Role of the ubiquitin-selective CDC48(UFD1/NPL4 )chaperone (segregase) in ERAD of OLE1 and other substrates

Abstract

The OLE pathway of yeast regulates the abundance of the ER-bound enzyme Delta-9 fatty acid desaturase OLE1, thereby controlling unsaturated fatty acid pools and membrane fluidity. Previously, we showed that this pathway is exquisitely regulated by the ubiquitin/proteasome system. Activation of the pathway involves proteasomal processing of a membrane-bound transcription factor and the subsequent mobilization of the cleaved, ubiquitylated transcription factor from its partner molecule by CDC48(UFD1/NPL4), a ubiquitin-selective chaperone-like enzyme. Here we report that the OLE1 protein itself is naturally short-lived and is degraded by ubiquitin/proteasome-dependent ER-associated degradation (ERAD). We found that CDC48(UFD1/NPL4) plays a second role in the OLE pathway by mediating ERAD of OLE1. Intriguingly, other ERAD substrates also require CDC48(UFD1/NPL4) for degradation, indicating that this enzyme is a novel, constitutive component of the ERAD machinery. We propose that CDC48(UFD1/NPL4) functions as a segregase that liberates ubiquitylated proteins from non-modified partners.

Fig. 1. OLE1 is short-lived in vivo. (A) Expression shut-off experiments with WT cells expressing ^mycOLE1 and the mutant variant ^mycole1 under the control of the GAL1-10 promoter. Cells were grown in YPGal to an OD₆₀₀ of 0.5 at 23°C and shifted for another 2 h to 37°C. The experiment was started by adding glucose and cycloheximide to the medium. At each time point indicated, the cellular level of both epitope-tagged OLE1 variants was analyzed by anti-myc immunoblots (upper panel). As a control, the blots were reprobed with an antibody against the stable ER membrane protein dolichol phosphate mannose synthase, DPM1 (lower panel). (B) Sequence comparison of Δ-9 fatty acid desaturases from different organisms (Sc, Saccharomyces cerevisiae; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Mm, Mus musculus; Hs, Homo sapiens). The two conserved His residues, which were replaced by Ala residues in the mutant ole1 variant, are shaded in gray. (C) Growth of the ole1Δ deletion strain expressing either none, ^mycOLE1 or mutant ^mycole1 in the presence and absence of oleic acid, respectively. The lethal phenotype of ole1Δ in the absence of unsaturated fatty acids in the growth medium can only be suppressed by expressing the functional desaturase.

Fig. 2. OLE1 turnover is mediated by ERAD. Expression shut-off experiments with different ERAD mutants expressing the mutant ^mycole1 variant under the control of the GAL1-10 promoter. Experimental procedures were identical to those described in Figure 1A. Shown are the protein levels for ^mycole1 (upper panels) and as a control for DPM1 (lower panels). The right panel shows the quantification of the ^mycole1 decay. The estimated half-lives of ^mycole1 in WT, ubc6Δ ubc7Δ, cue1Δ, hrd1Δ, cim5-1 and sec61-R2 strains were 25, 80, 80, 35, 55 and 70 min, respectively.

Fig. 3. Involvement of the CDC48^UFD1/NPL4 segregase in OLE1 degradation. (A) Expression shut-off experiments with the ts mutants ufd1-2, npl4-1, cdc48-6 and the deletion strain shp1Δ expressing the mutant ^mycole1 variant under the control of the GAL1-10 promoter in the absence of oleic acid (at 37°C). Shown are the protein levels for ^mycole1 (upper panels) and as a control for DPM1 (lower panels). The right panel shows the quantification of the ^mycole1 decay. The estimated half-lives of ^mycole1 in WT, ufd1-1, npl4-1, cdc48-6 and shp1Δ strains were 25, 275, 110, 60 and 27 min, respectively. (B) The same experiment as in (A) was performed in the presence of 0.2% oleic acid added 1 h prior to the 37°C shift to the medium. (C) Yeast two-hybrid interaction of CDC48 and SHP1. Open reading frames of SHP1 and CDC48 were cloned into pGAD424 and pGBD, respectively. Transformants were streaked onto SC-Leu-Trp-His plates to test for two-hybrid interaction. Empty vectors are indicated by ‘–’. (D) Co-immunoprecipitation of CDC48 with SHP1. Cytosolic fractions from yeast cells expressing epitope-tagged ^VSVCDC48 (indicated by ‘+’) and ^mycSHP1 were subjected to anti-myc and anti-IgG immunoprecipitation, respectively. The panel designated as ‘input’ shows 10% of the amount of protein used for immunoprecipitation.

Fig. 4. Accumulation of stabilized OLE1 at the membrane. WT and ufd1-2 cells expressing mutant ^mycole1 under the control of the GAL1-10 promoter were grown in YPGal to a final OD₆₀₀ of 1.0. An expression shut-off assay was performed by adding glucose and cycloheximide to the medium. After 30 min, cells corresponding to 50 OD₆₀₀ (for both yeast strains) were subjected to cell fractionation as described in Materials and methods. Equal amounts of total extract (T), soluble (S) and pellet (P) fraction were analyzed by immunoblotting with anti-myc (upper panel) and the ER membrane protein anti-DPM1 (lower panel) antibody, respectively.

Fig. 5. General role of the CDC48^UFD1/NPL4 segregase in ERAD. (A) Expression shut-off (cycloheximide chase) with WT and ufd1-2, npl4-1, cdc48-6 and the deletion strain shp1Δ expressing ^mycHMG2. Cells were grown in YPD to an OD₆₀₀ of 0.5 at 23°C and then shifted for another 2 h to 37°C. After adding cycloheximide to the medium, samples were taken at the time points indicated and protein extracts were prepared. The protein level of epitope-tagged ^mycHMG2 is shown by performing western blots with an anti-myc antibody (upper panel). As a control, blots were reprobed with an antibody against the stable ER membrane protein DPM1 (lower panel). The right panel shows the quantification of the ^mycHMG2 decay. The estimated half-lives of ^mycHMG2 in WT, ufd1-1, npl4-1, cdc48-6 and shp1Δ strains were 20, 175, 110, 70 and 30 minutes, respectively. (B) Steady state level of the unstable ^DEG1SEC62^FLAG variant in WT, ufd1-2, npl4-1, cdc48-6 cells and the deletion strain shp1Δ. Cells were grown in YPGal to an OD₆₀₀ of 0.5 at 23°C and after another 2 h of growth at 37°C, aliquots were taken and protein samples prepared. The steady state level of the epitope-tagged SEC62 variant was analyzed by using an anti-FLAG antibody (upper panel). As a control, blots were reprobed with an antibody against DPM1 (lower panel).