Protein aggregation has long been associated with disease states, such as Alzheimer’s, Huntington’s, and Parkinson’s diseases. This model is challenged by increasing recognition of functional protein aggregates. Our focus has been exploration of intra-aggregate organization, and investigation of the ability of ATP to mediate energy independent aggregate solubilization by acting as a hydrotrope. Furthering our understanding of the composition and regulation of these structures will provide insight into how their dysregulation gives rise to disease, and provide a template for the development of novel therapeutics. Sub-nuclear organelles are a class of non-membrane bound organelle. These compartments form through liquid-liquid phase separation of their components, resulting in a functional aggregate that demonstrates liquid properties. Many of the factors driving liquid-liquid phase separation also drive the formation of amyloid fibrils. In vitro studies have demonstrated that liquid-liquid phase separation can promote amyloid fibril formation, and that liquid-liquid phase separated droplets can mature through amyloid fibril formation. In vivo, functional amyloids are known to contribute to peptide hormone storage, melanin production, and maintenance of long-term memory. Despite these findings, there has been limited investigation into the relationship between liquid-liquid phase separated droplets and amyloid fibrils in vivo. Our exploration of intra-aggregate organization took advantage of the numerous protein aggregates found within Xenopus oocytes. Using a dye and antibodies specific to the defining cross-β structure of amyloid, we demonstrated that amyloids form as a normal part of Xenopus oogenesis. Amyloid was detected within nuclear and cytosolic aggregates. In the cytosol, amyloid was observed within yolk platelets and a population of cortical particles whose identity has yet to be determined. In the nucleus, multiple liquid-liquid phase separated sub-nuclear organelles, including those associated with RNA polymerase I, II and III transcription as well as RNA processing, were found to have an amyloid component. Proteomic analysis of aggregate enriched material revealed proteins associated with ribonucleoprotein complex biogenesis, DNA replication, RNA processing and DNA-templated transcription. Our analysis further demonstrated that nuclear amyloids were stable, remaining intact for hours post isolation, but rapidly destabilized following treatment with RNase. Recently, adenosine triphosphate (ATP) was demonstrated to act as a hydrotrope in vitro. Physiologic levels were shown to maintain protein solubility and solubilize recombinant liquid-liquid phase separated droplets, in the absence of ATP hydrolysis. These observations suggest that this previously unappreciated activity of ATP has a large influence on liquid-liquid phase separated droplet stability. However, these observations were made under conditions that diverge from those found in vivo. The liquid-liquid phase separated droplets tested were devoid of RNA and composed of a single protein. Amyloid containing aggregates required super physiological levels of ATP, suggesting the sub-nuclear organelles we previously described would be resistant to this activity. By observing nucleoli within isolated Xenopus oocyte nuclei, we demonstrate that ATP has the capacity to act as a hydrotrope in vivo. However, the hydrotropic action alone of ATP is not sufficient to solubilize nucleoli. Instead, solublization requires a sensitization step which is dependent on a soluble factor(s) and requires ATP hydrolysis. Our studies support an in vivo model of liquid-liquid phase separated aggregate solubilization where a soluble factor(s) sensitizes an aggregate in an energy dependent process. The susceptible aggregate is then solubilized by the hydrotropic action of ATP. We further speculate that liquid-liquid phase separated aggregate formation is a reversal of the above steps. These findings highlight a need to further understand the relationship between liquid-liquid phase separation and amyloid formation, and identify an urgent need to dissect the factors influencing intracellular ATP levels.

Proliferating cell nuclear antigen (PCNA) is a versatile protein involved in all pathways of DNA metabolism. It is best known as a processivity factor for classical polymerases, which synthesize DNA on non-damaged templates during DNA replication (ex: pol δ). Non-classical polymerases, on the other hand, are those that synthesize DNA on damaged templates (ex: pol η). PCNA also functions in repair, recombination, and most other DNA-dependent cellular processes. A number of separation of function mutant PCNA proteins have been identified, suggesting that PCNA could be a valuable target to manipulate DNA metabolism. This thesis focuses on the study of PCNA mutant proteins that affect translesion synthesis (TLS) and mismatch repair (MMR). During TLS, the process by which DNA polymerases replicate through DNA lesions, PCNA recruits and stabilizes polymerases at the replication fork. TLS requires the monoubiquitylation of PCNA, and PCNA and ubiquitin-modified PCNA (Ub-PCNA) stimulate TLS by classical and non-classical polymerases. Two mutant forms of yeast PCNA, one with an E113G substitution and one with a G178S substitution, support normal cell growth but inhibit TLS. To better understand the role of PCNA in TLS, I re-examined the structures of both mutant PCNA proteins and identified substantial disruptions of the subunit interface that forms the PCNA trimer. This resulted in reduced trimer stability in the mutant proteins. The mutant forms of PCNA and Ub-PCNA do not stimulate TLS of an abasic site by either classical pol δ or non-classical pol η. Normal replication by pol η was also impacted, but normal replication by pol δ was much less affected. These findings support a model in which reduced trimer stability causes these mutant PCNA proteins to occasionally undergo conformational changes that compromise their ability to stimulate TLS by both classical and non-classical polymerases. During MMR, PCNA recruits and coordinates proteins involved in the mismatch recognition, excision, and resynthesis steps. Previously, two mutant forms of PCNA were identified that cause defects in MMR with little if any other defects. These are the C22Y and C81R mutant PCNA proteins. In order to understand the structural and mechanistic basis by which these two substitutions in PCNA proteins block MMR, we solved the X-ray crystal structures of both mutant proteins and carried out further biochemical studies. I found that these amino acid substitutions lead to distinct structural changes in PCNA. The C22Y substitution alters the positions of the α-helices lining the central hole of the PCNA ring, whereas the C81R substitution creates a distortion in the β-sheet at the PCNA subunit interface. I conclude that the structural integrity of the α-helices lining the central hole and the β-sheet at the subunit interface are both necessary to form productive complexes with MutSα and mismatch-containing DNA. As described above, my studies focused on four amino acid substitutions in PCNA that disrupt TLS and MMR: the E113G and G178S substitutions cause defects in TLS while the C22Y and C81R substitutions cause defects in MMR. The structures of these mutant PCNA proteins revealed that three of the four substitutions caused disruptions near the subunit interface of PCNA. To further examine the importance of this region, we generated random mutations of the PCNA subunit interface and performed in vivo genetics assays and in vitro biochemical assays to examine their effects on TLS and MMR. We determined that the subunit interface of PCNA is very dynamic and that small changes at this interface can cause drastically different effects on TLS and MMR. Moreover, we suggest that the integrity of the subunit interface as well as the nearby β-strands in domain A are crucial for proper PCNA function in vivo and in vitro.

Hrr25 is a highly conserved serine/threonine protein kinase with diverse functions and it localizes to a number of intracellular sites. Hrr25 possesses an N-terminal kinase domain, a middle region, and a C-terminal proline/glutamine rich domain. In this thesis, we systematically characterized the roles of these three domains of Hrr25 on cell growth, cell morphology, and its cellular localizations. Additionally, we identified a novel target of Hrr25, Pin4. We show that Hrr25-dependent phosphorylation of Pin4 is required for cell wall integrity maintenance. We found that that Hrr25 and Pin4 physically interact in vivo and that the C-terminal domain of Hrr25 is required for this interaction and for maintaining cell wall integrity. Together, we identified a new target and a novel function for Hrr25 and provided insights into the structure-function relationship of Hrr25 domains.

Pdr5 and Yor1 are two ATP-binding cassette transporters regulated by the pleiotropic drug resistance (PDR) network in the yeast Saccharomyces cerevisiae. Recent work from another group demonstrated that a pdr5Δ yor1 strain confers remarkable resistance to a sphingolipid intermediate, phytosphingosine (PHS), which was surprising as loss of these transporters normally leads to elevated drug sensitivity. PHS is toxic to the cell at high levels due to mislocalization of nutrient permeases, such as the high affinity tryptophan transporter, Tat2. Although the above study suggested that this resistance was due to increased expression of Rsb1, a known mediator of PHS tolerance, this was not reproducible in our hands and we sought to identify other determinants for this phenotype. The work presented here demonstrates that the pdr5Δ yor1 strain exhibits delayed turnover of Tat2 and an increase in tryptophan uptake, which we postulate is due to changes membrane asymmetry resulting in decreased endocytosis. Conversely, cells lacking Rsb1 showed a decrease in tryptophan import and increased Tat2 turnover, independent of endogenous PHS levels. Rsb1 has a predicted 7 transmembrane (7TM) topology, which argues against the idea that Rsb1 functions directly in PHS transport, as there are currently no known transporters with this topology. These data suggest that Rsb1 and Pdr5/Yor1 function in regulation of endocytosis of Tat2, and possibly other membrane proteins. Ethyl methanesulfonate mutagenesis of the pdr5Δ yor1 strain and a candidate gene approach were alternative methods used to identify mediators of PHS tolerance in this strain. Inconsistent results from PHS selection led to the discovery that the pdr5Δ yor1 strain was also robustly resistant to the sphingolipid biosynthesis inhibitor, Aureobasidin A (AbA), which was subsequently used for analysis. These approaches revealed several genes, including Gda1, Mss4, and Ypk1 that are important for AbA tolerance in the pdr5Δ yor1 strain. Many of these determinants play a role in cell wall integrity, suggesting that loss of Pdr5 and Yor1 may lead to activated cell wall integrity pathways resulting in altered cell wall structure.

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Thesis (Ph. D.)--New York University, Graduate School of Arts and Science, 2005. / Typescript. Includes bibliographical references (leaves 188-191). Also available in electronic format on the World Wide Web. Access restricted to users affiliated with the licensed institutions.

Thesis (Ph. D.)--New York University, Graduate School of Arts and Science, 2005. / Typescript. Includes bibliographical references (leaves 148-184). Also available in electronic format on the World Wide Web. Access restricted to users affiliated with the licensed institutions.

Thesis (Ph. D.)--New York University, Graduate School of Arts and Science, 2005. / Typescript. Includes bibliographical references (leaves 114-125). Also available in electronic format on the World Wide Web. Access restricted to users affiliated with the licensed institutions.

Thesis (Ph.D.)--New York University, Graduate School of Arts and Science, 2007. / Typescript. Includes bibliographical references (leaves 182-227). Also available in electronic format on the World Wide Web. Access restricted to users affiliated with licensed institutions.