The transition between the closed and open conformations of the Sec61 complex permits nascent protein insertion into the translocation channel. A critical event in this structural transition is the opening of the lateral translocon gate that is formed by four transmembrane (TM) spans (TM2, TM3, TM7 and TM8 in Sec61p) to expose the signal sequence-binding (SSB) site. To gain mechanistic insight into lateral gate opening, mutations were introduced into a lumenal loop (L7) that connects TM7 and TM8. The sec61 L7 mutants were found to have defects in both the posttranslational and cotranslational translocation pathways due to a kinetic delay in channel gating. The translocation defect caused by L7 mutations could be suppressed by the prl class of sec61 alleles that reduce the fidelity of signal sequence recognition. The prl mutants are proposed to act by destabilizing the closed conformation of the translocation channel. Our results indicate that the equilibrium between the open and closed conformations of the protein translocation channel maintains a balance between translocation activity and signal sequence recognition fidelity. In the opening of the translocation channel, both the lateral and lumenal gate must open in a coordinated fashion for efficient protein translocation to occur. The lumenal gate is composed of a short helix of the loop preceding the second TM span, referred to as the plug helix, and six hydrophobic pore ring residues which form the constriction ring in the center of the channel. We identified three lateral gate polar residues and three hydrophobic residues from the plug domain that affect channel gating. Mutagenesis of the lateral gate polar cluster residues yields either a gain of function (prl phenotype) or a loss of function (translocation defect) phenotype. The combination of polar cluster mutations with each other or with plug domain mutations which cause a prl phenotype resulted in the mutually suppressive or additive phenotypes in double mutant strains. Cooperation between these residues is made possible through a structural link which connects the two translocation channel gates at their interface. The structural link provides a mechanism for the channel to coordinate the movement of multiple domains in the channel gating conformational change. Translocation assays demonstrated that this mechanism of gating regulation is particularly important for efficient protein translocation of substrates using the posttranslational translocation pathway. Our results indicate that residues from the plug and lateral gate domain form a regulatory cluster of residues responsible for efficient translocation channel gating.

Protruding from the apical surface of nearly every cell in our body lies a specialized sensory organelle—the primary cilium. Eukaryotic cells use these ubiquitous structures to monitor the extracellular environment, defects in which result in an ever-growing list of human maladies termed ciliopathies including obesity, retinal degeneration and polycystic kidney disease. The sensory functions of primary cilia rely on the unique complement of receptors concentrated within the ciliary membrane. Vital to the proper functioning of the cilium is the cell's ability to target specific proteins to the ciliary membrane yet little is known how a cell achieves this highly polarized distribution. IFT20, a subunit of the intraflagellar transport particle is localized to the Golgi complex that is hypothesized to sort proteins to the ciliary membrane. We show that IFT20 is anchored to the Golgi complex by the golgin protein GMAP-210 and mice lacking GMAP210 die at birth with a pleiotropic phenotype that includes growth restriction and heart defects. Cilia on GMAP210 mutant cells have reduced amounts of the membrane protein polycystin-2 localized to them suggesting IFT20 and GMAP-210 function together in the sorting or transport of proteins to the ciliary membrane. To better understand the mechanism of ciliary protein trafficking, we identify a ciliary targeting sequence (CTS) contained within fibrocystin, the gene mutated in autosomal recessive polycystic kidney disease, and investigate a series of proteins required for the delivery of this sequence to the primary cilium. We demonstrate the small G protein Rab8 interacts with the CTS of fibrocystin and controls the ciliary levels of the CTS. Arf4 is another small G protein deemed a key regulator of ciliary protein trafficking. We show Arf4 binds the CTS of fibrocystin but is not absolutely required for trafficking of the fibrocystin CTS to cilia. Arf4 mutant mice are embryonic lethal and die at mid-gestation likely due to defects in the non-ciliated visceral endoderm, where the lack of Arf4 caused defects in cell structure and apical protein localization. This suggests Arf4 is not only important for the efficient transport of fibrocystin to cilia, but also plays critical roles in non-ciliary processes. Together this work aims to elucidate the mechanisms of protein targeting to the ciliary membrane.

Chromosomal replication is an essential process in all life. This dissertation highlights regulatory roles for two critical protein complexes at the heart of the replication fork: 1) the sliding clamp, the major polymerase processivity factor, and 2) the sliding clamp loader, a spiral-shaped AAA+ ATPase, which loads the clamp onto DNA. The clamp is a promiscuous binding protein that interacts with at least 100 binding partners to orchestrate many processes on DNA, but spatiotemporal regulation of these binding interactions is unknown. Remarkably, a recent disease-causing mutant of the sliding clamp showed specific defects in DNA repair pathways. We aimed to use this mutant as a tool to understand the binding specificity of clamp interactions, and investigate the disease further. We solved three structures of the mutant, and biochemically showed perturbation of partnerbinding for some, but not all, ligands. Using a fission yeast model, we showed that mutant cells are sensitive to select DNA damaging agents. These data revealed significant flexibility within the binding site, which likely regulates partner binding. Before the clamp can act on DNA, the sliding clamp loader places the clamp onto DNA at primer/template (p/t) junctions. The clamp loader reaction couples p/t binding and subsequent ATP hydrolysis to clamp closure. Here we show that composition (RNA vs. DNA) of the primer strand affects clamp loader binding, and that the order of ATP hydrolysis around the spiral is likely sequential. These studies highlight additional details into the clamp loader mechanism, which further elucidate general mechanisms of AAA+ machinery.

The proper workings of an organism rely on the accurate expression of genes throughout its lifetime. An important determinant for protein production is the availability of template mRNA molecules, the net effect of which is governed by their rates of synthesis vs. their rates of degradation. Normal mRNAs are proposed to be relatively stable in the cytoplasm while present in a protective, circularized conformation – the closed loop – through eIF4G-bridged interactions with 3’-bound poly(A) binding protein (Pab1p) and 5’-bound eIF4E. Introduction of a premature nonsense codon into an otherwise normal mRNA results in its rapid destabilization in cells, suggesting that not all stop codons behave the same, and events at premature termination events that lead to accelerated degradation of nonsense-containing mRNAs likely differ from those at normal termination, in which normal decay rates are maintained. The enhanced degradation observed for nonsense-containing mRNAs occurs through an evolutionarily conserved pathway involving the products of the UPF1, UPF2/NMD2, and UPF3 genes, the precise biochemical roles of which have remained elusive. We have developed a yeast cell-free translation system that allows us to assay biochemical events occurring at premature termination codons, compare them to those occurring at normal terminators, and study the role of Upf1p in these events. We find that premature termination is an inefficient process compared to normal termination and that one outcome of termination at a premature termination codon (PTC) is reinitiation at a nearby start codon. This in vitro post-termination reinitiation phenotype is dependent on the presence of Upf1p, a finding we have recapitulated in vivo. We also developed biochemical assays to define a role for Upf1p in translation following premature termination in vitro and find that Upf1p is involved in post-termination ribosome dissociation and reutilization. Supporting this idea are our findings that Upf1p predominantly cosediments with purified 40S ribosomal subunits. Finally, using our in vitro translation/toeprinting system, we have further characterized events leading to the formation of the mRNA closed loop structure and find that two states of the closed loop exist. The first requires the preinitiation 48S complex and includes Pab1p, eIF4G, eIF4E, and eIF3, whereas the second is formed after 60S joining and additionally requires the translation termination factors eRF1 and eRF3.

White adipose plays a major role in the regulation of whole body metabolism through the storage and hydrolysis of triglycerides and by secretion of adipokines. The function of endocrine cells is highly dependent on the unfolded protein response (UPR), a homeostatic signaling mechanism that balances the protein folding capacity of the endoplasmic reticulum (ER) with the cell's secretory protein load. Here we demonstrate that the adipocyte UPR pathway is necessary for its secretory functions, and can thus play a crucial role in the control of whole body energy homeostasis. ER protein folding capacity is dependent both on the number of available chaperones as well as on their activity, which requires a sufficient ATP supply. In 3T3-L1 adipocytes, mitochondrial biogenesis occurred in parallel with induction of the UPR; therefore, we tested whether it was necessary for efficient ER function. Inhibition of mitochondrial ATP synthesis through depletion of Tfam, a mitochondrial transcription factor, or treatment with inhibitors of oxidative phosphorylation, demonstrate that ER function is sensitive to acute changes in adenine nucleotide levels. In addition, adenylate kinase 2 (AK2), which regulates mitochondrial adenine nucleotide interconversion, is markedly induced during adipocyte and B cell differentiation. AK2 depletion impairs induction of the UPR and secretion in both cell types. Interestingly, cytosolic adenylate kinase 1 (AK1) does not have the same effect upon UPR induction. We show that adenine nucleotides promote proper ER function and alterations in specific aspects of ATP synthesis can impair UPR signaling. Understanding the complex energetic regulation of the UPR may provide insight into the relationship between UPR and disease.

Haemophilus influenzae is a pathogenic Gram-negative bacterium that colonizes the upper respiratory tract of humans and can cause otitis media, upper and lower respiratory infections, and meningitis. Factors important for H. influenzae to colonize humans and cause disease are not fully understood. Different bacterial pathogens are armed with virulence mechanisms unique to their specific strategies for interacting with their hosts. Many of the proteins mediating these interactions are secreted and contain disulfide bonds required for function or stability. I postulated that identifying the set of secreted proteins in H. influenzae that require periplasmic disulfide bonds would provide better understanding of this bacterium's pathogenic mechanisms. In this thesis, the periplasmic disulfide bond oxidoreductase protein, DsbA, was found to be essential for colonization and virulence of H. influenzae. Mutants of dsbA were also found to be sensitive to the bactericidal effects of serum. However, the DsbA-dependent proteins important for pathogenesis of this organism have not been previously identified. To find them, putative targets of the periplasmic disulfide bond pathway were identified and examined for factors which might be important for mediating critical virulence aspects. By doing so, novel virulence factors were discovered including those important for heme and zinc acquisition, as well as resistance to complement. Overall, the work presented here provides insight into requirements for H. influenzae to survive within various host environments.

The utility of a model organism for studying biological processes is closely tied to its amenability to genome manipulation. Although tools for targeted genome engineering in mice have been available since 1987, most organisms including zebrafish have lacked efficient reverse genetic tools, which has stymied their broad implementation as a model system to study biological processes. The development of zinc finger nucleases (ZFNs) that can create double-strand breaks at desired sites in a genome has provided a universal platform for targeted genome modification. ZFNs are artificial restriction endonucleases that comprise of an array of 3- to 6-C2H2-zinc finger DNA-binding domains fused with the dimeric cleavage domain of the type IIs endonuclease FokI. C2H2-zinc fingers are the most common, naturally occurring DNA-binding domain, and their specificity can be engineered to recognize a variety of DNA sequences providing a strategy for targeting the appended nuclease domain to desired sites in a genome. The utility of ZFNs for gene editing relies on their activity and precision in vivo both of which depend on the generation of ZFPs that bind desired target sites high specificity and affinity. Although various methods are available that allow construction of ZFPs with novel specificities, ZFNs assembled using existing approaches often display negligible in vivo activity, presumably resulting from ZFPs with either low affinity or suboptimal specificity. A root cause of this deficiency is the presence of interfering interactions at the finger-finger interface upon assembly of multiple fingers. In this study we have employed bacterial-one-hybrid (B1H)-based selections to identify two-finger zinc finger units (2F-modules) containing optimized interface residues that can be combined with published finger archives to rapidly yield ZFNs that can target more than 95% of the zebrafish and human protein-coding genes while maintaining a success rate higher than that of ZFNs constructed using available methods. In addition to genome engineering in model organisms, this advancement in ZFN design will aid in the development of ZFN-based therapeutics. In the process of creating this archive, we have undertaken a broader study of zinc finger specificity to better understand fundamental aspects of DNA recognition. In the process we have created the largest protein-DNA interaction dataset for zinc fingers to be described that will facilitate the development of better predictive models of recognition. Ultimately, these predictive models would enable the rational design of synthetic zinc finger proteins for targeted gene regulation or genomic modification, and the prediction of genomic binding sites for naturally occurring zinc finger proteins for the construction of more accurate gene regulatory networks.

A key regulator of DNA damage-induced apoptosis is the tumor suppressor gene, p53. p53 is a transcription factor that upregulates genes involved in cell cycle arrest, apoptosis, and senescence. How p53 decides to activate one of these responses in response to DNA damage is largely unanswered. Many have hypothesized it is due to interaction with various signaling pathways and post-translational modification. The p53 tumor suppressor can be modified by SUMO-1 in mammalian cells, but the functional consequences of this modification are unclear. Conjugation to SUMO is a reversible post-translational modification that regulates several transcription factors involved in cell proliferation, differentiation, and disease. In Chapter II, we demonstrate that the Drosophila homolog of human p53 can be efficiently sumoylated in insect cells. We identify two lysine residues involved in SUMO attachment, one at the C-terminus, between the DNA binding and oligomerization domains, and one at the N-terminus of the protein. We find that sumoylation helps recruit Drosophila p53 to nuclear dot-like structures that can be marked by human PML and the Drosophila homologue of Daxx. We demonstrate that mutation of both sumoylation sites dramatically reduces the transcriptional activity of p53 and its ability to induce apoptosis in transgenic flies, providing in vivo evidence that sumoylation is critical for Drosophilap53 function. Many therapeutic cancer treatments rely on DNA-damaging agents to induce apoptosis in cancer cells. However, fifty percent of all human tumors lack functional p53 and p53 mutant cells are partially resistant to damage-induced apoptosis. Therefore, it is important to identify mechanisms to induce apoptosis independent of p53. Drosophila provides a good model system to study p53-independent apoptosis because it contains a single p53 homolog. In Chapter III, we describe a p53-independent mechanism that acts in parallel to the canonical DNA damage response pathway in Drosophila to activate apoptosis in response to inappropriately repaired chromosome breaks. Induction of chromosome aberrations by DNA damage followed by cell division results in segmental aneuploidy and reduced copy number of ribosomal protein genes. We find that activation of the pro-apoptotic gene hid by the JNK pathway acts in a p53-independent mechanism to induce apoptosis and limit the formation of aneuploid cells. Mutations in grp, the Drosophila Chk1 homolog, and puc, a negative regulator of the JNK pathway sensitize p53 mutant cells to IR-induced apoptosis. We propose a model in which the death of cells with reduced copy number of genes required for cell survival helps maintain genomic integrity following chromosome damage

In order to preserve its genomic integrity, an organism needs to detect and repair DNA double-strand breaks (DSBs) in a prompt and accurate fashion. This goal is accomplished by enabling an exquisitely sensitive DSB sensing apparatus as well as multiple and often overlapping pathways for repair. All of these processes are carried out on a highly organized and compacted chromatin substrate in the nucleus. An important question is whether chromatin plays an active role in the process and whether it helps in the signaling or repair of this damage. We have used Chromosome Conformation Capture (3C) to show that there are no large scale changes in chromosome structure at a single site-specific DNA double-strand break, although looping interactions between DSBs and donors can be detected. In a surprising result, we found that 3C detected a nucleus-wide decrease in interactions with the DSB. We have used a combination of 3C, fluorescence microscopy and chromatin immunoprecipitation to show that the decrease in interactions is a result of the relocalization of persistent DSB to the nuclear periphery. We also show that this is dependent on the recruitment of telomerase complex to the DSB, which then interacts with its natural partner in the Inner nuclear membrane, Mps3, and relocalizes the DSB to the periphery. Thus, a DSB that cannot be repaired is shunted into a pathway where the cell attempts to survive by putting a de novotelomere on the broken chromosome. Remarkably, this is not an irreversible phenomenon despite the recruitment of telomerase and the relocalization to the periphery. DSBs which are repaired slowly due to the presence of homology on a different chromosome, or merely usage of a kinetically slower form of repair, undergo this pathway switch, but can still recover and repair the DSB if homology is present. We also show that the role of the periphery is to ensure repair through de novotelomere formation or other non-canonical repair pathways. Indeed, loss of peripheral localization results in a dramatic suppression of the genomic instability of the Slx5/8 mutants, which have been implicated in the persistent DSB response at the Nuclear pores. Thus, the nuclear periphery is a special compartment where DSBs go after they cannot be repaired by canonical pathways. Specialized components such as telomerase, silencing proteins and components of the SUMO pathway, all seem to play roles in the healing of these chromosomes. Importantly, the SUN domain homologues of Mps3 have been shown to play roles similar to their yeast homologues in meiotic bouquet formation through their interactions with telomeres. Thus, they may represent a conserved mechanism for chromosome healing and telomere anchoring, despite the fact that mammalian telomeres are rarely found at the nuclear periphery. Such survival mechanisms may be expected to operate in cancer cells which may or may not have upregulated telomerase expression.

Neuronal nicotinic acetylcholine receptors (nAChRs) are involved in a plethora of fundamental biological processes ranging from muscle contraction to the formation of memories. The studies described in this work focus on the transcriptional regulation of the CHRNB4 gene, which encodes the ß4 subunit of neuronal nAChRs. We previously identified a regulatory sequence (5´– CCACCCCT –3´), or “CA box”, critical for CHRNB4 promoter activity in vitro. Here I report transcription factor interaction at the CA box along with an in vivo analysis of CA box transcriptional activity. My data indicate that Sp1, Sp3, Sox10 and c-Jun interact with the CHRNB4 CA box in the context of native chromatin. Using an in vivo transgenic approach in mice, I demonstrated that a 2.3-kb fragment of the CHRNB4 promoter region, containing the CA box, is capable of directing cell-type specific expression of a reporter gene to many of the brain regions that endogenously express the CHRNB4 gene. Site-directed mutagenesis was used to test the hypothesis that the CA box is critical for CHRNB4 promoter activity in vivo. Transgenic animals were generated in which LacZ expression is driven by a mutant form of the CA box. Reporter gene expression was not detected in any tissue or cell type at ED18.5. Similarly, I observed dramatically reduced reporter gene expression at PD30 when compared to wild type transgenic animals, indicating that the CA box is an important regulatory feature of the CHRNB4 promoter. ChIP analysis of brain tissue from mutant transgenic animals demonstrated that CA box mutation results in decreased interaction of the transcription factor Sp1 with the CHRNB4 promoter. I have also investigated transcription factor interaction at the CHRNB4 promoter CT box, (5´– ACCCTCCCCTCCCCTGTAA –3´) and demonstrated that hnRNP K interacts with the CHRNB4 promoter in an olfactory bulb derived cell line. Surprisingly, siRNA experiments demonstrated that hnRNP K knockdown has no impact on CHRNA5, CHRNA3 or CHRNB4 gene expression. Interestingly, knockdown of the transcription factor Purα results in significant decreases in CHRNA5, CHRNA3 and CHRNB4 mRNA levels. These data indicate that Purα can act to enhance expression of the clustered CHRNA5, CHRNA3 and CHRNB4 genes. Together, these results contribute to a more thorough understanding of the transcriptional regulatory mechanisms underlying expression of the CHRNB4 as well as the CHRNA5 and CHRNA3 genes, critical components of cholinergic signal transduction pathways in the nervous system.