Skip to main content
. 2022 Nov 9;42(4):e112275. doi: 10.15252/embj.2022112275

Figure 6. Orm2 degradation requires EGAD but not ERAD or INMAD pathway.

Figure 6

  1. E3 ligase Tul1 is required for Orm2 degradation. In the indicated strains, degradation of Orm2‐RFP was measured by CHX‐chase assay. Cells were analyzed by SDS–PAGE and immunoblotted for Orm2‐RFP with α‐RFP (3 biological replicates; n = 3). t = 0 was taken as 100% and data are represented as mean ± SEM.
  2. Cdc48 and the proteasome are required for Orm2 degradation. Same as (A), except cdc48‐2 and hrd2‐1 were analyzed for Orm2‐RFP degradation (3 biological replicates; n = 3). t = 0 was taken as 100% and data are represented as mean ± SEM.
  3. Dfm1 does not function in the post‐ubiquitination step of the Orm2 degradation pathway. Indicated strains expressing Orm2‐RFP were grown into the log phase. Cells were lysed, and microsomes were collected and immunoprecipitated with α‐RFP conjugated to agarose beads. Samples were then subjected to SDS–PAGE and immunoblot by α‐Ubiquitin and α‐RFP (3 biological replicates; n = 3).
  4. ERAD mutants do not rescue temperature‐sensitive lethality of tsc3Δ. Indicated strains were grown to log phase in SC and serially diluted cultures were plated on SC plates and incubated at 37°C and imaged on Day 2 (3 biological replicates, 2 technical replicates; n = 5).