Abstract
Quality control of endoplasmic reticulum proteins involves the identification and engagement of misfolded proteins, dislocation of the misfolded protein across the endoplasmic reticulum (ER) membrane, and ubiquitin-mediated targeting to the proteasome for degradation. Ancient ubiquitous protein 1 (AUP1) physically associates with the mammalian HRD1-SEL1L complex, and AUP1 depletion impairs degradation of misfolded ER proteins. One of the functions of AUP1 in ER quality control is to recruit the soluble E2 ubiquitin-conjugating enzyme UBE2G2. We further show that the CUE domain of AUP1 regulates polyubiquitylation and facilitates the interaction of AUP1 with the HRD1 complex and with dislocation substrates. AUP1 localizes both to the ER and to lipid droplets. The AUP1 expression level affects the abundance of cellular lipid droplets and as such represents the first protein with lipid droplet regulatory activity to be linked to ER quality control. These findings indicate a possible connection between ER protein quality control and lipid droplets.
Highlights
Serves as the E2 and HRD1 as the E3 [12]
We previously identified ancient ubiquitous protein 1 (AUP1) as a component of the HRD1-SEL1L endoplasmic reticulum (ER) quality control complex and showed that AUP1 is necessary for US11-mediated dislocation of class I MHC heavy chains [14]
AUP1 contributes to the degradation of misfolded ER proteins and affects the intracellular abundance of lipid droplets
Summary
Antibodies—The following antibodies have been described: anti-AUP1, anti-UBXD8, and anti-Ubc6e [14]; anti-SEL1L [35]; and anti-class I MHC HC (HC-70) [9]. Cells were incubated with oleic acid adsorbed to BSA [36], for the concentrations and times indicated. Samples were incubated for 3 h at 4 °C with the antibody and protein A-agarose beads or immobilized anti-HA as indicated. The immunoprecipitates were washed, boiled in reducing sample buffer, and run on SDS-PAGE. Proteins were transferred to PVDF membrane, and membranes were blocked in PBS/Tween/milk, incubated with primary and secondary antibodies, washed, and developed with Western Lighting Chemiluminescence Reagent Plus (PerkinElmer Life Sciences). Media containing cold cysteine and methionine were added, and equal numbers of cells were removed at the indicated chase times. Permeabilized cells were incubated with primary and secondary (Alexa Fluor 568-labeled) antibodies and washed with PBS before being mounted on a slide with Fluoromount-G (Southern Biotech). Lipid droplets were solubilized by incubation in a sonicating water bath for 2 h at 37 °C in 5% SDS
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