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

Degradation of Phosphorylated p53 by Viral Protein-ECS E3 Ligase Complex

p53-signaling is modulated by viruses to establish a host cellular environment advantageous for their propagation. The Epstein-Barr virus (EBV) lytic program induces phosphorylation of p53, which prevents interaction with MDM2. Here, we show that induction of EBV lytic program leads to degradation of p53 via an ubiquitin-proteasome pathway independent of MDM2. The BZLF1 protein directly functions as an adaptor component of the ECS (Elongin B/C-Cul2/5-SOCS-box protein) ubiquitin ligase complex targeting p53 for degradation. Intringuingly, C-terminal phosphorylation of p53 resulting from activated DNA damage response by viral lytic replication enhances its binding to BZLF1 protein. Purified BZLF1 protein-associated ECS could be shown to catalyze ubiquitination of phospho-mimetic p53 more efficiently than the wild-type in vitro. The compensation of p53 at middle and late stages of the lytic infection inhibits viral DNA replication and production during lytic infection, suggesting that the degradation of p53 is required for efficient viral propagation. Taken together, these findings demonstrate a role for the BZLF1 protein-associated ECS ligase complex in regulation of p53 phosphorylated by activated DNA damage signaling during viral lytic infection

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

E3 Ligases Regulate Organelle Inheritance in Yeast

Saccharomyces cerevisiae proliferates by budding, which includes the formation of a cytoplasmic protrusion called the ‘bud’, into which DNA, RNA, proteins, organelles, and other materials are transported. The transport of organelles into the growing bud must be strictly regulated for the proper inheritance of organelles by daughter cells. In yeast, the RING-type E3 ubiquitin ligases, Dma1 and Dma2, are involved in the proper inheritance of mitochondria, vacuoles, and presumably peroxisomes. These organelles are transported along actin filaments toward the tip of the growing bud by the myosin motor protein, Myo2. During organelle transport, organelle-specific adaptor proteins, namely Mmr1, Vac17, and Inp2 for mitochondria, vacuoles, and peroxisomes, respectively, bridge the organelles and myosin. After reaching the bud, the adaptor proteins are ubiquitinated by the E3 ubiquitin ligases and degraded by the proteasome. Targeted degradation of the adaptor proteins is necessary to unload vacuoles, mitochondria, and peroxisomes from the actin–myosin machinery. Impairment of the ubiquitination of adaptor proteins results in the failure of organelle release from myosin, which, in turn, leads to abnormal dynamics, morphology, and function of the inherited organelles, indicating the significance of proper organelle unloading from myosin. Herein, we summarize the role and regulation of E3 ubiquitin ligases during organelle inheritance in yeast

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

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