Browsing by Subject "Ubiquitins"
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Item PI(4)P-Dependent Recruitment of Clathrin Adaptors to the Trans-Golgi Network(2005-04-29) Wang, Jing; Yin, Helen L.The Trans Golgi Network (TGN) is the cell's central sorting station, and the complex trafficking patterns are organized by many types of trafficking adaptors. These include the heterotetrameric adaptor protein complexes (APs) and the monomeric Golgi-localized, gamma-ear containing, Arf-binding proteins (GGAs). The fundamental question of how these adaptors are recruited to TGN membrane remains unclear. Previous studies have shown that adaptor recruitment to the TGN is absolutely dependent on the small GTPase ADP ribosylation factor 1 (Arf1), but paradoxically, Arf1 has a broader intracellular distribution than these adaptors. We found that the Golgi is particularly enriched in phosphatidylinositol 4 phosphate [PI(4)P] and that the clathrin adaptor AP-1 binds PI(4)P directly, suggesting that PI(4)P binding may specify the TGN-specific recruitment in conjunction with Arf1. My studies showed that another monomeric clathrin adaptor GGA also binds PI(4)P and Arf1 independently. The C-terminal "triple helix bundle" of the GGA GAT domain is a polyfunctional module that interacts with multiple partners including PI(4)P and ubiquitin, and ubiquitin may provide a recognition signal for GGAs to control protein sorting. We found that PI(4)P increases wild type GAT binding to ubiquitin-conjugated agarose beads, but has no effect on a mutant GAT that does not bind PI(4)P. Therefore, PI(4)P may be an allosteric regulator of GGAs which enhances ubiquitin binding to GGAs. Based on these results, we conclude: (1) PI(4)P defines the TGN organelle identity by recruiting TGN-targeted adaptors; (2) TGN-enriched adaptors are recruited to the Golgi by binding to both PI(4)P and Arf1, and neither alone is sufficient; (3) PI(4)P acts as a scaffold, and may also be an allosteric regulator for GGAs that modulates GGA function with other ligands. We propose that the integration of combinatorial inputs from PI(4)P, Arf1 and ubiquitin may coordinately specify clathrin adaptor TGN recruitment through multiple low-affinity interactions.Item Variations in Mevalonate Pathway Flux in Human Cells with Familial Hypercholesterolemia(2020-05-01T05:00:00.000Z) Su, Shan; DeBose-Boyd, Russell A.; Liang, Guosheng; Radhakrishnan, ArunBACKGROUND: HMG-CoA reductase (HMGCR) is a membrane protein of the endoplasmic reticulum (ER) that catalyzes the reduction of HMG-CoA to mevalonate, a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids. Sterol and nonsterol isoprenoids exert stringent feedback control on HMGCR through multiple mechanisms. This ensures constant synthesis of essential nonsterol isoprenoids, while avoiding toxic overaccumulation of cholesterol. One regulator of HMGCR is UBIAD-1, a vitamin K2 biosynthetic enzyme. Individuals with familial hypercholesterolemia (FH) suffer from cholesterol excess due to the inability of cells to take up cholesterol from the environment, leading to a cholesterol depleted cellular state and an increase in cholesterol production. OBJECTIVE: In this study, we examine the effect of sterol and nonsterol isoprenoid depletion via statins followed by mevalonate treatment on the expression of genes and proteins in the mevalonate pathway and localization of UBIAD-1 in human fibroblasts. METHODS: Cells expressing FH mutations and control cells were grown on culture plates or coverslips and fed media containing FCS, or FCS plus compactin and 0.05 mM, 0.2 mM, 1 mM, 3 mM, or 10 mM mevalonate. After overnight feeding, cells were harvested for immunofluorescence visualization, and qRT-PCR and immunoblot analysis of genes and proteins related to cholesterol and nonsterol isoprenoid synthesis. RESULTS: Immunoblot analysis indicates that FH cells generally express higher amounts of sterol biosynthetic enzymes but lower amounts of CoQ10 biosynthetic enzymes than control cells. qRT-PCR showed that genes of the CoQ10 pathway in FH cells are expressed to a significantly less extent than in control cells, and that sterol synthetic genes are relatively unaffected in FH cells but upregulated in control cells fed compactin and mevalonate. Immunofluorescence and quantitation of UBIAD-1 Golgi localization indicate that compactin causes UBIAD-1 to migrate to the ER in both cells, and that FH cells require a greater concentration of mevalonate following the addition of compactin to restore Golgi localization. CONCLUSION: The FH phenotype causes a cellular deficiency of sterols, leading cells to upregulate mechanisms toward sterol synthesis at the expense of CoQ10 synthesis, which results in a relative CoQ10 deficiency in FH cells.