Skip to main content
. 2000 Oct 2;151(1):69–82. doi: 10.1083/jcb.151.1.69

Figure 2.

Figure 2

Dominant negative truncation mutants of Hrd1p. (a) hemi-Hrd1p expression stabilized Hmg2p-GFP similar to a RING-H2 deletion mutant of Hrd1p. Flow cytometry of log phase strains expressing Hmg2p-GFP (wt) or coexpressing hemi-Hrd1p or the RING-H2 motif deletion mutant of Hrd1p as indicated. The stabilizing effect of the hemi-HRD1 allele or the ΔH2-hrd1 allele was seen by the rightward shift in the fluorescence histogram compared with the wild-type strain. (b) Hrd3p overexpression reversed the hemi-HRD1 phenotype for 1myc-Hmg2p degradation. Isogenic strains expressing hemi-Hrd1p only (empty vector) or those coexpressing either the PTDH3-HRD3 or the PTDH3-HRD1 allele were assayed for 1myc-Hmg2p degradation by cycloheximide-chase assay, along with an isogenic wild-type strain (wt) without hemi-Hrd1p. Cell lysates were prepared at the indicated times after cycloheximide addition and immunoblotted using the 9E10 anti-myc mAb. (c) Hrd3p overexpression reversed the PTDH3-hemi-HRD1 phenotype for Hmg2p-GFP degradation. Flow cytometric analysis of strains expressing Hmg2p-GFP and the indicated alleles was performed as above in panel a. (d) RING-Hrd1p expression stabilized Hmg2p-GFP. Isogenic strains expressing Hmg2p-GFP and coexpressing either RING-Hrd1p (RING-HRD1), C399S-RING-Hrd1p (C399S-RING-HRD1), or full-length C399S-Hrd1p (C399S-hrd1) were analyzed by flow cytometry as in panel a. (e) Hrd3p overexpression did not suppress the RING-HRD1 phenotype for 1myc-Hmg2p degradation. Isogenic strains expressing RING-Hrd1p only (empty vector) or those coexpressing either the PTDH3-HRD3 allele or the PTDH3-HRD1 allele, were assayed for 1myc-Hmg2p degradation by cycloheximide chase assay along with a wild-type strain not expressing RING-Hrd1p. Cell lysates were prepared at the indicated times after cycloheximide addition and immunoblotted using the 9E10 anti-myc mAb. (f) Hrd3p overexpression did not reverse the PTDH3-RING-HRD1 phenotype for Hmg2p-GFP degradation. Flow cytometry of strains expressing Hmg2p-GFP and the indicated alleles was performed as in panel a.