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The understanding of the molecular mechanisms that affect the adaption of microbial species at different temperatures is essential to appreciate how biodiversity originates and how it can be maintained in a constantly changing environment. The Saccharomyces genus is an ideal group to study interspecific ecological traits, especially temperature, since it consists of a monophyletic group of species with high levels of sequence similarity within the Ascomycota phylum. The Saccharomyces genus is composed of eight species with a wide range of optimal growth temperatures and can be divided into thermo-tolerant, generalist and cold-tolerant. Saccharomyces species that share the same habitat (sympatry) may have optimum growth temperature, suggesting a partitioning of thermal niche possibly to avoid exploitative competition. In this study, the cellular role of a group of genes identified as key elements to provide optimal growth at cold temperatures by computational modelling and large-scale studies were investigated. Homozygote mutants of ADH3, GUT2, NMA1, YND1, ADH5 and FAA1, in all eight species belonging to the Saccharomyces genus were created. Upon deletion of these genes, S. kudriavzevii and S. arboricola displayed the most compromised fitness when compared with the other species. The level of expression of these genes in their native species at different temperature was determined. A significantly higher expression at cold for S. paradoxus, S. kudriavzevii and S. jurei compared to S. cerevisiae was observed. Different combinations of double mutants were also constructed, and the fitness scored in the different species. The double mutants ΔADH3/ΔADH3 and ΔYND1/ΔYND1 in S. paradoxus, S. uvarum and S. kudriavzevii, showed the greatest fitness impairment at cold. Interestingly, most of the genetic interactions in the double mutants showed positive and negative epistasis in S. cerevisiae and S. kudriavzevii, respectively. Also, interactions of ncRNAs with the genes under study to infer epigenetic mechanisms that are involved in cold-tolerance were evaluated. To tease apart the role of promoters (i.e. gene expression) from the allele type (i.e. genetic) in cold-adaptation, either the ADH3 and YND1 alelles or their promoters in S. cerevisiae, S. paradoxus, S. jurei and S. eubayanus with alelles or promoters belonging to the cold-tolerant species S. kudriavzevii were replaced independently. Fitness improvement was detected at cold temperature in the strains carrying S. kudriavzevii promoter for both ADH3 and YND1, whereas fitness was lower when the alleles were swapped, suggesting strength of expression is crucial in maintaining growth at cold temperatures over the type of allele. Additionally, different conditions for isolating yeast wild strains were explored, finding that cold-tolerant yeast species had higher fitness over thermotolerant and generalist in oak (Quercus) and spruce (Picea) infused media, and at low temperature S. eubayanus was the species which showed the best performance. Field studies were carried out to attempt to isolate S. eubayanus. The location of Scottish Highlands Forest was chosen based on maximum probability model based on climate data. No Saccharomyces species were isolated, instead species belonging to the genus Nakazawea were the most representative among the yeast isolates. Overall, this study contributes to understanding the importance of a non-essential group of genes in adaptation to cold temperatures and the role of promoter and allele type on cold adaptation in Saccharomyces species. It also, sheds light on the type of epistasis among the candidate genes in different species background.

Antimicrobial peptides (AMPs) are found throughout the animal kingdom and act as a natural defence against a broad spectrum of pathogens. These peptides are toxic to invading organisms without acting on host cells, so are of interest for their potential to act as potent new drugs against pathogenic organisms. AMPs traverse the cell wall and predominantly target the plasma membrane, resulting in destabilisation, leakage of intracellular components and cell death. In this thesis the mode of action of several AMPs was investigated. The role of the cell wall was studied and found to mediate peptide binding, the inhibition of certain cell wall components also increased peptide action, subsequent internalisation events were observed with varying localisation patterns and the effect of several genes that alter cell susceptibility to AMP were examined. Several Candida albicans mutants, each deficient in cell wall protein mannosylation, were tested in relation to their susceptibility to AMPs. It was discovered that cells lacking or deficient in the phosphomannan fraction, with a concomitant reduction in surface negative charge, correlated with reduced susceptibility to AMP action. To ascertain whether peptide binds to negatively charged phosphate, the effect of exogenous glucosamine 6-phosphate (but not glucosamine hydrochloride) was studied demonstrating that peptide efficacy was reduced due to the presence of exogenous phosphate. More specifically, sequestration of the truncated cationic AMP dermaseptin S3 (DsS3(1-16)) was reduced in these phosphomannan deficient mutants. Microscopy analysis of fluorescein tagged DsS3(1-16) also revealed the differential localisation patterns of this AMP: transiently binding to the plasma membrane, localisation to the vacuole or diffuse distribution throughout the cytoplasm. It is proposed that for these cationic AMPs to exert their full antifungal action they must first bind to the negatively charged phosphate. The echinocandins are a relatively new class of antifungal that function by inhibiting 1,3-β glucan synthase resulting in reduced 1,3-β glucan in the cell wall. As AMPs have to traverse the cell wall it was postulated that cells lacking this fraction would display increased AMP binding to the membrane. Clinical isolate strains of Candida and Cryptococcus spp. were acquired to test their susceptibility to AMP and echinocandin combinations. Comparing the fractional inhibitory concentration index (FICI) (supported by viable cell counts and on a solid surface using disc diffusion assays) synergy was observed between caspofungin, anidulafungin and several AMPs in vitro. In vitro toxicity assays revealed no increase in haemolytic or cytotoxic action on combination. These synergistic combinations could provide a novel treatment against fungal pathogens. The final area of study was based upon work that identified genes whose expression altered cell susceptibility to AMPs. Three genes were selected for investigation that upon deletion increased the action of DsS3(1-16) or magainin 2 on S. cerevisiae. Results from growth analysis, peptide sequestration and cell viability counts confirmed that deletion of HAL5, LDB7 or IMP2’ did increase susceptibility. Additionally, deletion of HAL5 increased the probability of cell depolarisation upon peptide exposure. Expression of GFP-tagged Imp2’ also increased when cells were exposed to DsS3(1-16). It was concluded that deletion of HAL5 increases depolarisation due to insufficient potassium efflux, leading to ion leakage and cell death facilitated by AMP action. Double strand break repair and DNA protection are probably compromised upon deletion of LDB7 and IMP2’, increasing the inhibitory action of DsS3(1-16) that has previously been shown to bind to DNA.

NADP-dependent aliphatic alcohol dehydrogenases had been previously identified in various strains of Acinetobacter calcoaceticus, including A. calcoaceticus NCIB 8250 and Acinetobacter sp. HO1-N. During the course of this study similar enzymes were identified in the yeast Saccharomyces cerevisiae D273-10B and in all of the other Gram negative and Gram positive eubacteria and yeasts that were examined. This thesis is chiefly concerned with purification and characterisation of these enzymes in A. calcoaceticus and S. cerevisiae and with studies on the physiological role of the enzyme of A. calcoaceticus.

A key goal of evolutionary biology is understanding how organisms build new traits. Many adaptations that distinguish taxa evolved so long ago that they are separated across species barriers, and many of these traits also have a complex genetic basis. In this thesis, I have leveraged advances in tools for interspecies genetics to illuminate the genetic underpinnings of thermotolerance evolution in Saccharomyces yeast, as a case study. We sought to improve the statistical power of screens to detect genetic determinants of this interspecific trait, both through computational advances in data processing and further development of the screening methodology. We wanted to know what traces of the putative ancient selective sweep(s) on thermotolerance factors might linger in the genomes of modern yeasts. We were also curious how initial conditions the evolutionary landscape might have differed from those further along during the evolutionary acquisition of thermotolerance. In Chapter 1, we introduce the field, and the questions within the field that we studied specifically. In Chapter 2, we investigate the population and comparative genetics of thermotolerance loci, as well as the mechanisms by which one key allele contributes to the phenotype. In Chapter 3, we use a barcoded version of the reciprocal hemizygosity analysis via sequencing screening technique to identify dozens of candidate genes involved in the thermotolerance phenotype. In Chapter 4, we delve into thermotolerance across a range of temperatures and discuss models for the early evolutionary landscape of the thermotolerance divergence. Lastly, in Chapter 5, we use directed evolution to gain insight into adaptations that allow a thermosensitive Saccharomyces yeast to acquire thermotolerance.

In order to better understand their impact on ethanol tolerance in Saccharomyces cerevisiae, six individual genes were overexpressed in two different commercial wine yeast strains, Montrachet and Elixir, and fermented in a high brix, defined, wine-like medium. These genes mainly consisted of those involved in the production of unsaturated fatty acids, although genes dealing with glycerol efflux and reactive oxygen species sequestration were also evaluated. The fermentations were monitored for brix, biomass, and nitrogen utilization. HPLC analysis of hexose sugars, glycerol, and ethanol was reserved for the most interesting of these genes, FPS1 and FAS1, in order to further understand the metabolism associated with these genetic changes. A final fermentation was then run using mutants lacking the FPS1 gene, and mutants overexpressing both components of the FAS complex, FAS1 and FAS2. The results showed a clear trend of greater ethanol tolerance, measured as the amount of ethanol produced before the cessation of fermentation, with less expression of FPS1 in Montrachet, but little result in Elixir. The FPS1 deletion mutant for the Montrachet strain developed higher biomass, utilized more sugar, and produced more ethanol compared to the control. Likewise, overexpression of the FAS complex showed a deleterious effect on the same ethanol tolerance proxies of Elixir, with more muted results in Montrachet. FAS overexpression caused reduced biomass and more sluggish fermentation. These responses demonstrate not only the potential of leveraging these genes for the production of novel commercial strains of S. cerevisiae, but also the interesting strain dependent responses associated with their differential expression.

Acetyl-CoA is an abundant metabolite in microorganisms and is a common precursor for many native and non-native products produced in microbial hosts. Acetyl-CoA is formed in four distinct locations in the yeast Saccharomyces cerevisiae creating three distinct pools of acetyl-CoA: the cytosolic pool, the peroxisomal pool, and the mitochondrial pool. Increasing access to the acetyl-CoA contained in those pools will increase the production of products synthesized from acetyl-CoA. One such product is the polyketide triacetic acid lactone (TAL) which is formed from acetyl-CoA and malonyl-CoA (also made from acetyl-CoA) via the enzyme 2-pyrone synthase (2PS) from Gerbera hybrida. This work focuses on improving production of TAL by enhancing access of 2PS to each distinct pool of acetyl-CoA in S. cerevisiae. A combinatorial CRISPR knockout approach was used to knockout random combinations of pathway and regulatory genes related to acetyl and malonyl-CoA utilization in the cell for the purpose of identifying gene knockout combinations that increase the cytosolic pool of these metabolites and the production of TAL in the cytosol. Conditions for both construction of guide RNA and cell cultivation were optimized to reduce bias. Expression of 2PS in the knockout library strains gave a distribution of TAL production with several strains producing significantly more TAL than the unmodified strain. To increase access to the peroxisomal pool of acetyl-CoA, formed from β-oxidation, three genes in the TAL production pathway, the acetylcarnitine transferase, Cat2; the modified acetyl-CoA carboxylase, Acc1S1157A; and 2PS, were anchored on the surface of the peroxisome using the native Pex15 anchoring motif. This method utilizes the advantages of both enzyme colocalization and the capture and rapid use of the β-oxidation produced acetyl-CoA, exported from the peroxisome in the form of acetylcarnitine. Anchoring all three enzymes on the peroxisomal membrane surface significantly improved TAL production. The mitochondria both creates acetyl-CoA and uses it in the TCA cycle, leading to a large mitochondrial acetyl-CoA pool. Targeting 2PS to the mitochondria allows access to that large pool in the confined volume of an organelle. Multiple variants of the mitochondrial leader sequence (MLS), CoxIV, were assessed for 2PS targeting in S. cerevisiae. The mitochondrial targeting method was expanded into the industrially relevant non-conventional yeast Kluyveromyces marxianus by targeting both 2PS and the S. cerevisiae Acc1S1157A.

The brewing of beer requires four main ingredients: malt, water, hops, and yeast. During the brewing process, the brewer creates a malt-derived sugar water, called wort, that is inoculated with yeast, causing fermentation to create carbon dioxide and alcohol from the sugars. Traditionally hops are added to the wort during the boiling process in order to balance the sweetness with isomerized alpha-acids that create bitterness. Hops also contain many other flavor-active compounds that brewers are attempting to harness for the creation of aromatic and complex beers. These flavor-active compounds can become even more desirable when hops are added to active fermentation in the presence of Saccharomyces yeast, a process known as dry-hopping. During this dry-hopping process, aromatic compounds from the hops can undergo biotransformation with glycosides of the yeast to create unique flavors, but these dry-hops also contain enzymes that are biochemically changing more than just aromatic compounds. The enzymes in the hops are breaking down residual oligosaccharides in the beer that are meant to create a fuller bodied beverage, known as dextrins, and converting them into simple, more fermentable sugars that the yeast can easily assimilate. The yeast ferments the newly hydrolyzed sugars, adding excess alcohol and possibly yeast derived off-flavors to the beer, a process termed “hop creep”. In this series of studies, the interactions between yeast and hops during the dry-hopping and fermentation process of beer was examined, as well as factors contributing to the hop creep phenomenon. Hop creep has been extensively studied from the back end of the brewing process, the hop perspective; this work intends to look at the phenomenon from the front end of fermentation, the yeast perspective. In the first part of this study, a bench top analysis was repeated to determine contribution to dry-hop creep from different hop cultivars. Previous research had determined a statistical difference amongst cultivars, but was unable to be confirmed here, so research set out to develop a bench method for hop creep analysis that brewers could perform easily with little laboratory skills. Following method development, a screening of Saccharomyces yeasts from the UC Davis Phaff Yeast Culture Collection commenced in order to identify yeasts with properties advantageous to brewing. Yeasts were screened for their ethanol tolerance, carbohydrate metabolism, as well as nitrogen and amino acid assimilation, all of which are important qualities for the brewer. The screening was successful and expanded to full pilot scale at Anheuser-Busch InBev Research Pilot Brewery at the Robert Mondavi Institute. Six non-cerevisiae and non-pastorianus Saccharomyces yeasts that had not previously been used to create ales were used in fermentations in duplicate, with one fermenter in each set receiving 10 g/L of dry-hops during fermentation. Beers were measure for alcohol, real degree of fermentation (RDF), gravity, calories, pH, and yeast viability during fermentation, and sensory analysis was performed on finished beer. All yeasts displayed unique characteristics that may offer great potential to a complexly evolving and consumer driven beer market. In addition to the six yeasts described above, twenty-four more Saccharomyces strains chosen for their typical use in the production of alcoholic beverages were also used in fermentation at 40 L pilot scale, totaling thirty unique yeasts used in this study. Yeasts from the Phaff Collection, as well as three commercial suppliers, were aseptically propagated from single cultures with the necessary cell volume for these pilot scale fermentations. Again, they were fermented in duplicate, with one fermenter receiving a dry-hop of 10 g/L, and fermentation was monitored until gravity was deemed terminal and the beer had fully attenuated. RDF, alcohol, and pH were tracked as fermentation progressed; the previously determined bench method was also utilized to determine the amount of hop creep expected. No Saccharomyces yeasts in this study presented themselves for the effective mitigation of hop creep, but analysis of this manner has never been performed before, so much insight was gained. More research remains on a correlation of flocculation and dry-hop creep, amino acid and diacetyl analysis on dry-hopped in comparison to non-hopped fermentations, and secondary metabolites from the fermentation of beer with these unique yeasts, especially in the presence of hops.

Regulatory phenomena occur in every phase of eukaryotic transcription. Recent improvements in the measurement of nascent RNA measurements have led to insights in elongation dynamics. Native elongating transcript sequencing (NET-seq) reports genome-wide RNA polymerase (RNAP) occupancy at nucleotide level resolution revealing phenomena associated with pausing, the control of anti-sense transcription elongation and others. Here we report that in S. cerevisiae elongation is characterized by regions of constant RNAP occupancy punctuated by abrupt reductions in occupancy. Furthermore, many of these locations are in the vicinity of covalent histone modifications, particularly H3K4 di- and tri-methylation. Additionally, these locations have a characteristic nucleotide type signature.

Nanotechnology takes advantage of cellular biology’s natural nanoscale operations by interacting with biomolecules differently than soluble or bulk materials, often altering normal cellular processes such as metabolism or growth. To gain a better understanding of how copper nanoparticles hybridized on cellulose fibers called carboxymethyl cellulose (CMC) affected growth of Saccharomyces cerevisiae, the mechanisms of toxicity were explored. Multiple methodologies covering genetics, proteomics, metallomics, and metabolomics were used during this investigation. The work that lead to this dissertation discovered that these cellulosic copper nanoparticles had a unique toxicity compared to copper. Further investigation suggested a possible ionic or molecular mimicry scenario with zinc, likely involving the Zrt family of zinc transporters and involving arrestin mediated endocytosis. Reactive oxygen species were generated by copper nanoparticles that induced lipid peroxidation, altering the phosphatidylcholine and phosphatidylethanolamine membrane composition, resulting in a disfigured cell surface. Following this study, I designed experiments aimed at furthering this dissertation's focus on metabolism by describing the metabolism of a novel species, Saccharomyces arboricola, thus filling a gap in knowledge in the industrial fermentation field. Low levels of endogenous amino/organic acids separated S. arboricola from the S. cerevisiae industrial strains and their interspecific hybrids showed a mosaic metabolic profile of parental strains. Overall, my dissertation research identified mechanisms of cellulosic copper nanotoxicity that included transport, metal homeostasis, reactive oxygen species, and the cellular membrane composition. Perturbations of Saccharomyces yeast by exogenous exposure to nanocopper or by interspecific hybridization had effects on the cellular metabolism.

Years of trait mapping studies have uncovered the genetic determinants underlying phenotypic evolution over both short and long timescales in a variety of organisms. A more general understanding of the mechanisms through which evolution produces phenotypic novelty, however, still remains out of reach. Many outstanding questions remain, among them how coding sequence divergence and gene expression divergence respectively contribute to trait evolution, as well as what role rare and large effect genetic variants play in the genetic networks underlying evolving phenotypes. Using Saccharomyces yeast as a genetically tractable model system in which to probe these questions, we identify and dissect several examples of trait divergence within and between yeast species. We first investigate an instance of intraspecific variation among yeasts isolated from a single population, finding that large effect rare variants at hypermutable loci are drivers of many common morphological and growth phenotypes. Next, in a comparison of both recently-diverged and long-diverged species, we investigate the role that gene expression variation plays in phenotypic evolution. We find evidence in multiple gene networks for complex and coherent regulatory evolution, and in one instance we succeed in identifying the loci that effect this change, as well as several phenotypic novelties they produce. Collectively, these investigations will contribute to the growing body of literature that describe the genetic mechanisms that underlie phenotypic change and seek, eventually, to achieve a greater understanding of basic evolutionary principles.

Genome-wide analysis of gene expression and regulation is important for elucidating basic principles of how cells function. In addition to gene expression, this dissertation will also discuss two methods of RNA-mediated RNA regulation: mRNA splicing and microRNAs. Three projects investigating gene expression and regulation using the Illumina platform are discussed here. The first project describes mRNA expression analysis of dozens of genes on the mating locus of the multicellular green alga, Volvox carteri in both the male and female mating types. The analysis describes sex-specific genes for both male and female mating types, and reveals the evolutionary history of the locus. The second project describes very low frequency splice products in a unicellular fungus with a fairly simple splicing landscape, Saccharomyces cerevisiae. These extremely rare splice events shed light on the mechanism of selecting splice sites in eukaryotic organisms. The third project describes an algorithm for predicting novel candidate microRNAs from small RNA sequence data. We describe a Naïve Bayes Classifier to differentiate microRNAs from contaminants and provide experimental validation of top candidates.

Cellular differentiation is an important process for the development and maintenance of an organism. Saccharomyces cerevisiae (yeast) has been successful as a tool for important discoveries relating to eukaryotic cellular processes. Stationary phase yeast cultures have been found to differentiate into two major cell types with distinct phenotypes. This genome-wide study using pooled samples of the yeast homozygous deletion set has identified over 400 genes which are important for differentiation of stationary phase yeast. These genes are predominantly involved in mitochondrial function thereby supporting our hypothesis that mitochondrial function is integral to the differentiation process. In addition, further experimentation focused on viability of yeast cultures revealed the importance of vacuolar proteins in survival of yeast cultures.

Sir proteins are responsible for maintaining stable transcriptional repression and silencing of telomeres, rDNA, and silent mating-type loci in S. cerevisiae. Decades of research identify a thorough list of the proteins required to establish and maintain silencing at HML and HMR, but despite many models being put forward, the mechanism by which Sir-proteins repress transcription has not been definitively determined. I sought to determine the mechanism of transcriptional silencing within heterochromatin in yeast. I tested models of transcriptional silencing using a variety of orthogonal in vivo approaches to build a more comprehensive model of the mechanism of silencing by Sir proteins. Specifically, I assayed for RNA Pol II in an engaged, DNA-melted form at the promoter of HMLα1 to conclusively test predictions of the downstream-inhibition model. I then adapted the single-subunit RNA polymerase of T7 phage and its cognate promoter to test the ability of a silenced template to be transcribed by a heterologous transcription assay in vivo. Finally, I adapted the thoroughly studied GAL1 promoter and Gal4 activator to test whether the extent of repression by Sir-proteins at HMR is sensitive to the nature of the transcription factor or to the promoter being repressed. These studies provided a framework for understanding the mechanism of Sir-protein repression. I propose a dynamic affinity-based competition model for transcriptional silencing where Sir proteins primarily restrict transcription before RNA Polymerase II has melted DNA at its target and competition between Sir proteins and gene-specific activators at promoters determines the efficiency of silencing.

The 2 micron plasmid of Saccharomyces cerevisiae is a multi-copy circular DNA genome that resides in the nucleus and exhibits nearly chromosome-like stability in host populations. Several host factors are required for equal plasmid segregation during cell division. One of them is cohesin (a multi-subunit protein complex) which mediates sister chromatid cohesion, a crucial mechanism for faithful segregation of replicated chromosomes in eukaryotes. The 2 micron plasmid mimics chromosomes in assembling cohesin at its partitioning locus. Studies on minichromosomes (centromere containing plasmids) reveal that cohesin forms a ring that embraces replicated sister centromeres topologically rather than physically. The functional similarities between chromosome and plasmid segregation prompted us to examine whether the topological mechanism proposed for centromere-mediated replicative cohesion is also true in the case of the plasmid. In the present study, we have characterized the nature and stoichiometry of cohesin's association with the 2 micron plasmid. Another host factor required for equal plasmid segregation is the CenH3 histone variant Cse4, so far considered to be uniquely associated with centromeric nucleosomes. Cse4 provides an epigenetic landmark at centromeres, and is required for assembly of the kinetochore complex. Surprisingly, Cse4 also interacts with the 2 micron plasmid partitioning locus. We have now functionally characterized this interaction, which can be preserved even in an ectopic, chromosomal context. The steady state level of Cse4 is highly limiting in yeast due to ubiquitin-mediated proteolysis. Only centromere-associated Cse4 is protected from this regulatory turnover control. We find that, in contrast to the situation with centromeres, association of Cse4 with the 2 micron plasmid is highly sub-stoichiometric but still promotes equal plasmid segregation. We also find that Cse4 induces an unusual right handed DNA writhe at the plasmid partitioning locus, as it does at the centromere. Our findings suggest that the plasmid has designed strategies to minimize the utilization of host factors that are in short supply. They signify the advantage of clustering and group behavior in the evolutionary success of a multi-copy selfish genome. Finally, they also suggest the possible emergence of the yeast centromere and the plasmid partitioning locus from a common ancestral sequence.

In quiescent yeast, the widespread reorganization of cytosolic proteins into punctate has been observed (Narayanaswamy et al. 2009). We seek to better understand and describe this reorganization, which we hypothesize to be a protein aggregation phenomenon. To test this hypothesis, we examined mutant proteins (Ade4p protein variants) in yeast with predicted non-native aggregation propensities and measured their punctate formation kinetics. Monitoring punctate formation kinetics involved the validation of an automated quantification technique using an Amnis ImageStream imaging flow cytometer. The automated punctate counts were strongly correlated with the manual punctate counts, with usual R² values of 0.99 or better, but evaluated 50-fold more cells per run. Fitness evaluations of the mutant yeast in the form of growth curves and batch competition experiments revealed the slowed growth of the Ade4-1286 strain and the functional inequality to the wild type strain of the Ade4-mtoin2034, Ade4-mtoin2105, and Ade4-2800 strains in competition experiments, especially when the mutants were forced to generate their own adenine. Subsequent structural analysis of the mutant proteins revealed destabilizing mutations for 4 of the 6 mutant proteins with 2 of the mutations classified as significantly destabilizing ([delta][delta]G >2 kcal/mol). We concluded that the reduction in protein fitness was likely due to the destabilizing effects of the mutations. Evaluation of the punctate formation kinetics revealed little difference between strains in the rate of punctate formation. Further examination revealed the wild type Ade4p and all of the mutants (with the exception of the Ade4-1286 mutant) were predicted to have similar aggregation propensities according to a secondary aggregation predicting algorithm (Zyggregator, Pawar et al. 2005). Additionally, solvent accessibility calculations estimate ~3-19% of the side chain surface area to be solvent accessible, which indicates proximity of mutations to the protein surface. However, mutating buried amino acids likely would have generated a greater disturbance (Matthews 1993, Tokuriki et al. 2007). We concluded that the mutations, although destabilizing, altered the aggregation propensity very little. Deletion of chaperone proteins (Hsp82p, Hsc82p, and Ssa1p) revealed no difference in the Ade4-GFP punctate formation kinetics, although a slight kinetic difference was detected in the chaperone (Hsp82p) knockout, Gln1-GFP strain and the wild type strain. While further workup is necessary in the chaperone knockout, Gln1-GFP work, the initial results are promising and suggest the involvement of protein folding machinery in punctate formation.

Autophagy is the major degradative process for recycling cytoplasmic constituents and eliminating unnecessary organelles in eukaryotic cells. In yeast autophagy is primarily a response to nutrient limitation for maintaining cellular homeostasis, and in higher eukaryotes it also plays a role in developmental processes and various pathophysiological states. Due to its essentially unlimited degradative capacity, it is critical that regulatory mechanisms are in place to modulate the timing and magnitude of the autophagic response. One set of autophagy-related (Atg) proteins that seems to function in this regard includes a complex that contains the Atg1 kinase. Aside from Atg1, the proteins in this complex participate primarily in either nonspecific autophagy or specific types of autophagy, including the cytoplasm to vacuole targeting (Cvt) pathway, which operates under vegetative conditions, and peroxisome degradation. Accordingly, these proteins are prime candidates for factors that regulate the conversion between these pathways, including the change in size of the sequestering vesicle, the most obvious morphological difference. Atg17 is identified as an autophagy-specific component based on a study of the atg17Delta mutant. It may regulate the magnitude of the autophagic response as part of the Atg1 complex. Particularly, a physiological function of Atg17 is proposed to be the regulation of the initial steps of vesicle formation in starvation conditions. Most Atg proteins are recruited to the phagophore assembly site (PAS), a proposed site for vesicle formation during either nonspecific or specific types of autophagy. Therefore, appropriate recruitment of Atg proteins to this site is critical for their function in autophagy. Atg11 facilitates PAS recruitment for the Cvt pathway that occurs under vegetative conditions. In contrast, Atg17 seems to play a similar role to Atg11 in PAS formation under autophagy-inducing conditions. Furthermore I propose that protein complexes containing the Atg1 kinase have two roles for PAS formation during nonspecific autophagy. The Atg1 C terminus mediates an interaction with Atg13 and Atg17, facilitating a structural role of Atg1 that is needed to efficiently organize an initial step of PAS assembly, whereas Atg1 kinase activity affects the dynamics of protein movement at the PAS involved in Atg protein cycling.

Cell homeostasis is maintained by the balance between biosynthesis and degradation. The lysosome/vacuole is one of the major cellular degradative sites. Multiple mechanisms are utilized by proteins to target to this organelle, including autophagy. As a highly conserved cellular process, autophagy can protect the cell from various stresses as well as promote cell death in certain conditions. Extensive studies of the molecular machinery of autophagy in the yeast Saccharomyces cerevisiae have revealed 31 autophagy-related proteins. Here, Trs85, a component of Transport Protein Particle (TRAPP) complex, was identified as being specifically involved in both specific- and non-specific autophagy and found to be required for autophagosome formation, suggesting it as a new autophagy-related protein. Another vacuolar targeting pathway is the multivesicular body pathway, which mediates membrane receptor downregulation and biosynthetic delivery of certain vacuolar hydrolases. My study indicated that the yeast acid trehalase, which hydrolyzes the disaccharide, trehalose, an important carbon reserve and membrane protectant against stress, is transported into the vacuole via the multivesicular body pathway. Further analysis uncovered a new sorting signal utilized by this protein to target into the multivesicular body in an ubiquin-independent manner.

Ribosome biogenesis in eukaryotes is a complex process that involves more than 170 trans-acting proteins. Many of these trans-acting factors are predicted to be enzymes that are important for assembly. One of these proteins, Saccharomyces cerevisiae Nog1p, is a nucleolar member of the Obg/CgtA family of monomeric GTPases. Depletion of Nog1p leads to defects in 60S ribosomal subunit assembly, rRNA processing, and 60S ribosomal subunit nuclear export, indicating that Nog1p is critical for the biogenesis of the 60S ribosomal subunit. This dissertation focuses on the characterization of mutants and truncated variants of Nog1p. Several mutations in conserved residues of the GTP-binding domain of Nog1p affect cell growth and 60S assembly, but do not affect the association of Nog1p with the pre-60S particle. A mutation in the GTP-binding domain, nog1P176V, was found to modestly affect the steady-state levels of rRNA precursors and the protein levels in pre-60S complexes. Studies utilizing truncated variants of Nog1p revealed that, of the 647 amino acids that comprise Nog1p, both residues 1-126 and the highly acidic region between 456 and 479 are necessary for function, but not for ribosome association. In contrast, however, amino acids 347-456 are critical for 60S ribosome association. Additionally, the last 168 amino acids of Nog1p (479-647) contribute to, but are not critical for Nog1p function. Clustered charge-to-alanine mutagenesis studies revealed several amino acids in both the N- and C-termini that are also important, but not critical for Nog1p function. Together these data suggest that Nog1p makes multiple contacts with interacting partners in the cell, and that these interactions are important for optimal Nog1p function.

The virtual 2-dimensional gel (V2DG) substitutes the size dimension of standard 2-dimensional electrophoretic gels by direct matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis of an immobilized pH gradient (IPG) gel, thereby increasing mass accuracy and resolution, and promising future advantages in automated proteomics. To understand the practical issues in the use of V2DG, this technology was applied to the detection of proteins encoded by the small Open Reading Frames (smORFs) of S. cerevisiae. The complete genome of this model organism has been sequenced, and most of the larger protein-coding ORFs have been detected and characterized. The protein products of many smORFs, however, continue to elude detection. The current study uses the identification of small proteins in S. cerevisiae as a means of pinpointing and addressing the challenges remaining for V2DG technology in 3 crucial areas: (1) processing of the IPG gel, (2) control of the mass spectrometer, and (3) software processing of mass spectrometric data files. Proteins of S. cerevisiae were focused on IPG strips, and the strips were introduced directly into both single MALDI-TOF MS and tandem MALDI-TOF-TOF MS instruments. Single stage MALDI-MS analysis was used to characterize signal quality on the gel surface. Tandem MALDI-MS analysis generated ion fragmentation spectra used to identify protein-coding ORFs in a database containing the full nucleotide sequence of S. cerevisiae. The unique contributions made by this research include: (1) showing that tandem MS can be used to make protein identifications directly from V2DGs, (2) development of a set of software programs dedicated specifically to the creation, manipulation, and analysis of V2DGs were developed, and (3) showing how the ion optics parameters of the MALDI mass spectrometer can be tuned to compensate for variation in the surface height of the IPG strip to generate ion fragmentation spectra with sufficient signal strength for database searches. One remaining technical challenge is the need to preserve the sharply focused proteins bands on the strip.

RNase P is an endoribonuclease responsible for cleavage of the 5' leader of precursor tRNAs (pre-tRNAs). Most forms of RNase P are ribonucleoprotein complexes containing RNA and protein subunits. Bacterial RNase P contains one RNA moiety and one protein subunit, with the RNA subunit by itself capable of cleaving pre-tRNAs in vitro. Eukaryotic nuclear RNase P also has a single RNA molecule, but many more protein subunits. The RNA subunits are relatively conserved in all three kingdoms of life, but structural elements of the RNA are identified that are conserved only in eukaryotes. Eukaryotic RNase P is closely related to another ribonucleoprotein enzyme, RNase MRP, which is involved in 5.8S rRNA maturation. Several protein subunits are common to RNases P and MRP. My thesis research focuses on functional characterization of the eukaryotic-specific features of nuclear RNase P in the yeast Saccharomyces cerevisiae . This work is illustrated in Chapters II and III of my dissertation. Chapter II describes characterization of the eukaryotic-specific conserved elements of the yeast RNase P RNA. Four eukaryotic-specific regions were mutated, and effects of the mutations on cell growth, enzyme function and biogenesis of RNase P were examined. Results suggest that most eukaryotic-specific conserved regions of RNase P RNA are important for holoenzyme assembly, localization and pre-tRNA processing. Chapter III focuses on characterization of the largest common protein subunit of yeast RNase P and MRP, Pop1p. Sequence alignment of Pop1p with its homologs has revealed four conserved regions. Mutations at the highly conserved positions were obtained by randomization mutagenesis, and were screened for conditional growth defects. Most Pop1p mutations affect both pre-tRNA and 5.8S rRNA processing, but a few mutations preferentially weaken only one of the two activities. In many cases, functional defects of RNase P and MRP are consistent with assembly defects of the holoenzymes. Mutations were also obtained that slow RNase P and MRP functions without notably affecting holoenzyme assembly. Taken together this study suggests that Pop1p plays a role in assembly and stability of the RNases P and MRP holoenzymes, but that it also has additional roles in the functions of the enzymes.

The Obg subfamily of GTPases is a novel family of GTP-binding proteins that are universally conserved and predicted to play a role in translation. Interestingly, all Obg proteins studied to date are involved in some aspect of ribosome function. In Chapters 2 and 3 of this study I demonstrate that the Saccharomyces cerevisiae GTPase, Mtg2p (Yhr168wp), is essential for mitochondrial ribosome function. Cells lacking MTG2 lose their mitochondrial DNA, giving rise to respiratory incompetent cells. In addition, cells expressing a temperature sensitive mgt2-1 allele are defective in mitochondrial protein synthesis and contain lower levels of mitochondrial ribosomal subunits. Significantly, elevated levels of Mtg2p partially suppress the thermosensitive loss of mitochondrial DNA in a 21S rRNA methyltransferase mutant, mrm2. Consistent with a role for Mtg2p in mitochondrial ribosome biogenesis, I show that Mtg2p is peripherally localized to the mitochondrial inner membrane and associates with the 54S large ribosomal subunit in a salt-dependent manner. The Obg subfamily of bacterial GTP-binding proteins are biochemically distinct from Ras-like proteins raising the possibility that they are not controlled by conventional guanine nucleotide exchange factors (GEFs) and/or guanine nucleotide activating proteins (GAPs). To test this hypothesis, in Chapter 4 we generated mutations in the Caulobacter crescentus obg gene, (cgtAC) which, in Ras-like proteins, would result in either activating or dominant negative phenotypes. I purified CgtACS173N (analogous to RasS17N) and CgtACP168V (analogous to RasG12V) protein and measured their guanine nucleotide affinities, exchange rates and half life of hydrolysis. In C. crescentus, a P168V mutant is not activating in vivo, although in vitro, the P168V protein showed a modest reduction in the affinity for GDP. Neither the S173N nor N280Y mutations resulted in a dominant negative phenotype. Further, the S173N was significantly impaired for GTP binding, consistent with a critical role of this residue in GTP binding. In general, conserved amino acids in the GTP-binding pocket were, however, important for function.

To survive extreme environmental conditions, pathological situations, and in response to certain developmental programs, eukaryotic organisms employ a degradative process called autophagy. This pathway targets cytosolic components and organelles for destruction in the vacuole/lysosome through engulfment by double-membrane vesicles that form in the cytosol. Despite our increased understanding of the significance of this pathway to eukaryotic development and disease, and the fact that many specific genetic components have been identified, the molecular mechanisms leading to the formation of these double-membrane vesicles remains largely unknown. Many advances have been made with the discovery of a pathway closely related to autophagy in the yeast Saccharomyces cerevisiae. This pathway, termed the Cytoplasm to Vacuole Targeting (Cvt) pathway, is a biosynthetic pathway to target residential vacuolar hydrolases properly; however, despite its metabolic role, it is both genetically and molecularly related to the catabolic autophagy pathway. This thesis examines the function of several molecular components involved in these pathways in S. cerevisiae. Chapters 2 and 3 describe the characterization of ATG23, a previously unidentified gene required for Cvt and efficient autophagy. Atg23 displays functional and physical interactions with Atg9, a protein of unknown function. This work has described new functions for five Atg proteins, as well as a previously unidentified step in Cvt and autophagy. In chapter 4, I present analysis of the Atg12-Atg5-Atg16 protein complex, whose function was previously unknown. I provide evidence consistent with the hypothesis that these proteins form a coat complex around the double membrane vesicle. This includes fluorescent microscopy images showing the Atg16-containing protein complex forming a structure around a growing vesicle. This is the first report of visualizing vesicle formation in vivo by fluorescence microscopy in S. cerevisiae. This technique can be expanded to visualize many Atg proteins during the formation of double membrane vesicles. These discoveries have advanced our understanding of these two pathways. There are many remaining questions, however; Chapter V describes work that remains to be done to advance on these discoveries.

The regulated transfer of protein and lipid cargo between organelles is essential to the maintenance of eukaryotic cell structure. Such processes are particularly critical at the trans Golgi network (TGN), where extensive, highly coordinated protein sorting must occur to mediate vesicular communication between the TGN and other organelles including the vacuole, endosomes, the plasma membrane and earlier Golgi compartments. To gain a better understanding of protein trafficking at the TGN, we use the baker's yeast, Saccharomyces cerevisiae as a model system to study the transmembrane TGN resident processing protease, Kex2p. Kex2p maintains steady-state localization to the TGN by cycles of transport between TGN and endocytic compartments, making it an excellent reporter for vesicular delivery between these compartments. Using Kex2p as a marker, we have begun to reconstitute TGN transport and fusion events in cell-free systems. Initial experiments identified a previously uncharacterized membrane fusion event, homotypic fusion of TGN compartments. Homotypic TGN fusion was found to require a t-SNARE complex consisting of Tlg2p, Tlg1p and Vti1p. Fusion was impaired between TGN membranes lacking function of the rab GTPase, Vps21p. A putative membrane tethering complex protein Pep3p was also required for fusion. As is the case for homotypic fusion of other endomembrane compartments, this fusion likely reflects a basic mechanism for maintenance of the integrity of the TGN. To further the analysis of Kex2p trafficking at the TGN, we developed a cell-free assay for the specific transport of Kex2p from the TGN to the late endosome/prevacuolar compartment (PVC). This transport step is critical for the biogenesis of the yeast vacuole/lysosome as it is the step in which hydrolytic enzyme precursors are sorted away from secretory protein traffic for delivery into the endosomal/lysosomal membrane system. Cell-free TGN to PVC transport requires Pep12p, a neuronal syntaxin homolog thought to mediate fusion of all incoming vesicular traffic at the prevacuolar compartment. This work enhances our understanding of TGN trafficking by illuminating a novel membrane fusion event, homotypic TGN fusion, that may be important for TGN maintenance and sorting, and sets the stage for biochemical dissection of TGN to late endosomal delivery of lysosomal precursor enzymes.

Previous reports have suggested that the Obg subfamily proteins are involved in ribosome function in both prokaryotes and eukaryotes. Rbg1p is an uncharacterized protein of Saccharomyces cerevisiae belonging to the Obg subfamily of GTP-binding proteins. We show that Rbg1p associates exclusively with translating ribosomes. Rbg2p, a related Obg protein, also partially associates with polyribosomes. In an effort to further delineate the cellular function of Rbg1p, a yeast 2-hybrid screen was conducted. Among the Rbg1p interacting proteins identified was a protein of unknown function, Gir2p. Gir2p contains a domain also found in Gcn2p (an essential factor of the general control pathway) called the GI domain. Interestingly, Gir2p, like Gcn2p, interacts with Gcn1p. The GI domain of Gir2p mediated this interaction. Overexpression of Gir2p in the presence of 3-amino triazole resulted in cell death as has been previously observed with the overexpression of Gcn2p, providing further evidence of an in vivo interaction between Gir2p and Gcn1p. Finally, Gir2p co-fractionated with polyribosomes and this fractionation pattern was partially dependent on the presence of Gcn1p. Based on these findings, we concluded that Rbg1p and its interacting partner Gir2p associate with ribosomes and with a key protein involved in stress response raising the possibility that Rbg1p is also involved in stress response. The studies presented here also describe the cellular localization of CgtAE. CgtAE is an essential Escherichia coli Obg protein. We show that CgtAE is associated predominantly with the 50S ribosomal subunit. Interestingly, CgtAE co-purifies with SpoT, a ppGpp hydrolase/synthetase involved in stress response. The interaction between CgtAE and SpoT was confirmed using reciprocal co-precipitation experiments and by two-hybrid assays. Consistent with an interaction with CgtAE, SpoT also co-fractionated with the large ribosomal subunit as well, suggesting a possible location of interaction for these two proteins. These studies raise the possibility that the ribosome-associated CgtA E is involved in SpoT mediated stress response.

Many essential biological functions, including apoptosis, blood coagulation, and hormonal signaling, are regulated, in part, by proteolysis. To serve as a means of regulation, proteolytic cleavage must be performed in an extremely specific fashion because aberrant cleavage of non-substrate proteins would have unwanted consequences. Members of the Kex2/furin family of processing proteases cleaves a variety of substrates in the eukaryotic secretory pathway. Although this family of proteases contain the same catalytic fold and similarly hydrolyze peptide bonds C-terminal to basic residues, the consensus cleavage sites of these well characterized proteases are distinct from one another. The substrate specificity of the prototypical member of this family, Kex2 from S. cerevisiae, was investigated using several strategies. First, the substrate specificity for P4 and P6 residues was analyzed by substituting basic residues into the putative P4 and P6 binding pockets by site directed mutagenesis. Whereas Kex2 shows little recognition for P6 Arg, one mutant, T252D Q283E-Kex2, exhibited 15-fold preference for Arg versus Ala at P6. Another mutant, E255I-Kex2 was predicted to reduce recognition for Arg at P4, and as expected, it showed decreased processing of P4 Arg peptide substrates in some contexts. In a second set of experiments, a series of Kex2 mutants with altered P2 specificity that were obtained in a genetic screen were analyzed biochemically. The processing of wild-type and the target specificity was less dramatic than observed in the genetic assay. However, they followed the same general trends in improved cleavage of non-native P2 substrates. Also, an attempt was made to redesign a genetic selection to obtain more robust Kex2 mutants with arbitrary P2 specificity, but this effort was hampered by a prohibitively high background. Finally, a novel member of the Kex2/furin family, Prt1 from the fungal pathogen P. carinii, was expressed in Sf9 cells and purified. N-terminal sequencing data indicated that the propeptide was not autocatalytically processed. It is possible that this protease needs an activating protein that is not expressed in insect cells or more physiological conditions for autoproteolysis of its propeptide.

Ribonuclease P (RNase P) is the enzyme responsible for a cleavage of the 5' leaders of pre-tRNAs in all living organisms. In the yeast Saccharomyces cerevisiae, nuclear RNase P consists of one RNA (RPR1 RNA) and nine protein subunits. RPR1 RNA is initially transcribed as a precursor and subsequently processed into a mature form. Defects in subunit assembly have been associated with a failure of RPR1 RNA to be processed from the precursor to mature form. In order to understand the holoenzyme subunit assembly, the studies in this dissertation address the nature of the precursor form of RNase P (pre-RNase P). Instead of using a long, multistep chromatographic purification of RNase P, which could lead to degradation and loss of the enzymatic activity, a rapid and specific affinity isolation of the enzyme is strongly preferred. However, the RNA affinity ligands for detection and isolation of RNAs or ribonucleoproteins have been lacking. In this study, two small RNA tags have been identified through in vitro selection. These tags bind specifically to Sephadex or streptavidin, and the binding to their targets can be disrupted under mild conditions with either dextran or d-biotin, respectively. This useful feature allows specific isolation and recovery of the tagged RNAs or ribonucleoproteins with intact structures and activity. To isolate RNase P, RPR1 RNA was tagged at various positions with the Sephadex-binding RNA, enabling a rapid and specific affinity isolation of either pre- or mature-RNase P. Pre-RNase P was found to be as active in pre-tRNA cleavage in vitro as mature-RNase P. Its protein subunit composition was also similar to that of mature-RNase P, except that very little Pop3p and Rpr2p could be detected relative to the other subunits. Further depletion of these two subunits did not affect RPR1 RNA maturation even though they are required for RNase P function in vivo. In situ hybridization showed that pre-RNase P RNA was localized primarily in the nucleolus, indicating that the assembly is likely to occur there. The finding that pre-RNase P was enzymatically active suggests that, in addition to being the precursor, it might have an in vivo catalytic role for either pre-tRNAs or other unidentified substrates.