title: Subcellular Fractionation Analysis of the Extraction of Ubiquitinated Polytopic Membrane Substrate during ER-Associated Degradation.
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.
authors: Division of Biological Sciences, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, Japan.
authors: Kunio
authors: K
authors: Nakatsukasa
authors: Division of Biological Sciences, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, Japan.
authors: Takumi
authors: T
authors: Kamura
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000302:isolation & purification / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D005591:Chemical Fractionation; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D060746:Endoplasmic Reticulum-Associated Degradation; D007425:Intracellular Membranes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013347:Subcellular Fractions; D054875:Ubiquitination; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism; D000074405:Valosin Containing Protein
chemical_list: D018528:ATP-Binding Cassette Transporters; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C100415:DSK2 protein, S cerevisiae; C490846:RAD23 protein, S cerevisiae; C489277:STE6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; D000251:Adenosine Triphosphatases; C000614177:CDC48 protein, S cerevisiae; D000074405:Valosin Containing Protein