Derlin-3 Is Required for Changes in ERAD Complex Formation under ER Stress

Endoplasmic reticulum (ER)-associated protein degradation (ERAD) is a quality control system that induces the degradation of ER terminally misfolded proteins. The ERAD system consists of complexes of multiple ER membrane-associated and luminal proteins that function cooperatively. We aimed to reveal the role of Derlin-3 in the ERAD system using the liver, pancreas, and kidney obtained from different mouse genotypes. We performed coimmunoprecipitation and sucrose density gradient centrifugation to unravel the dynamic nature of ERAD complexes. We observed that Derlin-3 is exclusively expressed in the pancreas, and its deficiency leads to the destabilization of Herp and accumulation of ERAD substrates. Under normal conditions, Complex-1a predominantly contains Herp, Derlin-2, HRD1, and SEL1L, and under ER stress, Complex-1b contains Herp, Derlin-3 (instead of Derlin-2), HRD1, and SEL1L. Complex-2 is upregulated under ER stress and contains Derlin-1, Derlin-2, p97, and VIMP. Derlin-3 deficiency suppresses the transition of Derlin-2 from Complex-1a to Complex-2 under ER stress. In the pancreas, Derlin-3 deficiency blocks Derlin-2 transition. In conclusion, the composition of ERAD complexes is tissue-specific and changes in response to ER stress in a Derlin-3-dependent manner. Derlin-3 may play a key role in changing ERAD complex compositions to overcome ER stress

Derlin-1 Deficiency Is Embryonic Lethal, Derlin-3 Deficiency Appears Normal, and Herp Deficiency Is Intolerant to Glucose Load and Ischemia in Mice

<div><p>Accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) causes a cellular condition called ER stress. To overcome ER stress, unfolded proteins are eliminated by an ER-associated degradation (ERAD) system. To explore the physiological requirements for ERAD-related membrane proteins in mammals, we generated Derlin-1–, Derlin-3–, and Herp-deficient mice by gene targeting. Complete loss of Derlin-1 caused embryonic lethality at around E7–E8 (early somite stages). In contrast, Derlin-3– and Herp-deficient mice were born alive with the expected Mendelian frequency, and were superficially indistinguishable from wild-type mice. However, in the Derlin-3– and Herp-deficient mouse organs, the expression levels of ERAD-related proteins were affected under both normal and ER stress conditions; specific effects differed among the organs. Degradation of ERAD substrates was reduced in the Herp-deficient liver, and Herp-deficient mice exhibited impaired glucose tolerance and vulnerability to brain ischemic injury, both of which are known to be implicated in ER stress. Our findings indicate that ERAD or uncharacterized functions involving Derlin-1 are essential in early embryonic development. Derlin-3– and Herp-deficient mice may become useful model animals for investigations of the physiological contribution of ERAD under stressful or pathological conditions.</p> </div

Genotype of offspring from the cross of heterozygous mice.

a<p>Chi-square test.</p

Effect of Herp deficiency on NHK-GFP degradation.

<p>(<b>A</b>) NHK-GFP was expressed in the livers of <i>Herpud1</i>^+/+ mice in the absence or presence of epoxomicin (<i>n</i> = 2), and detected by Western blotting using anti-GFP antibodies. (<b>B</b>) NHK-GFP was expressed in the livers of <i>Herpud1</i>^+/+ and <i>Herpud1</i>^−/− mice (<i>n</i> = 2), and detected by Western blotting using anti-GFP antibodies.</p

Genotype of embryos from the cross of <i>Derl1</i>+/− heterozygous mice.

<p>The heterozygous mice were mated and the embryos in uteri were genotyped. Resorbed and rudimentary embryos were not genotyped.</p>a<p>Chi-square test of +/+, +/−, and −/−, not including resorbed embryos.</p>b<p>Chi-square test using +/+, +/−, and −/−, counting resorbed embryos as −/−.</p>c<p>C57BL/6JJcl wild-type mice.</p>d<p>Jcl:ICR wild-type mice.</p

Glucose tolerance and insulin response tests.

<p>Blood glucose levels were examined at the indicated time points after intraperitoneal injection of glucose (<b>A</b> and <b>B</b>) or insulin (<b>C</b>) into <i>Derl3</i>^−/− (<b>A</b>, open symbols), <i>Derl3</i>^+/+ (<b>A</b>, filled symbols), <i>Herpud1</i>^−/− (<b>B</b> and <b>C</b>, open symbols), and <i>Herpud1</i>^+/+ mice (<b>B</b> and <b>C</b>, filled symbols). Data are expressed as the means with error bars of standard deviation (<b>A</b>, <i>n</i> = 12; <b>B</b>, <i>n</i> = 18; <b>C</b>, <i>n</i> = 16). Asterisks indicate <i>p</i><0.01 (t-test) for <i>Herpud1</i>^−/− vs. <i>Herpud1</i>^+/+ mice.</p

Targeted disruption of Derlin-1, Derlin-3, and Herp.

<p>Structures of the mouse <i>Derl1</i>, <i>Derl3</i>, and <i>Herpud1</i> loci, and the targeting vectors for disrupting each gene. Neo; neomycin resistance gene, IRES; internal ribosome entry site, DTA; diphtheria toxin A chain.</p

Effects of Derlin-3 and Herp deficiency on the levels of ERAD-related proteins.

<p>WT, <i>Derl3</i>^−/−, and <i>Herpud1</i>^−/− mice were intraperitoneally injected with PBS as control (C) or Tm (2 µg/g body weight) 12 h before sacrifice. Liver, pancreas, and kidney homogenates were subjected to Western blotting using the indicated antibodies. Quantitative data are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034298#pone.0034298.s002" target="_blank">Figure S2</a>. Total RNAs prepared from the organs were subjected to RT-PCR in order to analyze the splicing of XBP1 (lowest panels). Quantitative data on the ratios (spliced/unspliced+spliced) are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034298#pone-0034298-g004" target="_blank">Figure 4</a>. *unspliced and **spliced forms of XBP1 mRNA.</p

Stereomicroscopic appearance of Derlin-1–deficient mouse embryos.

<p><i>Derl1</i>^−/− embryos at E8.5 exhibited developmental delay and resorption, respectively, whereas <i>Derl1</i>^+/+ and <i>Derl1</i>^+/− embryos were normal. Bars, 50 µm.</p