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Endoplasmic reticulum degradation requires lumen to cytosol signaling. Transmembrane control of Hrd1p by Hrd3p.

Gardner RG, Swarbrick GM, Bays NW, Cronin SR, Wilhovsky S, Seelig L, Kim C, Hampton RY - J. Cell Biol. (2000)

Bottom Line: Endoplasmic reticulum (ER)-associated degradation (ERAD) is required for ubiquitin-mediated destruction of numerous proteins.Our studies show that Hrd1p and Hrd3p form a stoichiometric complex with ERAD determinants in both the lumen and the cytosol.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, University of California at San Diego, La Jolla, California 92093, USA.

ABSTRACT
Endoplasmic reticulum (ER)-associated degradation (ERAD) is required for ubiquitin-mediated destruction of numerous proteins. ERAD occurs by processes on both sides of the ER membrane, including lumenal substrate scanning and cytosolic destruction by the proteasome. The ER resident membrane proteins Hrd1p and Hrd3p play central roles in ERAD. 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. Rigorous reevaluation of Hrd1p topology demonstrated that the Hrd1p RING-H2 domain is located and functions in the cytosol. 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. Additionally, we identified a lumenal region of Hrd3p dispensable for regulation of Hrd1p stability, but absolutely required for normal ERAD. Our studies show that Hrd1p and Hrd3p form a stoichiometric complex with ERAD determinants in both the lumen and the cytosol. 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.

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Orientation of Hrd1p proteins and complementation of a hrd1Δ allele by expression of both the RING-HRD1 and hemi-HRD1 alleles. (a) RING-Hrd1p-GFP was localized to the ER. Fluorescence microscopy was performed on otherwise isogenic ubc7Δ strains expressing RING-Hrd1p-GFP, Hrd1p-GFP, hemi-Hrd1p-GFP or C399S-Hrd1p-GFP. Cells were grown to log phase and observed by fluorescence microscopy. Position of the nucleus in each cell was visualized by DAPI staining (data not shown). Arrows indicate the perinuclear, ER localization of the cellular GFP fluorescence, which in all cases is similar to the ER localization of Hmg2p-GFP (right). (b) COOH-terminal RING-H2 domain of Hrd1p was exposed to the cytosol. Protease protection assay using isolated intact microsomes from strains expressing either 3HA-RING-Hrd1p, 3HA-Hrd1p, or 3HA-Hrd3p and 1myc-Hmg2p. Microsomes were treated with the indicated concentrations of trypsin in the absence or presence of Triton X-100 (X). Levels of each protein were assessed by immunoblotting using antibodies specific for the fused epitope tags or for the native protein in the case of Kar2p. For each protein observed, total amount in the cell was comparable to that found only in the crude microsomes (compare Lys lane with 0 lane). (c) Protease protection of untagged Hrd1p. Same experiment as in panel b, using both trypsin (left) or proteinase K (right). Hrd1p was immunoblotted with antisera against the 203 residue COOH-terminal region of Hrd1p. On the left, the black arrow indicates the trypsin-insensitive fragment of full-length Hrd1p, and the gray arrow indicates the trypsin-insensitive fragment for RING-Hrd1p. For the proteinase K experiment, the blot for 3HA-Hrd1p (second from left) was stripped and reprobed with an anti-HA mAb to detect both 3HA-Hrd1p and 3HA-Hrd3p (far right). (d) Coexpression of RING-Hrd1p and hemi-Hrd1p complemented the hrd1Δ allele for mevalonate pathway–regulated Hmg2p-GFP degradation. Cells containing the hrd1Δ allele and expressing Hmg2p-GFP and either RING-Hrd1p, hemi-Hrd1p, or both were grown to log phase and analyzed by flow cytometry (left). To assess proper regulation of Hmg2p-GFP degradation, zaragozic acid (10 μg/ml) or lovastatin (25 μg/ml) was added to cells early in log phase and cells were allowed to grow for 4 h before flow cytometric analysis (middle and right). (e) Coexpression of RING-Hrd1p and hemi-Hrd1p complemented the hrd1Δ allele for constitutive 6myc-Hmg2p degradation. Cells containing the hrd1Δ allele and expressing 6myc-Hmg2p and either RING-Hrd1p, hemi-Hrd1p, or both were subject to a cycloheximide chase assay. Cell lysates were prepared at the indicated times after cycloheximide addition and immunoblotted using the 9E10 anti-myc mAb.
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Figure 4: Orientation of Hrd1p proteins and complementation of a hrd1Δ allele by expression of both the RING-HRD1 and hemi-HRD1 alleles. (a) RING-Hrd1p-GFP was localized to the ER. Fluorescence microscopy was performed on otherwise isogenic ubc7Δ strains expressing RING-Hrd1p-GFP, Hrd1p-GFP, hemi-Hrd1p-GFP or C399S-Hrd1p-GFP. Cells were grown to log phase and observed by fluorescence microscopy. Position of the nucleus in each cell was visualized by DAPI staining (data not shown). Arrows indicate the perinuclear, ER localization of the cellular GFP fluorescence, which in all cases is similar to the ER localization of Hmg2p-GFP (right). (b) COOH-terminal RING-H2 domain of Hrd1p was exposed to the cytosol. Protease protection assay using isolated intact microsomes from strains expressing either 3HA-RING-Hrd1p, 3HA-Hrd1p, or 3HA-Hrd3p and 1myc-Hmg2p. Microsomes were treated with the indicated concentrations of trypsin in the absence or presence of Triton X-100 (X). Levels of each protein were assessed by immunoblotting using antibodies specific for the fused epitope tags or for the native protein in the case of Kar2p. For each protein observed, total amount in the cell was comparable to that found only in the crude microsomes (compare Lys lane with 0 lane). (c) Protease protection of untagged Hrd1p. Same experiment as in panel b, using both trypsin (left) or proteinase K (right). Hrd1p was immunoblotted with antisera against the 203 residue COOH-terminal region of Hrd1p. On the left, the black arrow indicates the trypsin-insensitive fragment of full-length Hrd1p, and the gray arrow indicates the trypsin-insensitive fragment for RING-Hrd1p. For the proteinase K experiment, the blot for 3HA-Hrd1p (second from left) was stripped and reprobed with an anti-HA mAb to detect both 3HA-Hrd1p and 3HA-Hrd3p (far right). (d) Coexpression of RING-Hrd1p and hemi-Hrd1p complemented the hrd1Δ allele for mevalonate pathway–regulated Hmg2p-GFP degradation. Cells containing the hrd1Δ allele and expressing Hmg2p-GFP and either RING-Hrd1p, hemi-Hrd1p, or both were grown to log phase and analyzed by flow cytometry (left). To assess proper regulation of Hmg2p-GFP degradation, zaragozic acid (10 μg/ml) or lovastatin (25 μg/ml) was added to cells early in log phase and cells were allowed to grow for 4 h before flow cytometric analysis (middle and right). (e) Coexpression of RING-Hrd1p and hemi-Hrd1p complemented the hrd1Δ allele for constitutive 6myc-Hmg2p degradation. Cells containing the hrd1Δ allele and expressing 6myc-Hmg2p and either RING-Hrd1p, hemi-Hrd1p, or both were subject to a cycloheximide chase assay. Cell lysates were prepared at the indicated times after cycloheximide addition and immunoblotted using the 9E10 anti-myc mAb.

Mentions: First, we tested the localization of intact Hrd1p and its truncation mutants by using the GFP fusions described in the interaction studies above. To aid visibility, all GFP fusions were expressed in strains with the ubc7Δ allele, since both the Hrd1p-GFP and the RING-Hrd1p-GFP were subject to degradation in a wild-type background (data not shown, and see Fig. 3). When expressed from the TDH3 promoter, both hemi-Hrd1p-GFP and RING-Hrd1p-GFP demonstrated identical cellular localization of GFP fluorescence (Fig. 4 a). The GFP fluorescence was primarily localized to a perinuclear band, as observed by 4,6-diamino-2-phenylindole (DAPI) staining of the nucleus (data not shown), with some GFP fluorescence localized to the periphery of the cell. This type of cellular localization is common for ER-localized membrane proteins (Parlati et al. 1995; Hampton et al. 1996b; Saito et al. 1999). The cellular localization of either hemi-Hrd1p-GFP or RING-Hrd1p-GFP was identical to that observed for full-length Hrd1p-GFP (Fig. 4 a), which has been previously determined to be ER localized (Bordallo et al. 1998). C399S-Hrd1p-GFP had identical ER localization as Hrd1p-GFP, as expected (Fig. 4 a). Furthermore, the cellular localization of each GFP construct was similar to Hmg2p-GFP (Fig. 4 a), a known ER-localized membrane protein (Hampton et al. 1996b).


Endoplasmic reticulum degradation requires lumen to cytosol signaling. Transmembrane control of Hrd1p by Hrd3p.

Gardner RG, Swarbrick GM, Bays NW, Cronin SR, Wilhovsky S, Seelig L, Kim C, Hampton RY - J. Cell Biol. (2000)

Orientation of Hrd1p proteins and complementation of a hrd1Δ allele by expression of both the RING-HRD1 and hemi-HRD1 alleles. (a) RING-Hrd1p-GFP was localized to the ER. Fluorescence microscopy was performed on otherwise isogenic ubc7Δ strains expressing RING-Hrd1p-GFP, Hrd1p-GFP, hemi-Hrd1p-GFP or C399S-Hrd1p-GFP. Cells were grown to log phase and observed by fluorescence microscopy. Position of the nucleus in each cell was visualized by DAPI staining (data not shown). Arrows indicate the perinuclear, ER localization of the cellular GFP fluorescence, which in all cases is similar to the ER localization of Hmg2p-GFP (right). (b) COOH-terminal RING-H2 domain of Hrd1p was exposed to the cytosol. Protease protection assay using isolated intact microsomes from strains expressing either 3HA-RING-Hrd1p, 3HA-Hrd1p, or 3HA-Hrd3p and 1myc-Hmg2p. Microsomes were treated with the indicated concentrations of trypsin in the absence or presence of Triton X-100 (X). Levels of each protein were assessed by immunoblotting using antibodies specific for the fused epitope tags or for the native protein in the case of Kar2p. For each protein observed, total amount in the cell was comparable to that found only in the crude microsomes (compare Lys lane with 0 lane). (c) Protease protection of untagged Hrd1p. Same experiment as in panel b, using both trypsin (left) or proteinase K (right). Hrd1p was immunoblotted with antisera against the 203 residue COOH-terminal region of Hrd1p. On the left, the black arrow indicates the trypsin-insensitive fragment of full-length Hrd1p, and the gray arrow indicates the trypsin-insensitive fragment for RING-Hrd1p. For the proteinase K experiment, the blot for 3HA-Hrd1p (second from left) was stripped and reprobed with an anti-HA mAb to detect both 3HA-Hrd1p and 3HA-Hrd3p (far right). (d) Coexpression of RING-Hrd1p and hemi-Hrd1p complemented the hrd1Δ allele for mevalonate pathway–regulated Hmg2p-GFP degradation. Cells containing the hrd1Δ allele and expressing Hmg2p-GFP and either RING-Hrd1p, hemi-Hrd1p, or both were grown to log phase and analyzed by flow cytometry (left). To assess proper regulation of Hmg2p-GFP degradation, zaragozic acid (10 μg/ml) or lovastatin (25 μg/ml) was added to cells early in log phase and cells were allowed to grow for 4 h before flow cytometric analysis (middle and right). (e) Coexpression of RING-Hrd1p and hemi-Hrd1p complemented the hrd1Δ allele for constitutive 6myc-Hmg2p degradation. Cells containing the hrd1Δ allele and expressing 6myc-Hmg2p and either RING-Hrd1p, hemi-Hrd1p, or both were subject to a cycloheximide chase assay. Cell lysates were prepared at the indicated times after cycloheximide addition and immunoblotted using the 9E10 anti-myc mAb.
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Figure 4: Orientation of Hrd1p proteins and complementation of a hrd1Δ allele by expression of both the RING-HRD1 and hemi-HRD1 alleles. (a) RING-Hrd1p-GFP was localized to the ER. Fluorescence microscopy was performed on otherwise isogenic ubc7Δ strains expressing RING-Hrd1p-GFP, Hrd1p-GFP, hemi-Hrd1p-GFP or C399S-Hrd1p-GFP. Cells were grown to log phase and observed by fluorescence microscopy. Position of the nucleus in each cell was visualized by DAPI staining (data not shown). Arrows indicate the perinuclear, ER localization of the cellular GFP fluorescence, which in all cases is similar to the ER localization of Hmg2p-GFP (right). (b) COOH-terminal RING-H2 domain of Hrd1p was exposed to the cytosol. Protease protection assay using isolated intact microsomes from strains expressing either 3HA-RING-Hrd1p, 3HA-Hrd1p, or 3HA-Hrd3p and 1myc-Hmg2p. Microsomes were treated with the indicated concentrations of trypsin in the absence or presence of Triton X-100 (X). Levels of each protein were assessed by immunoblotting using antibodies specific for the fused epitope tags or for the native protein in the case of Kar2p. For each protein observed, total amount in the cell was comparable to that found only in the crude microsomes (compare Lys lane with 0 lane). (c) Protease protection of untagged Hrd1p. Same experiment as in panel b, using both trypsin (left) or proteinase K (right). Hrd1p was immunoblotted with antisera against the 203 residue COOH-terminal region of Hrd1p. On the left, the black arrow indicates the trypsin-insensitive fragment of full-length Hrd1p, and the gray arrow indicates the trypsin-insensitive fragment for RING-Hrd1p. For the proteinase K experiment, the blot for 3HA-Hrd1p (second from left) was stripped and reprobed with an anti-HA mAb to detect both 3HA-Hrd1p and 3HA-Hrd3p (far right). (d) Coexpression of RING-Hrd1p and hemi-Hrd1p complemented the hrd1Δ allele for mevalonate pathway–regulated Hmg2p-GFP degradation. Cells containing the hrd1Δ allele and expressing Hmg2p-GFP and either RING-Hrd1p, hemi-Hrd1p, or both were grown to log phase and analyzed by flow cytometry (left). To assess proper regulation of Hmg2p-GFP degradation, zaragozic acid (10 μg/ml) or lovastatin (25 μg/ml) was added to cells early in log phase and cells were allowed to grow for 4 h before flow cytometric analysis (middle and right). (e) Coexpression of RING-Hrd1p and hemi-Hrd1p complemented the hrd1Δ allele for constitutive 6myc-Hmg2p degradation. Cells containing the hrd1Δ allele and expressing 6myc-Hmg2p and either RING-Hrd1p, hemi-Hrd1p, or both were subject to a cycloheximide chase assay. Cell lysates were prepared at the indicated times after cycloheximide addition and immunoblotted using the 9E10 anti-myc mAb.
Mentions: First, we tested the localization of intact Hrd1p and its truncation mutants by using the GFP fusions described in the interaction studies above. To aid visibility, all GFP fusions were expressed in strains with the ubc7Δ allele, since both the Hrd1p-GFP and the RING-Hrd1p-GFP were subject to degradation in a wild-type background (data not shown, and see Fig. 3). When expressed from the TDH3 promoter, both hemi-Hrd1p-GFP and RING-Hrd1p-GFP demonstrated identical cellular localization of GFP fluorescence (Fig. 4 a). The GFP fluorescence was primarily localized to a perinuclear band, as observed by 4,6-diamino-2-phenylindole (DAPI) staining of the nucleus (data not shown), with some GFP fluorescence localized to the periphery of the cell. This type of cellular localization is common for ER-localized membrane proteins (Parlati et al. 1995; Hampton et al. 1996b; Saito et al. 1999). The cellular localization of either hemi-Hrd1p-GFP or RING-Hrd1p-GFP was identical to that observed for full-length Hrd1p-GFP (Fig. 4 a), which has been previously determined to be ER localized (Bordallo et al. 1998). C399S-Hrd1p-GFP had identical ER localization as Hrd1p-GFP, as expected (Fig. 4 a). Furthermore, the cellular localization of each GFP construct was similar to Hmg2p-GFP (Fig. 4 a), a known ER-localized membrane protein (Hampton et al. 1996b).

Bottom Line: Endoplasmic reticulum (ER)-associated degradation (ERAD) is required for ubiquitin-mediated destruction of numerous proteins.Our studies show that Hrd1p and Hrd3p form a stoichiometric complex with ERAD determinants in both the lumen and the cytosol.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, University of California at San Diego, La Jolla, California 92093, USA.

ABSTRACT
Endoplasmic reticulum (ER)-associated degradation (ERAD) is required for ubiquitin-mediated destruction of numerous proteins. ERAD occurs by processes on both sides of the ER membrane, including lumenal substrate scanning and cytosolic destruction by the proteasome. The ER resident membrane proteins Hrd1p and Hrd3p play central roles in ERAD. 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. Rigorous reevaluation of Hrd1p topology demonstrated that the Hrd1p RING-H2 domain is located and functions in the cytosol. 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. Additionally, we identified a lumenal region of Hrd3p dispensable for regulation of Hrd1p stability, but absolutely required for normal ERAD. Our studies show that Hrd1p and Hrd3p form a stoichiometric complex with ERAD determinants in both the lumen and the cytosol. 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.

Show MeSH
Related in: MedlinePlus