Overexpression of the Tumor Autocrine Motility Factor Receptor Gp78, a Ubiquitin Protein Ligase, Results in Increased Ubiquitinylation and Decreased Secretion of Apolipoprotein B100 in HepG2 Cells

Abstract

Apolipoprotein B100 (apoB) is a large (520-kDa) complex secretory protein; its secretion is regulated posttranscriptionally by several degradation pathways. The best described of these degradative processes is cotranslational ubiquitinylation and proteasomal degradation of nascent apoB, involving the 70-and 90-kDa heat shock proteins and the multiple components of the proteasomal pathway. Ubiquitinylation involves several proteins, including ligases called E3s, that coordinate the covalent binding of ubiquitin to target proteins. The recent discovery that tumor autocrine motility factor receptor, also known as gp78, is an endoplasmic reticulum (ER)-associated E3, raised the possibility that this E3 might be involved in the ER-associated degradation of nascent apoB. In a series of experiments in HepG2 cells, we demonstrated that overexpression of gp78 was sufficient for increased ubiquitinylation and proteasomal degradation of apoB, with reduced secretion of apoB-lipoproteins. This action of gp78 was specific: overexpression of the protein did not affect secretion of either albumin or apolipoprotein AI. Furthermore, overexpression of a cytosolic E3, Itch, had no effect on apoB secretion. Finally, using an in vitro translation system, we demonstrated that gp78 led to increased ubiquitinylation and proteasomal degradation of apoB48. Together, these results indicate that an ER-associated protein, gp78, is a bona fide E3 ligase in the apoB ERassociated degradation pathway.Apolipoprotein B100 (apoB) 1 is the essential protein component of atherogenic very low density and low density lipoproteins (1), and overproduction of these lipoproteins is a common feature of human dyslipidemia (2). Extensive studies of cultured primary hepatocytes and hepatoma cells have established that significant control over apoB secretion can be achieved at the post-transcriptional level by degradation. One form of presecretory degradation, namely the ER-associated degradation (ERAD) of apoB, is regulated by the availability of newly synthesized core lipids (triglyceride, cholesterol ester) and microsomal triglyceride transfer protein activity (1, 3, 4). When lipid availability or microsomal triglyceride transfer protein activity is limited, translocation of apoB is incomplete, and apoB is ubiquitinylated co-translationally and degraded by the cytosolic ubiquitin-proteasome pathway (5-9).Cytosolic and nuclear proteins are targeted for proteasomal degradation by the addition of multi-ubiquitin chains. The specificity of this process is largely conferred by ubiquitin (Ub) protein ligases (E3s) (10 -12). E3s interact directly or indirectly with substrate and mediate the transfer of Ub from Ub-conjugating enzymes to target proteins. Two major E3 classes have been identified: HECT domain E3s and RING finger E3s (10 -12). HECT domain E3s are homologous to E6-AP C-terminal domain E3s, which accept Ub from Ub-conjugating enzymes, thereby forming thiol-ester intermediates with Ub. The Ub is then transferred to the target protein. In contrast, RING ...

Recruitment of the p97 ATPase and ubiquitin ligases to the site of retrotranslocation at the endoplasmic reticulum membrane

“…FLAG-tagged gp78͞AMFR, another membrane-anchored ubiquitin ligase implicated in retrotranslocation (21)(22)(23), also associated with Derlin-1 and p97 (Fig. 1c, lane 5), whereas FLAG-tagged Parkin, an E3 ligase involved in the degradation of misfolded Pael receptor (24), coprecipitated Ͻ5% of Derlin-1 (Fig.…”

Gp78 Cooperates with RMA1 in Endoplasmic Reticulum-associated Degradation of CFTRΔF508

“…Subsequently, it was shown that gp78 localizes not only to the cell surface but also to the ER (Wang et al, 1997). Similar to Hrd1p, gp78 is a RING finger-dependent ubiquitin ligase, an integral ER membrane protein, and it is involved in ERAD, in cooperation with Ube2g2/UBC7, the mammalian counterpart of yeast Ubc7p (Fang et al, 2001;Liang et al, 2003;Song et al, 2005;Lee et al, 2006). Recently, Chen et al (2006) demonstrated that both the E2 binding site and the coupling of ubiquitin to ER degradation (CUE) domain of gp78 are essential for its ubiquitin ligase activity.…”

The activity of a human endoplasmic reticulum-associated degradation E3, gp78, requires its Cue domain, RING finger, and an E2-binding site

“…This protein, also known as the tumor autocrine motility factor receptor (21), includes a Ϸ350-aa cytoplasmic tail that contains a RING finger, which is essential for E3 activity. When overexpressed, gp78 targets itself and heterologous substrates, such as CD3-␦ and apolipoprotein B, for degradation (22,23). Most recently, gp78 has been implicated in the regulated degradation of hydroxymethylglutaryl-CoA reductase (24).…”