-disrupted murine alleles. Studies in fission yeast may therefore help to explain the ways in which Int-6 function can be perturbed during MMTV-induced mammary tumorigenesis.

The role of the yeast cleavage and polyadenylation factor subunit Ydh1p/Cft2p in pre-mRNA 3'-end formation

Cleavage and polyadenylation factor (CPF) is a multi‐protein complex that functions in pre‐mRNA 3′‐end formation and in the RNA polymerase II (RNAP II) transcription cycle. Ydh1p/Cft2p is an essential component of CPF but its precise role in 3′‐end processing remained unclear. We found that mutations in YDH1 inhibited both the cleavage and the polyadenylation steps of the 3′‐end formation reaction in vitro. Recently, we demonstrated that an important function of CPF lies in the recognition of poly(A) site sequences and RNA binding analyses suggesting that Ydh1p/Cft2p interacts with the poly(A) site region. Here we show that mutant ydh1 strains are deficient in the recognition of the ACT1 cleavage site in vivo. The C‐terminal domain (CTD) of RNAP II plays a major role in coupling 3′‐end processing and transcription. We provide evidence that Ydh1p/Cft2p interacts with the CTD of RNAP II, several other subunits of CPF and with Pcf11p, a component of CF IA. We propose that Ydh1p/Cft2p contributes to the formation of important interaction surfaces that mediate the dynamic association of CPF with RNAP II, the recognition of poly(A) site sequences and the assembly of the polyadenylation machinery on the RNA substrate.