Ancient Ubiquitous Protein-1 Mediates Sterol-Induced Ubiquitination of HMG CoA Reductase in Lipid Droplet-Associated Endoplasmic Reticulum Membranes.

From the Howard Hughes Medical Institute and the Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390-9046.
Molecular biology of the cell (Impact Factor: 4.47). 12/2012; 24(3). DOI: 10.1091/mbc.E12-07-0564
Source: PubMed


Sterol-induced binding to Insigs in endoplasmic reticulum (ER) membranes triggers ubiquitination of the cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl CoA reductase. This ubiquitination, which is mediated by Insig-associated ubiquitin ligases gp78 and Trc8, is obligatory for extraction of reductase from lipid droplet-associated ER membranes into the cytosol for proteasome-mediated ER-associated degradation (ERAD). Here, we identify lipid droplet-associated ancient ubiquitous protein-1 (Aup1) as one of several proteins that copurify with gp78. RNA interference (RNAi) studies show that Aup1 recruits the ubiquitin-conjugating enzyme Ubc7 to lipid droplets and facilitates its binding to both gp78 and Trc8. The functional significance of these interactions is revealed by the observation that RNAi-mediated knockdown of Aup1 blunts sterol-accelerated ubiquitination of reductase, which appears to occur in lipid droplet-associated membranes, and subsequent ERAD of the enzyme. In addition, Aup1 knockdown inhibits ERAD of Insig-1, another substrate for gp78, as well as that of membrane-bound precursor forms of sterol regulatory element-binding proteins-1 and 2, transcription factors that modulate expression of genes encoding enzymes required for cholesterol synthesis. Considered together, these findings not only implicate a role for Aup1 in maintenance of intracellular cholesterol homeostasis, but they also highlight the close connection between ERAD, lipid droplets, and lipid droplet-associated proteins.

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Available from: Youngah Jo, Oct 14, 2014
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    • "Through the cascade reactions cooperated with E1, E2 (Ubc7), and E3 (gp78), as well as other cofactors, such as Ufd1 [5], the reductase was modified by Lys-48 linkage ubiquitin chains at the K248 (the dominant ubiquitination site) and K89 [15]. With assistance of at least two proteins associated with gp78, p97/VCP, and Aup1 [4,16], the ubiquitinated reductase was translocated to lipid droplet-associated ER membrane and dislocated from membrane into cytosol for proteasomal degradation [16,17]. This postubiquitination process can be promoted by geranylgeraniol (GG-OH) or its metabolically active geranylgeranyl-pyrophosphate (GG-PP) [15]. "
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    ABSTRACT: To maintain cholesterol homeostasis, the processes of cholesterol metabolism are regulated at multiple levels including transcription, translation, and enzymatic activity. Recently, the regulation of protein stability of some key players in cholesterol metabolism has been characterized. More and more ubiquitin ligases have been identified including gp78, Hrd1, TRC8, TEB4, Fbw7, and inducible degrader of low density lipoprotein receptor. Their working mechanisms and physiological functions are becoming revealed. Here, we summarize the structure, substrates and function of these ubiquitin ligases. Their potential application in drug discovery is also discussed.
    Diabetes & metabolism journal 06/2014; 38(3):171-80. DOI:10.4093/dmj.2014.38.3.171
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    • "The ubiquitination machinery also involves E3 ubiquitin ligases that use adaptor proteins for substrate recognition. Ancient ubiquitous protein 1 (AUP1), a highly conserved protein that localizes to ER and LDs34,38 and is found in LD proteoms,8 acts as an adaptor recruiting different ubiquitin ligases such as E2 conjugase Ube2g2,144 an E3 ligase AMFR/gp78.138,145,146 "
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    ABSTRACT: The main cells of the adipose tissue of animals, adipocytes, are characterized by the presence of large cytosolic lipid droplets (LDs), which store triglyceride (TG) and cholesterol. However, most cells have LDs and the ability to store lipids. LDs have a well-known central role in storage and provision of fatty acids and cholesterol. However, the complexity of the regulation of lipid metabolism on the surface of the LDs is still a matter of intense study. Beyond this role, a number of recent studies have suggested that LDs have major functions in other cellular processes, such as protein storage and degradation, and infection and immunity. Thus, our perception of LDs, from simple globules of fat to highly dynamic organelles of unexpected complexity, has been radically transformed. Here we compiled some recent evidence supporting the emerging view that LDs act as platforms connecting a number of relevant metabolic and cellular functions.
    Lipid Insights 05/2014; 7(1):7-16. DOI:10.4137/LPI.S11128
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    • "We previously described AUP1 as a monotopic membrane protein localizing to both, LDs and ER membranes [27], [28]. It was also shown that AUP1 can be ubiquitinated [29], binds the E2 ligase Ube2g2 via a C-terminally located G2BR domain [27], [29] and that the AUP1 CUE domain binds dislocation substrates and components of the ER quality control machinery [29], [30]. Here, we present evidence that AUP1 promotes LD clustering and show that modification of AUP1 by a single ubiquitin moiety is sufficient to induce LD clustering. "
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    ABSTRACT: Lipid droplets, the intracellular storage organelles for neutral lipids, exist in a wide range of sizes and of morphologically distinct organization, from loosely dispersed lipid droplets to tightly packed lipid droplet clusters. We show that the lipid droplet protein AUP1 induces cluster formation. A fraction of AUP1 is monoubiquitinated at various lysine residues. This process depends on its internal CUE domain, which is a known ubiquitin-binding domain. AUP1 with a deleted or point mutagenized CUE domain, as well as a lysine-free mutant, are not ubiquitinated and do not induce lipid droplet clustering. When such ubiquitination deficient mutants are fused to ubiquitin, clustering is restored. AUP1 mutants with defective droplet targeting fail to induce clustering. Also, another lipid droplet protein, NSDHL, with a fused ubiquitin does not induce clustering. The data indicate that monoubiquitinated AUP1 on the lipid droplet surface specifically induces clustering, and suggest a homophilic interaction with a second AUP1 molecule or a heterophilic interaction with another ubiquitin-binding protein.
    PLoS ONE 09/2013; 8(9):e72453. DOI:10.1371/journal.pone.0072453 · 3.23 Impact Factor
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