Mutant Yeast Strains Research Articles

Evaluation of Antifungal and DNA-Damaging Activities of Alkaloids from Branches of Porcelia macrocarpa

Bioactivity-guided fractionation of the alkaloidal fractions of the CH2Cl2 extract from branches of Porcelia macrocarpa using mutant yeast strains of Saccharomyces cerevisiae and fungi Cladosporium cladosporioides and C. sphaerospermum led to the isolation of bioactive alkaloids: two tetrahydrobenzylisoquinolines (1 and 2), two aporphines (3 and 4), one proaporphine (5), one oxoaporphine (6), four azantraquinones (7-10) and four azafluorenones (11-14). The alkaloids cleistopholine (7) and 6-methoxycleistopholine (8) showed the highest fungitoxic activity while the mixture of 6- and 7-methoxyonychine (12+13) and 6,7-dimethoxyonychine (14) showed a weak DNA-damaging potential.

Elevated copy number of L-A virus in yeast mutant strains defective in ribosomal stalk

The eukaryotic ribosomal stalk, composed of the P-proteins, is a part of the GTPase-associated-center which is directly responsible for stimulation of translation-factor-dependent GTP hydrolysis. Here we report that yeast mutant strains lacking P1/P2-proteins show high propagation of the yeast L-A virus. Affinity-capture-MS analysis of a protein complex isolated from a yeast mutant strain lacking the P1A/P2B proteins using anti-P0 antibodies showed that the Gag protein, the major coat protein of the L-A capsid, is associated with the ribosomal stalk. Proteomic analysis revealed that the elongation factor eEF1A was also present in the isolated complex. Additionally, yeast strains lacking the P1/P2-proteins are hypersensitive to paromomycin and hygromycin B, underscoring the fact that structural perturbations in the stalk strongly influence the ribosome function, especially at the level of elongation.

Metal induction of a Paxillus involutus metallothionein and its heterologous expression in Hebeloma cylindrosporum

* Metallothioneins are small polypeptides involved in metal tolerance of many eukaryotes. Here we characterized the Pimt1 gene, coding for a metallothionein from the ectomycorrhizal fungus Paxillus involutus. * Expression of Pimt1 in P. involutus under metal stress conditions was measured by northern blot and RT-PCR analyses. The full-length cDNA was used to perform functional complementation in yeast mutant strains and agrotransformation of Hebeloma cylindrosporum. * Heterologous expression in yeast showed that PiMT1 was able to complement the hypersensitivity of mutant strains to cadmium (Cd) and copper (Cu), but not to zinc (Zn). Transcripts were almost undetectable under control conditions, whereas Cu and Cd, but not Zn, strongly induced Pimt1 expression in P. involutus. Constitutive overexpression of Pimt1 in H. cylindrosporum conferred a higher copper tolerance. * The present study identified PiMT1 as a potential determinant in the response of mycorrhizal fungi to Cu and Cd stress. Additionally, we demonstrated the usefulness of mycorrhizal fungi transformation using Agrobacterium technology to approach gene function.

Chemosensitisation of drug-resistant and drug-sensitive yeast cells to antifungals

Multidrug resistance in yeast results from overexpression of genes encoding drug efflux transporters owing to gain-of-function mutations in transcription factors regulating their expression. We have screened a library of synthetic compounds for modulators of drug resistance using the multidrug-resistant Saccharomyces cerevisiae pdr3-9 mutant strain. One of the compounds, 7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine (CTBT), displayed weak antifungal activity and strongly inhibited the growth of yeast cells in combination with subinhibitory concentrations of other antifungals with a different mode of action. Biological activity of CTBT was demonstrated in Saccharomyces, Kluyveromyces and Candida yeast species grown on solid and in liquid media. The chemosensitising effect of CTBT, manifested as increased antifungal activity of fluconazole, was demonstrated in yeast mutant strains with deleted genes encoding the major multidrug resistance transcription factors Yap1p, Pdr1p and Pdr3p as well as the drug efflux pumps Pdr5p and Snq2p in S. cerevisiae or their counterparts in Candida albicans and Candida glabrata, named Cdr1p and Mdr1p, respectively. Importantly, CTBT also increased the sensitivity to fluconazole in multidrug-resistant cells overexpressing the efflux pumps. Yeast cells grown in the presence of subinhibitory concentrations of CTBT exhibited an altered sterol composition and a slightly enhanced accumulation of Rhodamine 6G, which suggests that the plasma membrane plays a role in sensitisation. This novel chemosensitisation by CTBT that can overcome multidrug resistance in yeast may prove useful in combined treatment of infections caused by drug-resistant fungal pathogens.

A Cadmium-transporting P1B-type ATPase in Yeast Saccharomyces cerevisiae

Detoxification and homeostatic acquisition of metal ions are vital for all living organisms. We have identified PCA1 in yeast Saccharomyces cerevisiae as an overexpression suppressor of copper toxicity. PCA1 possesses signatures of a P1B-type heavy metal-transporting ATPase that is widely distributed from bacteria to humans. Copper resistance conferred by PCA1 is not dependent on catalytic activity, but it appears that a cysteine-rich region located in the N terminus sequesters copper. Unexpectedly, when compared with two independent natural isolates and an industrial S. cerevisiae strain, the PCA1 allele of the common laboratory strains we have examined possesses a missense mutation in a predicted ATP-binding residue conserved in P1B-type ATPases. Consistent with a previous report that identifies an equivalent mutation in a copper-transporting P1B-type ATPase of a Wilson disease patient, the PCA1 allele found in laboratory yeast strains is nonfunctional. Overexpression or deletion of the functional allele in yeast demonstrates that PCA1 is a cadmium efflux pump. Cadmium as well as copper and silver, but not other metals examined, dramatically increase PCA1 protein expression through post-transcriptional regulation and promote subcellular localization to the plasma membrane. Our study has revealed a novel metal detoxification mechanism in yeast mediated by a P1B-type ATPase that is unique in structure, substrate specificity, and mode of regulation.

Cytotoxic C-Methylated Chalcones from Syzygium samarangense.

The flavonoids 2′-hydroxy-4′,6′-dimethoxy-3′-methylchalcone (1), 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (2), 2′,4′-dihydroxy-6′-methoxy-3′-methylchalcone (3), 2′,4′-dihydroxy-6′-methoxy-3′-methyldihydrochalcone (4) and 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethyldihydrochalcone (5), isolated from Syzygium samarangense. (Blume) Merr. & L.M. Perry (Myrtaceae), were subjected to cytotoxicity testing using the dimethylthiazoldiphenyl tetrazolium (MTT) assay. The cell lines used were the Chinese hamster ovarian (CHO-AA8) and the human mammary adenocarcinoma, (MCF-7 and SKBR-3). Among the test compounds, 2 exhibited significant differential cytotoxicity against the MCF-7 cell line with an IC50 of 0.0015 ± 0.0001 nM. It was also cytotoxic against the SKBR-3 cell line with an IC50 of 0.0128 ± 0.0006 nM. Doxorubicin, the positive control, had an IC50 of 2.60 ± 0.28 × 10−4 nM against the MCF-7 cell line and an IC50 of 2.76 ± 0.52 × 10−5 nM against the SKBR-3 cell line. When tested in a mechanism-based yeast bioassay for detecting DNA-damaging agents using genetically engineered Saccharomyces cerevisiae. RS322Y (RAD52) mutant strain and (LF15/11) (RAD+) wild-type strain, 2 showed significant selective cytotoxicity against the RAD52 yeast mutant strain. It had an IC12 of 0.1482 nM, as compared with the positive control, streptonigrin, which had an IC12 of 0.0134 nM. Hence, 2 is a cytotoxic natural product with potential anticancer application.

TcrPDEA1, a cAMP-specific phosphodiesterase with atypical pharmacological properties from Trypanosoma cruzi

Cyclic nucleotide phosphodiesterases (PDEs) catalyze the degradation of cAMP and cGMP, and regulate a variety of cellular processes by controlling the levels of these second messengers. We have previously described the presence of both a calcium-stimulated adenylyl cyclase and two membrane-bound cAMP-specific PDEs (one of them strongly associated to the flagellum and the other one with a possible vesicular localization) in Trypanosoma cruzi. Here we report the identification and characterization of TcrPDEA1, a singular phosphodiesterase of T. cruzi which is resistant to the typical phosphodiesterase inhibitors, such as IBMX, papaverine and theofylline. TcrPDEA1 is a single copy gene that encodes a 620-amino acid protein, which is grouped with PDE1 family members, mainly with its kinetoplastid orthologs. TcrPDEA1 was able to complement a mutant yeast strain deficient in PDE genes, demonstrating that this enzyme is a functional phosphodiesterase. TcrPDEA1 is specific for cAMP with a high Km value (191.1±6.5μM). Cyclic GMP neither activates the enzyme nor competes as a substrate. In addition, calcium-calmodulin did not affect the kinetic parameters and, as its counterpart in T. brucei, magnesium showed to be crucial for its activity and stability. Although TcrPDEA1 function remains unclear, its presence points out the high complexity of the cAMP signaling in trypanosomatids and the possible compartmentalization of the enzymes involved in the cAMP pathway.

Global analysis of functional surfaces of core histones with comprehensive point mutants

The core histones are essential components of the nucleosome that act as global negative regulators of DNA-mediated reactions including transcription, DNA replication and DNA repair. Modified residues in the N-terminal tails are well characterized in transcription, but not in DNA replication and DNA repair. In addition, roles of residues in the core globular domains are not yet well characterized in any DNA-mediated reactions. To comprehensively understand the functional surface(s) of a core histone, we constructed 320 yeast mutant strains, each of which has a point mutation in a core histone, and identified 42 residues responsible for the suppressor of Ty (Spt(-)) phenotypes, and 8, 30 and 61 residues for sensitivities to 6-azauracil (6AU), hydroxyurea (HU) and methyl-methanesulfonate (MMS), respectively. In addition to residues that affect one specific assay, residues involved in multiple reactions were found, and surprisingly, about half of them were clustered at either the nucleosome entry site, the surface required for nucleosome-nucleosome interactions in crystal packing or their surroundings. This comprehensive mutation approach was proved to be powerful for identification of the functional surfaces of a core histone in a variety of DNA-mediated reactions and could be an effective strategy for characterizing other evolutionarily conserved hub-like factors for which surface structural information is available.

Investigating the Qn Site of the Cytochrome bc1 Complex in Saccharomyces cerevisiae with Mutants Resistant to Ilicicolin H, a Novel Qn Site Inhibitor

The cytochrome bc1 complex resides in the inner membrane of mitochondria and transfers electrons from ubiquinol to cytochrome c. This electron transfer is coupled to the translocation of protons across the membrane by the protonmotive Q cycle mechanism. This mechanism topographically separates reduction of quinone and reoxidation of quinol at sites on opposite sites of the membrane, referred to as center N (Qn site) and center P (Qp site), respectively. Both are located on cytochrome b, a transmembrane protein of the bc1 complex that is encoded on the mitochondrial genome. To better understand the parameters that affect ligand binding at the Qn site, we applied the Qn site inhibitor ilicicolin H to select for mutations conferring resistance in Saccharomyces cerevisiae. The screen resulted in seven different single amino acid substitutions in cytochrome b rendering the yeast resistant to the inhibitor. Six of the seven mutations have not been previously linked to inhibitor resistance. Ubiquinol-cytochrome c reductase activities of mitochondrial membranes isolated from the mutants confirmed that the differences in sensitivity toward ilicicolin H originated in the cytochrome bc1 complex. Comparative in vivo studies using the known Qn site inhibitors antimycin and funiculosin showed little cross-resistance, indicating different modes of binding of these inhibitors at center N of the bc1 complex.

Transcriptional Profiling of the Protein Phosphatase 2C Family in Yeast Provides Insights into the Unique Functional Roles of Ptc1

Type 2C protein phosphatases are encoded in Saccharomyces cerevisiae by several related genes (PTC1-5 and PTC7). To gain insight into the functions attributable to specific members of this gene family, we have investigated the transcriptional profiles of ptc1-5 mutants. Two main patterns were obtained as follows: the one generated by the ptc1 mutation and the one resulting from the lack of Ptc2-5. ptc4 and ptc5 profiles were quite similar, whereas that of ptc2 was less related to this group. Mutation of PTC1 resulted in increased expression of numerous genes that are also induced by cell wall damage, such as YKL161c, SED1, or CRH1, as well as in higher amounts of active Slt2 mitogen-activated protein kinase, indicating that lack of the phosphatase activates the cell wall integrity pathway. ptc1 cells were even more sensitive than slt2 mutants to a number of cell wall-damaging agents, and both mutations had additive effects. The sensitivity of ptc1 cells was not dependent on Hog1. Besides these phenotypes, we observed that calcineurin was hyperactivated in ptc1 cells, which were also highly sensitive to calcium ions, heavy metals, and alkaline pH, and exhibited a random haploid budding pattern. Remarkably, many of these traits are found in certain mutants with impaired vacuolar function. As ptc1 cells also display fragmented vacuoles, we hypothesized that lack of Ptc1 would primarily cause vacuolar malfunction, from which other phenotypes would derive. In agreement with this scenario, overexpression of VPS73, a gene of unknown function involved in vacuolar protein sorting, largely rescues not only vacuolar fragmentation but also sensitivity to cell wall damage, high calcium, alkaline pH, as well as other ptc1-specific phenotypes.

The Middle Domain of Hsp90 Acts as a Discriminator between Different Types of Client Proteins

The mechanism of client protein activation by Hsp90 is enigmatic, and it is uncertain whether Hsp90 employs a common route for all proteins. Using a mutational analysis approach, we investigated the activation of two types of client proteins, glucocorticoid receptor (GR) and the kinase v-Src by the middle domain of Hsp90 (Hsp90M) in vivo. Remarkably, the overall cellular activity of v-Src was highly elevated in a W300A mutant yeast strain due to a 10-fold increase in cellular protein levels of the kinase. In contrast, the cellular activity of GR remained almost unaffected by the W300A mutation but was dramatically sensitive to S485Y and T525I exchanges. In addition, we show that mutations S485Y and T525I in Hsp90M reduce the ATP hydrolysis rate, suggesting that Hsp90 ATPase is more tightly regulated than assumed previously. Therefore, the activation of GR and v-Src has various demands on Hsp90 biochemistry and is dependent on separate functional regions of Hsp90M. Thus, Hsp90M seems to discriminate between different substrate types and to adjust the molecular chaperone for proper substrate activation.

Synthesis, characterization and in vitro biological activity studies of Cu–M (M = Cu 2+, Co 2+, Ni 2+, Mn 2+, Zn 2+) bimetallic complexes

Six new bimetallic complexes of the type CuCu, CuCo, CuNi, CuZn and CuMn were prepared. The structures of these complexes and the ligand have been proposed on the basis of FAB mass, elemental analysis, UV–vis, IR, EPR and CV studies. All the complexes completely cleave pBS (SK−) DNA at a concentration of 10 μM; however, even at lower concentrations of 2 μM and 0.1 μM, the complexes ( I and Ia) showed partial cleavage. The results of the fluorescence binding studies of the metal complexes with CT-DNA showed that the presence of aliphatic ligands added additional binding effects including electrostatic, hydrogen binding and vander Waals interactions. Complexes ( I, Ia) showed 50% inhibition of COX-1 and COX-2 activities at as low a concentration as 12.5 μM, 13.5 μM, 14 μM and 14.5 μM. Inhibition assay of top I and top II by different complexes in mutant yeast strains (JN394, JN394 t −1 and JN394 t 2–5) with all the complexes showed significant inhibition of topoisomerase at 5 μM concentration. Complexes I and Ia exhibited good anti-microbial activities against all human pathogens tested except Klebsiella pneumoniae. The following studies showed that among the synthesized bimetallic complexes, complexes I and Ia seem to be promising candidates possessing DNA cleavage activities besides anti-microbial and anti-inflammatory properties to serve as chemical nucleases and chemotherapeutic agents.

Non-electrostatic interactions between cultured Saccharomyces cerevisiae yeast cells and adsorbent beads in expanded bed adsorption: Influence of cell wall properties

The present work focused on the importance of performing EBA experiments under real process conditions. To this end, a high ionic strength medium was used. The influence of cultivating Saccharomyces cerevisiae yeast cells on their subsequent adsorption on a Q HyperZ anion-exchanger was investigated. Two types of industrial yeast cells were used. Once cultured, both types of cells presented similar hydrophilic surface properties and identical adsorption profiles on the anion-exchanger. This was significantly different from the results obtained in previous work on the same yeast cells, just rehydrated in saline buffer, a biological model widely used in the literature. It was postulated that unavoidable “sticky” compounds, initially present in the culture medium or formed during the drying process, were strongly adsorbed on the cell wall and could not be completely removed during the successive washings of the rehydrated cell suspension before use. This could dramatically alter the yeast surface properties and modify the biomass/adsorbent interactions and the bed hydrodynamics, thus demonstrating the necessity to work with yeast coming from fresh cultures. Using biologically active yeast cells allowed to really elucidating the main physico-chemical mechanisms involved in cell adsorption by focusing on the role of the non-electrostatic interactions. Two laboratory mutant yeast strains in which the protein-related cell wall biogenesis was affected, were tested. No significant differences were observed between hydrophilic and hydrophobic yeast cells: the bed remained stable at its initial value and there was a low adsorption ratio in a narrow range between 10% and 17%. This clearly demonstrated that non-electrostatic interactions play a minor role on the affinity of yeast to anion-exchanger adsorbents.

An ATP‐binding cassette transporter related to yeast vacuolar ScYCF1 is important for Cd sequestration in Chlamydomonas reinhardtii

We generated a Cd-sensitive insertional mutant, Cds18, in Chlamydomonas reinhardtii and elucidated the deletion of a 10 kb fragment containing the promoter and a portion of the coding region for CrMRP2 gene that silenced the transcription of CrMRP2 in mutant Cds18. The association between CrMRP2 and Cd sensitivity was confirmed by complementing mutant Cds18 with a cloned genomic DNA fragment containing the promoter and complete coding sequence for CrMRP2. The genomic region and the full-length cDNA for CrMRP2 were cloned and sequenced. Computer searches detected the significant resemblance of CrMRP2 with HsMRP1, AtMRP3 and ScYCF1, in Homo sapiens, Arabidopsis thaliana and Saccharomyces cerevisiae, respectively. All are members of the multidrug resistance-associated protein (MRP)/cystic fibrosis transmembrane conductance regulator (CFTR) subfamily of ATP-binding cassette (ABC) transporters. When the cDNA of CrMRP2 was cloned into the yeast expression vector pEGKT and transformed into the yeast mutant strain DTY168 lacking ScYCF1, it restored the function of ScYCF1, a yeast vacuolar glutathione (GSH)-conjugate ABC transporter. A putative vacuolar-targeting motif (T/I/K)LP(L/K/I) was detected in the N-terminal part of CrMRP2. In wild-type C. reinhardtii, CrMRP2 transcription was significantly up-regulated upon Cd treatment. Comparing with mutant Cds18, the wild-type algal cells accumulated and sequestered more Cd in the stable high molecular weight (HMW) phytochelatin (PC)-Cd complex; the labile low molecular weight (LMW) PC-Cd complex was detected in mutant Cds18 at an earlier stage of Cd treatment. This study demonstrated the expression of CrMRP2 in C. reinhardtii and implicated its function in the formation/accumulation of stable HMW PC-Cd complex.

REV1 Protein Interacts with PCNA: Significance of the REV1 BRCT Domain In Vitro and In Vivo

REV1 protein, a eukaryotic member of the Y family of DNA polymerases, is involved in the tolerance of DNA damage by translesion DNA synthesis. It is unclear how REV1 is recruited to replication foci in cells. Here, we report that mouse REV1 can bind directly to PCNA and that monoubiquitylation of PCNA enhances this interaction. The interaction between REV1 protein and PCNA requires a functional BRCT domain located near the N terminus of the former protein. Deletion or mutational inactivation of the BRCT domain abolishes the targeting of REV1 to replication foci in unirradiated cells, but not in UV-irradiated cells. In vivo studies in both chicken DT40 cells and yeast directly support the requirement of the BRCT domain of REV1 for cell survival and DNA damage-induced mutagenesis.

Effects of Human Deafness γ-Actin Mutations (DFNA20/26) on Actin Function

Six point mutations in non-muscle gamma-actin at the DFNA20/26 locus cause autosomal dominant nonsyndromic hearing loss. The molecular basis for the hearing loss is unknown. We have engineered each gamma-actin mutation into yeast actin to investigate the effects of these mutations on actin function in vivo and in vitro. Cells expressing each of the mutant actins as the sole actin in the cell were viable. Four of the six mutant strains exhibited significant growth deficiencies in complete medium and an inability to grow on glycerol as the sole carbon source, implying a mitochondrial defect(s). These four strains exhibited abnormal mitochondrial morphology, although the mtDNA was retained. All of the mutant cells exhibited an abnormally high percentage of fragmented/non-polarized actin cables or randomly distributed actin patches. Five of the six mutants displayed strain-specific vacuole morphological abnormalities. Two of the purified mutant actins exhibited decreased thermal stability and increased rates of nucleotide exchange, indicative of increased protein flexibility. V370A actin alone polymerized abnormally. It aggregated in low ionic strength buffer and polymerized faster than wild-type actin, probably in part because of enhanced nucleation. Mixtures of wild-type and V370A actins displayed kinetic properties in proportion to the mole fraction of each actin in the mixture. No dominant effect of the mutant actin was observed. Our results suggest that a major factor in the deafness caused by these mutations is an altered ability of the actin filaments to be properly regulated by actin-binding proteins rather than an inability to polymerize.

Reconstitution of human hypoxia inducible factor HIF-1 in yeast: A simple in vivo system to identify and characterize HIF-1α effectors

Hypoxia inducible factor 1 (HIF-1), the master regulator of hypoxia-activated genes, is involved in many diseases and is a valid drug target. In order to develop a simple and genetically tractable in vivo system for HIF-1 analysis, we tested the inducible expression of both human HIF-1 subunits (HIF-1α and ARNT) in the yeast Saccharomyces cerevisiae and showed the formation of transcriptionally active HIF-1. The use of this system for the identification and characterization of HIF-1 effectors was first validated by showing that two chemical Hsp90 inhibitors, geldanamycin and radicicol, impaired the activity of HIF-1 in yeast. By applying this system in mutant yeast strains, we then identified Hsp90 co-chaperones, which were required for HIF-1 activity. Furthermore, using yeast strains co-expressing truncated forms of HIF-1α with ARNT or both HIF-1α and ARNT, we characterized fragments of HIF-1α that acted as dominant negative mutants and suppressed HIF-1 activity.

Pathway of sugar transport in germinating wheat seeds.

Three homeologous genes encoding a sucrose (Suc) transporter (SUT) in hexaploid wheat (Triticum aestivum), TaSUT1A, 1B, and 1D, were expressed in germinating seeds, where their function is unknown. All three TaSUT1 proteins were confirmed to be capable of transporting both Suc and maltose by complementation tests with the SUSY7/ura3 yeast (Saccharomyces cerevisiae) mutant strain. The role of Suc transporters in germinating grain was examined by combining in situ hybridization, immunolocalization, fluorescent dye tracer movement, and metabolite assays. TaSUT1 transcript and SUT protein were detected in cells of the aleurone layer, scutellar epidermis, scutellar ground cells, and sieve element-companion cell complexes located in the scutellum, shoot, and root. Ester loading of the membrane-impermeable fluorescent dye carboxyfluorescein into the scutellum epidermal cells of germinating seeds showed that a symplasmic pathway connects the scutellum to the shoot and root via the phloem. However, the scutellar epidermis provides an apoplasmic barrier to solute movement from endosperm tissue. Measurements of sugars in the root, shoot, endosperm, and scutellum suggest that, following degradation of endosperm starch, the resulting hexoses are converted to Suc in the scutellum. Suc was found to be the major sugar present in the endosperm early in germination, whereas maltose and glucose predominate during the later stage. It is proposed that loading the scutellar phloem in germinating wheat seeds can proceed by symplasmic and apoplasmic pathways, the latter facilitated by SUT activity. In addition, SUTs may function to transport Suc into the scutellum from the endosperm early in germination and later transport maltose.

Global Analysis of Protein Palmitoylation in Yeast

Protein palmitoylation is a reversible lipid modification that regulates membrane tethering for key proteins in cell signaling, cancer, neuronal transmission, and membrane trafficking. Palmitoylation has proven to be a difficult study: Specifying consensuses for predicting palmitoylation remain unavailable, and first-example palmitoylation enzymes--i.e., protein acyltransferases (PATs)--were identified only recently. Here, we use a new proteomic methodology that purifies and identifies palmitoylated proteins to characterize the palmitoyl proteome of the yeast Saccharomyces cerevisiae. Thirty-five new palmitoyl proteins are identified, including many SNARE proteins and amino acid permeases as well as many other participants in cellular signaling and membrane trafficking. Analysis of mutant yeast strains defective for members of the DHHC protein family, a putative PAT family, allows a matching of substrate palmitoyl proteins to modifying PATs and reveals the DHHC family to be a family of diverse PAT specificities responsible for most of the palmitoylation within the cell.

FLO11 mediated filamentous growth of the yeast Saccharomyces cerevisiae depends on the expression of the ribosomal RPS26 genes

The RPS26A and RPS26B isogenes of Saccharomyces cerevisiae encode two almost identical proteins of the small 40S ribosomal subunit, which differ by only two amino acid residues. Growth of an rps26BDelta mutant strain is normal, whereas an rps26ADelta strain displays a reduced growth rate and increased sensitivity towards the specific translational inhibitor paromomycin. An rps26ADelta rps26BDelta double mutant strain is inviable. RPS26A but not RPS26B is required for haploid adhesive and diploid pseudohyphal growth mediated by FLO11, which encodes an adhesion. The RPS26A and RPS26B transcripts make up about 70 and 30% of the cellular RPS26 mRNA, respectively. Overexpression of RPS26B, as well as an RPS26B open reading frame driven by the RPS26A promoter, complements the rps26ADelta deletion and restores haploid invasive growth as well as diploid pseudohyphal growth. These results suggest that the two proteins are functionally interchangeable. FLO11-lacZ activity is not present in haploid rps26ADelta yeast mutant strains, even though FLO11 mRNA levels are not reduced. This suggests that the amount of Rps26p is critical for accurate translation of the FLO11 mRNA, and therefore for the dimorphic switch of the bakera9s yeast from a single cell yeast to an adhesive filamentous growth form.