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. (a) hemi-Hrd1p expression stabilized Hmg2p-GFP similar to a RING-H2 deletion mutant of Hrd1p. Each lysate (right) was immunoblotted with an anti-myc mAb to detect total 1myc-hemi-Hrd1p (bottom) or with an anti-HA mAb to detect total Hrd3p (top).ĭominant negative truncation mutants of Hrd1p. Cells expressing both proteins were mixed with an equal number of empty cells to ensure an equal protein load and lysed (cartoon). Cells expressing each protein individually were mixed in equal quantities and lysed. Same experiment as in panel d, except 1myc-hemi-Hrd1p and 3HA-Hrd3p were expressed either in the same cell or in separate cells (sep). (f) hemi-Hrd1p coimmunoprecipitated with Hrd3p only when expressed in the same cell. Immunoprecipitates were immunoblotted with the appropriate mAb to detect coimmunoprecipitated 1myc-hemi-Hrd1p (bottom) or immunoprecipitated 3HA-Hrd3p (top). Cells expressing either 1myc-hemi-Hrd1p, 3HA-Hrd3p, or both proteins were lysed under nondenaturing conditions and immunoprecipitated with anti-HA polyclonal antisera. (e) Native coimmunoprecipitation of hemi-Hrd1p with Hrd3p. 4 a), four times less lysate was used in the control lanes so that all lanes had identical amounts of immunoprecipitated Hrd1p. To compensate for lower Hrd1p in the hemi-Hrd1p cells (see Fig. The same cross-linking assay in panel a was performed with cells expressing 3HA-Hrd3p with or without the P TDH3- hemi-HRD1 allele. (d) hemi-Hrd1p expression inhibited Hrd3p cross-linking Hrd1p. Precipitated proteins were immunoblotted with an anti-HA mAb to detect coimmunoprecipitated 3HA-Hrd3p (top), or with an anti-GFP mAb to detect immunoprecipitated Hrd1p-GFP fusions (bottom). Cells coexpressing 3HA-Hrd3p and the indicated Hrd1p-GFP fusion were subject to the cross-linking assay using anti-GFP antisera to immunoprecipitate the Hrd1p-GFP fusions from the lysates. (c) Hrd3p cross-linked to hemi-Hrd1p-GFP, but not RING-Hrd1p-GFP. Bottom row, cartoon representations of the various Hrd1p constructs including wt-Hrd1p, only the Hrd1p transmembrane domain (hemi-Hrd1p), and only the COOH-terminal RING-H2 domain (termed RING-Hrd1p). The start and end of each domain are indicated by the number of the corresponding amino acid residue below. Top row, linear representation of Hrd1p domains. Precipitated proteins were next immunoblotted with an anti-HA mAb to detect coimmunoprecipitated 3HA-Hrd3p (top) or with an anti-Hrd1p polyclonal antisera to verify equal amounts of immunoprecipitated Hrd1p (bottom). Log phase cells expressing the indicated 3HA epitope–tagged proteins were treated with the indicated concentrations of DSP, lysed, and immunoprecipitated with anti-Hrd1p antisera. Hrd1p and Hrd3p interacted via the Hrd1p NH 2-terminal transmembrane domain. The HRD complex engages in lumen to cytosol communication required for regulation of Hrd1p stability and the coordination of ERAD events on both sides of the ER membrane. Our studies show that Hrd1p and Hrd3p form a stoichiometric complex with ERAD determinants in both the lumen and the cytosol. Additionally, we identified a lumenal region of Hrd3p dispensable for regulation of Hrd1p stability, but absolutely required for normal ERAD. An engineered, completely lumenal, truncated version of Hrd3p functioned normally in both ERAD and Hrd1p stabilization, indicating that the lumenal domain of Hrd3p regulates the cytosolic Hrd1p RING-H2 domain by signaling through the Hrd1p transmembrane domain. Rigorous reevaluation of Hrd1p topology demonstrated that the Hrd1p RING-H2 domain is located and functions in the cytosol. We show that these two proteins directly interact through the Hrd1p transmembrane domain, allowing Hrd1p stability by Hrd3p-dependent control of the Hrd1p RING-H2 domain activity. The ER resident membrane proteins Hrd1p and Hrd3p play central roles in ERAD. ERAD occurs by processes on both sides of the ER membrane, including lumenal substrate scanning and cytosolic destruction by the proteasome. Endoplasmic reticulum (ER)-associated degradation (ERAD) is required for ubiquitin-mediated destruction of numerous proteins.
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