Browsing by Subject "Saccharomyces cerevisiae Proteins"
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Item Characterization of Class D VPS Proteins(2003-03-11) Friedberg, Andrew Seth; Sternweis, PaulThe vacuole of the yeast Saccharomyces cerevisiae is functionally similar to the mammalian lysosome. The components of the VPS (vacuolar protein sorting) system are responsible for proper delivery of vacuolar biosythetic enzymes. Efforts to dissect the genetics of this system have revealed several classes of mutants, each defective in one transport step in the VPS pathway. The Class D VPS proteins are thought to control anterograde traffic between the late Golgi and late endosome. Although most of these proteins have homologues of known function in other systems, two exceptions are the Vps3p and Vps8p proteins. Analysis of Vps3p reveals that it is associated with a highdensity structure, possibly a coated vesicle or a large protein complex. The Vps8p protein contains a C-terminal H2 RING finger motif, a domain often associated with E3 ubiquitin ligase activity. In vitro analysis reveals that a Vps8p fragment containing this domain has this activity. Deletion of the RING finger reveals that the endocytic marker Ste3p accumulates in an abnormally large late-endosome-derived structure, but that sorting of the soluble vacuolar cargo CPY is relatively unaffected. These results suggest a division of function within the Vps8p molecule.Item The Conserved Oligomeric Golgi (COG) complex is Required for Normal Import of Fatty Acids in Saccharomyces Cerevisiae(2004-08-19) Ballard, Johnathan L.; Goodman, Joel M.The goal of my work was to elucidate aspects of the mechanism of trafficking of membrane proteins to peroxisomes. The work described in this document centers around one protein from Saccharomyces cerevisiae, Cog7p. Cog7p is part of the conserved oligomeric Golgi (COG) complex. Results describing a basic function of Cog7p were published well after I began studying this protein. Nevertheless, I use the nomenclature outlined in that work. Cog7p functions in intra-Golgi vesicular transport in concert with seven other proteins. This protein complex is found in both yeast and mammals. We found Cog7p in a different context through a screen to identify proteins that function in the trafficking of membrane proteins to peroxisomes. In the screen a portion of Cog7p was found to interact with the membrane peroxisomal targeting sequence, mPTS, of the Candida boidinii peroxisomal membrane protein, Pmp47. I studied peroxisomal biogenesis in a strain of Saccharomyces cerevisiae in which the COG7 gene had been deleted. I showed that Cog7p was not required for peroxisomal biogenesis, but in so doing, established that Cog7p was required for the proper metabolism of fatty acids in a peroxisome-independent manner. I showed that Cog7p was required for the normal import of fatty acids; without Cog7p, yeast cells imported abnormally high amounts of free fatty acid from the environment. My results are consistent with the hypothesis that one or more protein(s) involved in fatty acid import require the COG complex for proper processing. My work ends before such a protein was identified, but I provide leads that if pursued would contribute to understanding the regulation of fatty acid import into yeast cells.Item Elucidating the Role of Yeast Lipin (PAH1) in Lipid Droplet Biogenesis(2012-07-17) Adeyo, Oludotun; Goodman, Joel M.Lipid droplets are unique organelles important for a host of cellular functions including the storage of neutral lipids, but factors that regulate the biogenesis and maintenance of these organelles remain relatively unknown. The primary focus of this dissertation will be to understand the role of the phosphatidic acid hydrolase (Pah1p) in the biogenesis of lipid droplets in Saccharomyces cerevisiae. Pah1p is an enzyme that converts phosphatidic acid to diacylglycerol, and its absence or elimination of its catalytic activity results in the accumulation of neutral lipids within membranes of the endoplasmic reticulum. Furthermore, lipid droplet formation is facilitated by diacylglycerol through a mechanism that appears to be independent of diacylglycrol’s role as a substrate for triglyceride biosynthesis. Finally, lipid droplets originated from regions of the endoplasmic reticulum where the Pah1p activators were located. The second part of this dissertation will focus on the lipodystrophy related protein Fld1p and its association with lipid droplets. Droplets always associate with Fld1p, and in the absence of lipid droplets, Fld1p is localized as patches distributed throughout the endoplasmic reticulum. In addition, induced lipid droplets originate from these Fld1 patches. I conclude from this work that diacylglycerol facilitates lipid droplet formation and that Fld1p is somehow involved in the biogenesis or maintenance of these organelles. [Keywords: Lipid droplet, Diacylglycerol, lipin, steryl esters, endoplasmic reticulum, Triacylglycerol, phosphatidic acid]Item Recognition Mechanisms of Nuclear Localization and Export Signals(2009-06-19) Suel, Katherine Elizabeth; Chook, Yuh MinThe transport of proteins between the nucleus and cytoplasm of cells is mediated by the Karyopherin beta family of proteins. Karyopherin betas recognize their substrates through either a nuclear localization or export signal depending on the direction of transport. Even though there are ten yeast import Karyopherin betas, for the past thirteen years there was only one well characterized nuclear localization signal, the classical nuclear localization signal. However, a second signal, the proline-tyrosine nuclear localization signal recognized by Karyopherin beta2, was recently identified through X-ray crystallography and biochemical studies of Karyopherin beta2 bound to one of its substrates. These studies identified rules for the recognition of the proline-tyrosine nuclear localization signal by Karyopherin beta2. The signal must have overall basic charge, structural disorder and a weak consensus sequence of an amino-terminal basic or hydrophobic-enriched region followed by a carboxyl-terminal arginine residue separated from a proline and tyrosine residue by two to five residues. The proline-tyrosine nuclear localization signal is also recognized by the Saccharomyces cerevisiae homolog of Karyopherin beta2, Karyopherin 104, demonstrating the generality of this import mechanism across eukaryotes. Thermodynamic analyses of the two known substrates of Karyopherin 104, Hrp1p and Nab2p, revealed physical properties governing its binding. The proline-tyrosine nuclear localization signal is an extended signal with significant sequence diversity. The signal is comprised of three binding epitopes, each of which can have varying energetic strengths in different substrates. The multivalent nature of the signal increases the diversity of the signal as well as the difficulty of identifying new substrates. A bioinformatics search identified putative proline-tyrosine nuclear localization signals which were validated through biochemical studies. Additionally, one of the proteins identified, Tfg2p, was verified as a bona fide Karyopherin 104 substrate. Analysis of Tfg2p's cellular localization revealed that its nuclear localization was not solely determined by the presence of a nuclear localization signal, but was also dependent on its retention in the nucleus. Furthermore, crystallographic studies of substrate Snurportin1 bound to the export karyopherin CRM1 revealed that its nuclear export signal has two binding epitopes implying that the multivalent nature of targeting signals may not be limited to the proline-tyrosine nuclear localization signal.Item Seipin Promotes Lipid Droplet Biogenesis(2013-01-17) Hilton, Christopher Lee; Goodman, Joel, Ph.D.Seipin is an ER membrane protein that is required for adipogenesis in mammalians. Humans lacking functional seipin have virtually no visible adipose tissue. Seipin has been shown to be essential for the later stages of the adipogenic program in mouse pre-adipocytes. In yeast, the absence of seipin (Fld1p) leads to clusters of tiny lipid droplets or “supersized” ones, suggesting a role of the protein in droplet formation. To determine if this is true we created yeast strains that allowed us to “turn on” lipid droplet synthesis by the regulated expression of enzymes that create either triacylglycerol (TAG) or sterol ester (SE), the main neutral lipid components of droplets, in a droplet-null background with seipin (4KO) or without it (4KOfld1Δ). Using fluorescence microscopy, I showed that the number of newly formed TAG fluorescent bodies (individual droplets or clusters of unresolvable small droplets) decreased but their size increased in the absence of seipin. The large fluorescent bodies in 4KOfld1Δ were fluorescently dimmer and had an irregular perimeter compared to those in the 4KO strain, while their intracellular membranes stained with BODIPY had brighter fluorescence, suggesting that seipin is involved in the packaging of TAG. Electron microscopy showed that the TAG fluorescent bodies were clusters of small droplets. Levels of whole-cell TAG were generally similar during droplet formation, although somewhat lower at early time points. Seipin deletion had a milder effect on formation of SE fluorescent bodies. We conclude that seipin plays a direct role in normal lipid droplet assembly. Finally, in several side projects, I leaned about a possible role of seipin in droplet protein composition, the effects of different detergents on the seipin homo-oligomer, and the lack of a role of seipin in ER stress.Item The Yeast Transcription Factor GAL4: A Model for Understanding Eukaryotic Transcription(2009-06-18) O'Neal, Melissa Ann; Kodadek, ThomasThe 26S proteasome regulates numerous cellular pathways, including transcription, through proteolytic and non-proteolytic methods. The Kodadek and Johnston laboratories recently established a novel function for the proteasomal ATPases: the destabilization of activator-DNA complexes. This is independent of proteolysis but requires direct activator-ATPase interactions as well as ATP hydrolysis. The Gal4 mutant Gap71, which is hyper-sensitive to destabilization from a GAL promoter, was instrumental to this discovery. Gal4, but not Gap71, was mono-ubiquitylated in a HeLa nuclear extract and in vivo, suggesting that mono-ubiquitylation of an activator is critical to resisting destabilization by the proteasomal ATPases. To gain a better understanding of these events, the three amino acid substitutions in the Gap71 DNA-binding domain were individually cloned and analyzed for their contributions to the function of Gal4. The data showed that Serine 22 and Lysine 23 but not Lysine 25 were important for the efficiency of the activator. The charge at Lysine 23 was found to be important for Gal4-based transcription and subsequent in vitro work revealed that Gal4 was not only phosphorylated at Serine 22 but that this phosphorylation event was essential for the function of the activator. Many times a phosphorylation event precedes a mono-ubiquitylation event on an activator. Knowing the kinase and ligase machinery that modifies Gal4 would permit us to further test our model. As a result, I designed selection screens in an attempt to isolate the kinase and/or ligase machinery components that modify Gal4. While these particular enzymes were not identified, other novel genes were found to negatively affect the galactose utilization pathway, MSU1 and SPS1. Altogether, the data demonstrated that two post-translation events, phosphorylation and mono-ubiquitylation, prevent an activator-DNA complex from being disrupted, leading to an elegant model in which the proteasomal ATPases act as an important check point in transcription.