RRC ID 36191
著者 Nakatsukasa K, Kamura T.
タイトル Subcellular Fractionation Analysis of the Extraction of Ubiquitinated Polytopic Membrane Substrate during ER-Associated Degradation.
ジャーナル PLoS One
Abstract 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.
巻・号 11(2)
ページ e0148327
公開日 2016-1-1
DOI 10.1371/journal.pone.0148327
PII PONE-D-15-29051
PMID 26849222
PMC PMC4743956
MeSH ATP-Binding Cassette Transporters / isolation & purification ATP-Binding Cassette Transporters / metabolism* Adenosine Triphosphatases / metabolism Cell Cycle Proteins / genetics Cell Cycle Proteins / metabolism Chemical Fractionation Cytosol / chemistry Cytosol / metabolism DNA-Binding Proteins / genetics DNA-Binding Proteins / metabolism Endoplasmic Reticulum-Associated Degradation* Intracellular Membranes / metabolism Saccharomyces cerevisiae / metabolism Saccharomyces cerevisiae Proteins / genetics Saccharomyces cerevisiae Proteins / isolation & purification Saccharomyces cerevisiae Proteins / metabolism* Subcellular Fractions Ubiquitination Ubiquitins / genetics Ubiquitins / metabolism Valosin Containing Protein
IF 2.74
引用数 12
WOS 分野 CELL BIOLOGY
リソース情報
酵母 BYP1804