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.

Fig 1. Subcellular fractionation assay reveals that the extraction of ubiquitinated Ste6* in vivo depends on Cdc48 and Ubx2.

(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.

Fig 2. Rad23 and Dsk2 are dispensable for the extraction of ubiquitinated Ste6*.

(A) Cycloheximide chase analysis of Ste6*-3HA was performed in rad23∆dsk2∆ 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 Fig 1. 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 rad23∆dsk2∆ cells (lane 4, upper panel).

Fig 3. Lipid droplet formation is dispensable for the degradation of ERAD-C substrate.

(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.∆ (dga1∆lro1∆are1∆are2∆) cells. L.D.∆ or its isogenic wild-type cells were transformed with a plasmid encoding Ste6*-3HA under the control of the GAL1 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.).

Fig 4. Ubiquitinated Ste6* is extracted from P20 fraction and sequestered into P100 fraction in a Cdc48-dependent manner.

(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 cdc48-3 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 Fig 1A. 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.

Fig 5. Flotation analysis of ubiquitinated Ste6*.

(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 Fig 1A 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.