Browsing by Subject "GTP-Binding Protein gamma Subunits"
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Item Dissecting the Role of the Lipodystrophy Protein Seipin in the Biogenesis of the Lipid Droplet Organelle(2014-07-14) Cartwright, Bethany Rose; Sternweis, Paul C.; Chook, Yuh Min; Bickel, PerryLong thought to be little more than inert storage depots, lipid droplets have recently become recognized as unique, dynamic, regulated organelles that play an essential role in fat storage. Despite this increased interest, much remains unknown. Lipid droplets have been observed to emerge from the endoplasmic reticulum, but the available models for lipid droplet biogenesis are largely conceptual, with little to no evidence for specific mechanisms of droplet formation. Debate even continues within the field as to whether lipid droplet formation is a spontaneous process, driven by physicochemical and hydrophobic forces, or a regulated process driven by protein factors. The Goodman laboratory previously found evidence to suggest that seipin, mutated in the most severe cases of congenital generalized lipodystrophy, may be a key factor in the early stages of lipid droplet formation. Seipin resides at the junction between lipid droplets and the endoplasmic reticulum, and deletion of seipin results in both a drastic impediment to de novo droplet formation and a striking disorganization of droplet morphology. For my thesis work, I have explored several aspects of seipin’s role at the lipid droplet. I have studied the effects of seipin deletion on protein targeting to abnormal lipid droplets, through which I identified a unique effect of seipin on the regulation of lipase targeting. I have also analyzed the topology of the seipin complex itself through a series of deletion mutants, identifying regions that contribute to the localization, membrane association, and stability of the seipin complex. Furthermore, these studies have led to novel insights on the function of seipin, through the characterization of a remarkable N-terminal seipin mutation that presents with defects in droplet initiation but homogenous droplet morphology. I have therefore concluded that seipin plays two dissectible roles in lipid droplet formation: 1) promoting lipid droplet initiation and 2) regulating subsequent droplet morphology. Finally, I suggest hypotheses on the mechanisms by which seipin exerts these effects, proposing that the N-terminus of seipin may regulate lipin, a mouse lipodystrophy protein, to effect droplet initiation, while the bulk of the protein may serve to regulate the access of phospholipids to the lipid droplet surface.Item Dual Regulation of Phospholipase C-beta by G betagamma(2016-03-29) Kadamur Bhavani, Ganesh; Rosen, Michael K.; Ross, Elliott M.; Albanesi, Joseph P.; Zhang, XuewuAgonist-bound G protein coupled receptors (GPCRs) activate G protein heterotrimers by catalyzing release of GDP and binding of GTP to the G alpha subunit (Ga), releasing active Ga and G betagamma (Gbg) subunits. Activated alpha subunits of the Gq family and betagamma subunits of the Gi family stimulate phospholipase C-beta (PLC-b) isoforms to catalyze hydrolysis of phosphatidylinositol-1,2-bisphosphate (PIP2) generating the second messengers, inositol trisphosphate (IP3) and diacylglycerol (DAG). PLC-b isoforms are also GTPase activating proteins (GAPs) for Gaq, and Gbg subunits inhibit the GAP activity of PLC-b. Coordinated regulation of these activities is essential for sustained signaling at steady state. Regulation of PLC-b by Gaq and Gbg is well studied but details of the mechanism are still lacking. Activation of PLC-b simultaneously by G protein pathways has been suggested based on observations in cells, but it is not known if scaffolding proteins or other factors are necessary for simultaneous stimulation of PLC-b by G protein subunits. The binding interface between Gbg and PLC-b is unclear, and so is the mechanism of PLC-b GAP inhibition by Gbg. To enable the study of these mechanisms in vitro, I developed a new method to purify Gaq subunits based on observations from the Tall group. This method combined Ric8A-mediated enhancement of Gaq expression and the traditional method of using detergents to isolate functional Ga from membrane bound G protein heterotrimers, resulting in 3- to 4-fold increase in yields of Gaq. Using purified proteins and working with other members of the lab, I showed that the PLC-b3 isoform is synergistically activated by Gaq and Gbg subunits. The observed synergism is up to 10-fold, quantitatively consistent with cellular observations, thus establishing that no additional proteins or pathways are required. Next, I developed a FRET-based binding assay between Gbg and PLC-b and identified the pleckstrin homology (PH) domain in PLC-b as the Gbg binding site. Using structural and biochemical analyses, I showed that Gbg-PLC-b requires intrinsic motion of the PH domain. This led to the proposal for a new conformation of PLC-b not observed in crystal structures and a new model for Gbg-PLC-b binding. Subsequent studies suggested that Gbg inhibits PLC-b GAP activity by a mechanism that does not require Gbg-PLC-b binding.Item Seipin Promotes Lipid Droplet Biogenesis(2013-01-17) Hilton, Christopher Lee; Goodman, Joel M.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.