The Role of Elongin BC-Containing Ubiquitin Ligases

The Elongin complex was originally identified as a positive regulator of RNA polymerase II and is composed of a transcriptionally active subunit (A) and two regulatory subunits (B and C). The Elongin BC complex enhances the transcriptional activity of Elongin A. “Classical” SOCS box-containing proteins interact with the Elongin BC complex and have ubiquitin ligase activity. They also interact with the scaffold protein Cullin (Cul) and the RING domain protein Rbx and thereby are members of the Cullin RING ligase (CRL) superfamily. The Elongin BC complex acts as an adaptor connecting Cul and SOCS box proteins. Recently, it was demonstrated that classical SOCS box proteins can be further divided into two groups, Cul2- and Cul5-type proteins. The classical SOCS box-containing protein pVHL is now classified as a Cul2-type protein. The Elongin BC complex containing CRL family is now considered two distinct protein assemblies, which play an important role in regulating a variety of cellular processes such as tumorigenesis, signal transduction, cell motility, and differentiation

Potential Physiological Relevance of ERAD to the Biosynthesis of GPI-Anchored Proteins in Yeast

Misfolded and/or unassembled secretory and membrane proteins in the endoplasmic reticulum (ER) may be retro-translocated into the cytoplasm, where they undergo ER-associated degradation, or ERAD. The mechanisms by which misfolded proteins are recognized and degraded through this pathway have been studied extensively; however, our understanding of the physiological role of ERAD remains limited. This review describes the biosynthesis and quality control of glycosylphosphatidylinositol (GPI)-anchored proteins and briefly summarizes the relevance of ERAD to these processes. While recent studies suggest that ERAD functions as a fail-safe mechanism for the degradation of misfolded GPI-anchored proteins, several pieces of evidence suggest an intimate interaction between ERAD and the biosynthesis of GPI-anchored proteins

Subcellular Fractionation Analysis of the Extraction of Ubiquitinated Polytopic Membrane Substrate during ER-Associated Degradation.

During ER-associated degradation (ERAD), misfolded polytopic membrane proteins are ubiquitinated and retrotranslocated to the cytosol for proteasomal degradation. However, our understanding as to how polytopic membrane proteins are extracted from the ER to the cytosol remains largely unclear. To better define the localization and physical properties of ubiquitinated polytopic membrane substrates in vivo, we performed subcellular fractionation analysis of Ste6*, a twelve transmembrane protein that is ubiquitinated primarily by Doa10 E3 ligase in yeast. Consistent with previous in vitro studies, ubiquitinated Ste6* was extracted from P20 (20,000 g pellet) fraction to S20 (20,000 g supernatant) fraction in a Cdc48/p97-dependent manner. Similarly, Ubx2p, which recruits Cdc48/p97 to the ER, facilitated the extraction of Ste6*. By contrast, lipid droplet formation, which was suggested to be dispensable for the degradation of Hrd1-substrates in yeast, was not required for the degradation of Ste6*. Intriguingly, we found that ubiquitinated Ste6* in the S20 fraction could be enriched by further centrifugation at 100,000 g. Although it is currently uncertain whether ubiquitinated Ste6* in P100 fraction is completely free from any lipids, membrane flotation analysis suggested the existence of two distinct populations of ubiquitinated Ste6* with different states of membrane association. Together, these results imply that ubiquitinated Ste6* may be sequestered into a putative quality control sub-structure by Cdc48/p97. Fractionation assays developed in the present study provide a means to further dissect the ill-defined post-ubiquitination step during ERAD of polytopic membrane substrates

Subcellular fractionation assay reveals that the extraction of ubiquitinated Ste6* <i>in vivo</i> depends on Cdc48 and Ubx2.

<p>(A) Membrane (P20) and supernatant (S20) fractions were prepared from cells expressing Ste6*-3HA and subjected to immunoprecipitation under denaturing conditions with anti-HA antibody. Proteins were separated by SDS-PAGE and immunoblotted with anti-ubiquitin antibody or anti-HA antibody. Arrowhead indicates the position of unmodified Ste6*. (B) Membrane (P20) and supernatant (S20) fractions were prepared as above and analyzed with anti-HA (Ste6*), Sec61 (ER), Dpm1 (ER), and Pgk1 (cytosol) antibodies by western blotting.</p

Rad23 and Dsk2 are dispensable for the extraction of ubiquitinated Ste6*.

<p>(A) Cycloheximide chase analysis of Ste6*-3HA was performed in <i>rad23</i>∆<i>dsk2</i>∆ cells. Quantification of three independent results was shown as a graph (error bars, S.D.). (B) Membrane (P20) and supernatant (S20) fractions were prepared from cells expressing Ste6*-3HA and processed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148327#pone.0148327.g001" target="_blank">Fig 1</a>. Arrowhead indicates the position of unmodified Ste6*. Smeared band below the position of unmodified Ste6* may be due to a partial degradation of ubiquitinated Ste6* in <i>rad23</i>∆<i>dsk2</i>∆ cells (lane 4, upper panel).</p

Ubiquitinated Ste6* is extracted from P20 fraction and sequestered into P100 fraction in a Cdc48-dependent manner.

<p>(A) Crude lysate (S3) prepared from wild-type cells was layered onto a 20–48% sucrose step gradient. After ultracentrifugation, 15 fractions were collected from top to bottom. Ubiquitinated Ste6* was immunoprecipitated from each fraction with anti-HA antibody under denaturing conditions, and its distribution was analyzed by western blotting with anti-ubiquitin antibody. The remaining aliquot from each fraction was analyzed by western blotting with anti-HA antibody (unmodified Ste6*) and with antibodies against organelle marker proteins (Kex2 (Golgi), Pma1 (plasma membrane), Pep12 (endosome), Dpm1 (ER), and Pgk1 (cytosol)). (B) Crude lysate (S3) was prepared from <i>cdc48-3</i> cells and analyzed as in (A). Arrowhead indicates the position of unmodified Ste6*. (C) Membrane (P20) and supernatant (S20) fractions were prepared from cells expressing Ste6*-3HA as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148327#pone.0148327.g001" target="_blank">Fig 1A</a>. The resulting S20 fraction was further subjected to the centrifugation at 100,000 g for 1 hour to give P100 and S100 fractions. The aliquot from each fraction was directly analyzed by western blotting with anti-ubiquitin antibody, anti-HA antibody (unmodified Ste6*), and anti-Pgk1 antibody. Note that less amount of ubiquitinated proteins were found in S100 fraction (lanes 3, 6, and 9) than in P20 fraction (lanes 1, 4, 7), although the amount of total proteins was higher in S100 fraction than in P20 fraction (see Coomassie Brilliant Blue R-250 (CBB)-stained membrane). Arrowhead indicates the position of unmodified Ste6*. (D) Ubiquitinated Ste6* was immunoprecipitated from each fraction prepared in (C) with anti-HA antibody under denaturing conditions and analyzed by western blotting with anti-ubiquitin antibody or anti-HA antibody.</p

Lipid droplet formation is dispensable for the degradation of ERAD-C substrate.

<p>(A) Schematic representation of lipid droplet formation. (B) Fluorescence microscopic analysis of BODIPY 493/503-stained lipid droplets in wild-type and lipid droplet deficient quadruple mutant strains. (C) Cycloheximide chase analysis of Ste6*-3HA was performed in L.D.∆ (<i>dga1</i>∆<i>lro1</i>∆<i>are1</i>∆<i>are2</i>∆) cells. L.D.∆ or its isogenic wild-type cells were transformed with a plasmid encoding Ste6*-3HA under the control of the <i>GAL1</i> promoter. Cells were first cultured in synthetic complete media supplemented with 2% raffinose and shifted to YPGalactose media for 6 h to induce the expression of Ste6*-3HA. After translation was terminated with cycloheximide, cells were collected at the indicated time points and subjected to western blotting with anti-HA antibody. Because the expression of Ste6* was significantly lower in L.D.∆ cells than in wild-type cells, the blots for Ste6* in L.D.∆ cells were exposed longer than those for wild-type. Quantification of three independent experiments was shown as a graph (error bars, S.D.).</p

Flotation analysis of ubiquitinated Ste6*.

<p>(A) Crude lysate (S3) was prepared from cells expressing Ste6*-3HA and further separated using sucrose flotation gradient ultracentrifugation as described under “Materials and methods”. Aliquots were removed from the top to the bottom of the gradient. A portion of each fraction was directly analyzed by western-blotting with antibodies against organelle marker proteins. The remaining samples were subjected to the immunoprecipitation with anti-HA antibody under denaturing condition as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148327#pone.0148327.g001" target="_blank">Fig 1A</a> to detect ubiquitinated Ste6*. Asterisk indicates a non-specific protein(s) reacted with anti-Sec61 antibodies. Arrowhead indicates the position of unmodified Ste6*. Smeared band below the position of unmodified Ste6* may be due to a partial degradation of ubiquitinated Ste6* in cells treated with proteasome inhibitor MG132. (B) Model for the extraction of ubiquitinated Ste6* during ERAD. Ubiquitinated Ste6* is extracted from the ER membrane-enriched P20 fraction to P100 fraction in a Cdc48-dependent manner.</p

Hypoxia-inducible factor-2α stabilizes the von Hippel-Lindau (VHL) disease suppressor, Myb-related protein 2.

Ubiquitin ligase von Hippel-Lindau tumor suppressor (pVHL) negatively regulates protein levels of hypoxia-inducible factor-α (HIF-α). Loss of pVHL causes HIF-α accumulation, which contributes to the pathogenesis of von Hippel-Lindau (VHL) disease. In contrast, v-Myb avian myeloblastosis viral oncogene homolog-like 2 (MYBL2; B-Myb), a transcription factor, prevents VHL pathogenesis by regulating gene expression of HIF-independent pathways. Both HIF-α and B-Myb are targets of pVHL-mediated polyubiquitination and proteasomal degradation. Here, we show that knockdown of HIF-2α induces downregulation of B-Myb in 786-O cells, which are deficient in pVHL, and this downregulation is prevented by proteasome inhibition. In the presence of pVHL and under hypoxia-like conditions, B-Myb and HIF-2α are both upregulated, and the upregulation of B-Myb requires expression of HIF-2α. We also show that HIF-2α and B-Myb interact in the nucleus, and this interaction is mediated by the central region of HIF-2α and the C-terminal region of B-Myb. These data indicate that oncogenic HIF-2α stabilizes B-Myb to suppress VHL pathogenesis

Dissecting the ER-Associated Degradation of a Misfolded Polytopic Membrane Protein

SummaryIt remains unclear how misfolded membrane proteins are selected and destroyed during endoplasmic reticulum-associated degradation (ERAD). For example, chaperones are thought to solubilize aggregation-prone motifs, and some data suggest that these proteins are degraded at the ER. To better define how membrane proteins are destroyed, the ERAD of Ste6p∗, a 12 transmembrane protein, was reconstituted. We found that specific Hsp70/40s act before ubiquitination and facilitate Ste6p∗ association with an E3 ubiquitin ligase, suggesting an active role for chaperones. Furthermore, polyubiquitination was a prerequisite for retrotranslocation, which required the Cdc48 complex and ATP. Surprisingly, the substrate was soluble, and extraction was independent of a ubiquitin chain extension enzyme (Ufd2p). However, Ufd2p increased the degree of ubiquitination and facilitated degradation. These data indicate that polytopic membrane proteins can be extracted from the ER, and define the point of action of chaperones and the requirement for Ufd2p during membrane protein quality control