Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Not embargoed. Vita. Bibliography: 120-126.

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 100 pages. Includes Vita. Includes bibliographical references.

α-Factor receptors from Saccharomyces cerevisiae are G-protein-coupled receptors containing seven transmembrane segments. The ability of α-factor receptors to form oligomeric complexes with each other and with other proteins was investigated. Both in vivo and in vitroevidence was obtained that suggests homo-oligomerization of receptors in the plasma membrane. When the membranes from cells coexpressing two differentially-tagged receptors were solubilized with detergent and subjected to immunoprecipitation, the antibodies specific for either epitope tag resulted in precipitation of both tagged species. Treatment of cultures with α-factor had little effect on the extent of oligomerization as judged by the sedimentation behavior of the receptor complexes and by the efficiency of coimmunoprecipitation. The ability of receptor complexes to undergo ligand-mediated endocytosis was evaluated by using membrane fractionation and fluorescence microscopy. Mutant receptors that fail to bind α-factor (Ste2-S184R) or lack the endocytosis signal (Ste2-T326) became competent for ligand-mediated endocytosis when they were expressed in cells containing wild-type receptors. Coimmunoprecipitation experiments indicated that the C-terminal cytoplasmic domain and intermolecular disulfide bonds were unnecessary for oligomer formation. Therefore, α-factor receptors form homo-oligomers and that these complexes are subject to ligand-mediated endocytosis. A crosslinking and immunoprecipitation strategy was used to capture and characterize the transient complexes that contain the α-factor receptor Ste2. Tagged receptors were crosslinked to form at least three high molecular weight complexes and the complexes were immunoprecipitated with antibodies against the tag. Western blotting analysis of the precipitated material revealed the presence of β and γ subunits of the heterotrimeric G protein, Ste4 and Stel8. Similar results were obtained when the cultures had been treated with α-factor prior to analysis. A truncated receptor missing most of the cytoplasmic C-terminal tail was also active in binding Ste4. Overall, these results constitute the first biochemical evidence for a physical association between the α-factor receptor and its cognate G-protein. Endocytic signals in the C-terminal tail (residues 297-431) of the α-factor receptor were analyzed. One signaling element, SINNDAKSS, (residues 331-339) is known to be sufficient (but not necessary) for endocytosis. Internal deletions of the STE2 gene were constructed that remove sequences encoding SINNDAKSS and selected regions of the C-terminal tail. Strains containing these alelles were then assayed for endocytosis in the presence and absence of α-factor. Residues from 360 to 431 were sufficient to mediate both constitutive and ligand-mediated endocytosis of the receptor even though 63 residues including the SINNDAKSS motif had been removed. Structural features of this region that were investigated further were the highly-ubiquitinated Lys374, the neighboring Lys387, and the GPFAD motif (residues 392-396). Lys374 and Lys387 were unnecessary for the element to promote exit from the plasma membrane; however, Lys374 may play some role in intracellular trafficking. The GPFAD motif was not sufficient to promote endocytosis, since the residues 360-399 provided no detectable endocytic activity. Overall, these results suggest that a new region in the C-terminal of the α-factor receptor, redundant with the SINNDAKSS motif, is sufficient to mediate the constitutive endocytosis as well as the ligand-mediated endocytosis of the receptor.

Modulating chromatin structure is an important step in maintaining control over the eukaryotic genome. SWI/SNF, one of the complexes belonging to the growing family of ATP-dependent chromatin remodeling enzymes, is involved in controlling the expression of a number of inducible genes whose proper regulation is vital for metabolism and progression through mitosis. The mechanism by which SWI/SNF modulates chromatin structure at the nucleosome level is an important aspect of this regulation. The work in this dissertation focuses on how the Saccharomyces cerevisiae SWI/SNF complex uses the energy of ATP-hydrolysis to alter DNA-histone contacts in nucleosomes. This has been approached in a two part fashion. First, the three-dimensional structure and subunit composition of SWI/SNF complex has been determined. From this study we have identified a potential region of the SWI/SNF complex that might [be] a site for nucleosomal interaction. Second, functional analysis of the ATPase domain of Swi2p, the catalytic subunit of SWI/SNF, has revealed that a specific conserved motif is involved in coupling ATP hydrolysis to the mechanism of chromatin remodeling. These results provide a potential model for the function of the SWI/SNF chromatin remodeling complex at the nucleosome level.

This study investigates endocytosis and ER export signals of the yeast α-factor receptor and the role that receptor oligomerization plays in these processes. The α-factor receptor contains signal sequences in the cytoplasmic C-terminal domain that are essential for ligand-mediated endocytosis. In an endocytosis complementation assay, I found that oligomeric complexes of the receptor undergo ligand-mediated endocytosis when the α-factor binding site and the endocytosis signal sequences are located in different receptors. Both in vitro and in vivo assays strongly suggested that ligand-induced conformational changes in one Ste2 subunit do not affect neighboring subunits. Therefore, the recognition of endocytosis signal sequence and the recognition of the ligand-induced conformational change are likely to be two independent events, where the signal sequence plays only a passive role in the ligand-induced endocytosis. Four amino acid substitutions (C59R, H94P, S141P and S145P) in TM domains I, II and III were identified that resulted in the accumulation of truncated receptors in the ER but did not block ER export of full-length receptors. The two DXE motifs in the C-terminal tail were required for export of the mutant receptors from the ER; however DXE was not essential for proper cell surface expression of wild-type receptors apparently because the receptors contain redundant ER export signals. An assay for oligomerization of receptors in the ER was developed based on the ability of truncated mutant receptors to exit the ER. The four substitutions (C59R, H94P, S141P and S145P) that caused DXE-dependent ER export failed to form homo-oligomers, suggesting that the DXE motifs and receptor oligomerization serve as independent ER export signals. Consistent with this view, two of the substitutions (S141P and S145P), when coexpressed, with wild-type receptors, formed hetero-oligomers that exited the ER. Finally, the full-length oligomer-defective mutant Ste2-S141P was sensitive to α-factor, suggesting that receptor monomers that reach the cell surface are able to activate the heterotrimeric G protein. The potential roles that TM1, 2 and 3 play in receptor oligomerization are discussed.

Chromatin remodeling is crucial to transcriptional regulation in vivo and a number of protein complexes capable of altering genomic architecture in the budding yeast Saccaromyces cerevisiaehave been identified. Among these, the SWI/SNF complex, a 2 MDa, eleven subunit protein assembly, has been the most extensively characterized. The SWI/SNF complex is required for the proper expression of a number of genes in yeast, although it is completely dispensable for the expression of others. Likewise, some, but not all, transcriptional activator proteins require SWI/SNF activity in order to function in vivo. The goal of this thesis work was to identify those components of the transcription process which dictate this dependence on SWI/SNF activity. Using the well characterized UASGALsystem, we have determined that one of these components is the nucleosome state of activator binding sites within a promoter. We find that while SWI/SNF activity is not required for the GAL4 activator to bind to and activate transcription from nucleosome-free binding sites, the complex is required for GAL4 to bind and function at low affinity, nucleosomal binding sites in vivo. The SWI/SNF -dependence of these nucleosomal binding sites can be overcome by 1) replacing the low affinity sites with higher affinity, consensus GAL4 binding sequences, or 2) placing the low affinity sites into a nucleosme-free region. These results provide the first in vivo evidence that the SWI/SNF complex can regulate gene expression by modulating the DNA binding of a transcriptional activator protein. To determine whether specific components of the GAL4 protein are necessary in order for the SWI/SNF complex to modulate binding to nucleosomal sites in our model system, we tested the SWI/SNF-dependent DNA binding of various derivative GAL4 proteins. We find that a functional activation domain is not required for SWI/SNF to modulate GAL4 binding in vivo. Interestingly, like the full length protein, GAL4 derivatives in which the activation domain has been mutated are able to partially occupy nucleosomal sites in the absence of SWI/SNF (binding in the absence of SWI/SNF is at least forty percent lower than in the presence of SWI/SNF), indicating the activation domain is also not required for SWI/SNF-independent DNA binding. These results support a model in which the SWI/SNF-dependence of a gene reflects the nucleosomal context of its important regulatory sequences, e.g. binding sites for transcriptional regulatory proteins. Although nucleosomal promoter regions have been correlated with SWI/SNF-dependence in the past, there has of yet been no gene at which nucleosome location has correlated with a specific genetic function. In the final part of this thesis work, we initiated a search for an endogenous SWI/SNF-dependent gene for which the nucleosome state of activator binding sites could be determined.

Cotranslational translocation is initiated by targeting of a ribosome-bound nascent polypeptide chain (RNC) to the endoplasmic reticulum (ER) membrane. The targeting reaction is coordinated by the signal recognition particle (SRP) through its interaction with the RNC and the membrane-bound SRP receptor (SR). A vacant translocon is a prerequisite for the subsequent nascent chain release from SRP-SR-RNC complex. It has been proposed that the protease-accessible cytosolic domains of the Sec61p complex play an important role in posttargeting steps by providing the binding site for the ribosome or interacting with the SR to initiate the signal sequence releasing. In this study, we have investigated the detailed mechanism that allows transfer of the ribosome-nascent chain (RNC) from the SRP-SR complex to the translocon using yeast S. cerevisiaeas the model system. Point mutations in cytoplasmic loops six (L6) and eight (L8) of yeast Sec61p cause reductions in growth rates and defects in translocation of nascent polypeptides that utilize the cotranslational translocation pathway. Sec61 heterotrimers isolated from the L8 sec61 mutants have a greatly reduced affinity for 80S ribosomes. Cytoplasmic accumulation of protein precursors demonstrates that the initial contact between the large ribosomal subunit and the Sec61 complex is important for efficient insertion of a nascent polypeptide into the translocation pore. In contrast, point mutations in L6 of Sec61p inhibit cotranslational translocation without significantly reducing the ribosome binding activity, indicating that the L6 and L8 sec61mutants impact different steps in the cotranslational translocation pathway. An interaction between the signal recognition particle receptor (SR) and the Sec61 complex has been proposed to facilitate transfer of the ribosome-nascent chain (RNC) complex to an unoccupied translocon. The slow growth and cotranslational translocation defects caused by deletion of the transmembrane span of yeast SRβ (srp102pΔTMD) are exaggerated upon disruption of the SSH1 gene, which encodes the pore subunit of a cotranslational translocation channel. Disruption of the SBH2 gene, which encodes the β-subunit of the Ssh1p complex, likewise causes a synthetic growth defect when combined with srp102pΔTMD. The in vivo kinetics of translocon gating by RNCs were slow and inefficient in the ssh1Δ srp102pΔTMD mutant. A critical role for translocon β-subunits in SR recognition is supported by the observation that deletion of both translocon β-subunits causes a block in the cotranslational targeting pathway that resembles elimination of either subunit of the SR, and could be partially suppressed by expression of carboxy-terminal Sbh2p fragments.

In eukaryotic cells, membrane-bound vesicles carry cargo between intracellular compartments, to and from the cell surface, and to the extracellular environment. Many conserved families of proteins are required for properly localized vesicle fusion, including the multi-subunit tethering complexes and the SNARE complexes. These protein complexes work together to promote proper vesicle fusion in other trafficking pathways. Contrary to these other pathways, our lab previously suggested that the exocyst subunit Sec6, a component of the exocytosis-specific tethering complex, inhibited Sec9:Sso1 SNARE complex assembly due to interactions in vitro with the SNARE protein Sec9 (Sivaram et al., 2005). My goal for this project was to test the hypothesis that Sec6 inhibited SNARE complex assembly in vivo. I therefore chose to generate Sec6:Sec9 loss-of-binding mutants, and study their effect both in vitro and in vivo. I identified a patch of residues on Sec9 that, when mutated, are sufficient to disrupt the novel Sec6-SNARE interaction. Additionally, I found that the previous inhibitory role for Sec6 in SNARE assembly was due to a data mis-interpretation; my re-interpretation of the data shows that Sec6 has a mild, if any, inhibitory effect on SNARE assembly. My results suggest a potential positive role for Sec6 in SNARE complex assembly, similar to the role observed for other tether-SNARE interactions.

There are two copies of each core histone in a nucleosome, however, it is unclear whether post-translational modifications on each molecule function redundantly or if symmetrical modifications are required to properly regulate gene expression. We tried to address this question by breaking nucleosomal symmetry and measuring its impact on gene expression. Our strategy includes re-engineering specific residues at the H3-H3 interface, generating pairs of mutant proteins, which were predicted by computational methods to form obligate heterodimers. Using S. cerevisiae as a model system, we tested the viability of strains with mutant histones, and analyzed the interaction between by co-immunoprecipitation from mononucleosome preparations. We also measured the changes of gene expression in the strains bearing single-tailed or tailless H3 heterodimers. The data suggested that the best computationally-derived H3 pair was frequently, but not exclusively heterodimeric in vivo. In order to obtain a more stringent H3 heterodimer, random mutagenesis was performed on four codons in the original computational design, and then genetic screening of the mutant libraries was performed.

No description available.