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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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Hrd1p and Hrd3p interacted via the Hrd1p NH2-terminal transmembrane domain. (a) Hrd3p cross-linked to Hrd1p. 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. 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). (b) Cartoon depicting Hrd1p topology. Top row, linear representation of Hrd1p domains. The start and end of each domain are indicated by the number of the corresponding amino acid residue below. 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). (c) Hrd3p cross-linked to hemi-Hrd1p-GFP, but not RING-Hrd1p-GFP. 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. 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). (d) hemi-Hrd1p expression inhibited Hrd3p cross-linking Hrd1p. The same cross-linking assay in panel a was performed with cells expressing 3HA-Hrd3p with or without the PTDH3-hemi-HRD1 allele. To compensate for lower Hrd1p in the hemi-Hrd1p cells (see Fig. 4 a), four times less lysate was used in the control lanes so that all lanes had identical amounts of immunoprecipitated Hrd1p. (e) Native coimmunoprecipitation of hemi-Hrd1p with Hrd3p. Cells expressing either 1myc-hemi-Hrd1p, 3HA-Hrd3p, or both proteins were lysed under nondenaturing conditions and immunoprecipitated with anti-HA polyclonal antisera. Immunoprecipitates were immunoblotted with the appropriate mAb to detect coimmunoprecipitated 1myc-hemi-Hrd1p (bottom) or immunoprecipitated 3HA-Hrd3p (top). (f) hemi-Hrd1p coimmunoprecipitated with Hrd3p only when expressed in the same cell. 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). Cells expressing each protein individually were mixed in equal quantities and lysed. Cells expressing both proteins were mixed with an equal number of empty cells to ensure an equal protein load and lysed (cartoon). 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).
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Figure 1: Hrd1p and Hrd3p interacted via the Hrd1p NH2-terminal transmembrane domain. (a) Hrd3p cross-linked to Hrd1p. 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. 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). (b) Cartoon depicting Hrd1p topology. Top row, linear representation of Hrd1p domains. The start and end of each domain are indicated by the number of the corresponding amino acid residue below. 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). (c) Hrd3p cross-linked to hemi-Hrd1p-GFP, but not RING-Hrd1p-GFP. 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. 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). (d) hemi-Hrd1p expression inhibited Hrd3p cross-linking Hrd1p. The same cross-linking assay in panel a was performed with cells expressing 3HA-Hrd3p with or without the PTDH3-hemi-HRD1 allele. To compensate for lower Hrd1p in the hemi-Hrd1p cells (see Fig. 4 a), four times less lysate was used in the control lanes so that all lanes had identical amounts of immunoprecipitated Hrd1p. (e) Native coimmunoprecipitation of hemi-Hrd1p with Hrd3p. Cells expressing either 1myc-hemi-Hrd1p, 3HA-Hrd3p, or both proteins were lysed under nondenaturing conditions and immunoprecipitated with anti-HA polyclonal antisera. Immunoprecipitates were immunoblotted with the appropriate mAb to detect coimmunoprecipitated 1myc-hemi-Hrd1p (bottom) or immunoprecipitated 3HA-Hrd3p (top). (f) hemi-Hrd1p coimmunoprecipitated with Hrd3p only when expressed in the same cell. 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). Cells expressing each protein individually were mixed in equal quantities and lysed. Cells expressing both proteins were mixed with an equal number of empty cells to ensure an equal protein load and lysed (cartoon). 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).

Mentions: Physical interactions between Hrd1p and Hrd3p were determined using an in vivo cross-linking assay modified from a procedure for cell lysates (Marcusson et al. 1994). The strains used for the cross-linking assays expressed fully functional, triple HA epitope–tagged versions of Hrd1p and/or Hrd3p from their native promoters. Each modified protein completely complemented the respective alleles for ERAD when expressed in single copy from their native promoters (data not shown). When lysates were derived from cells treated with increasing amounts of cross-linker and Hrd1p was immunoprecipitated using Hrd1p-specific antisera, Hrd3p coimmunoprecipitated in a cross-linker concentration–dependent fashion (Fig. 1 a, top left). In the absence of cellular Hrd1p, no cross-linker–dependent Hrd3p coimmunoprecipitation was observed (Fig. 1 a, top right). The small amount of Hrd3p that coimmunoprecipitated with Hrd1p in the absence of cross-linker was roughly equivalent to that seen in the absence of Hrd1p, indicating that this amount was nonspecific.


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)

Hrd1p and Hrd3p interacted via the Hrd1p NH2-terminal transmembrane domain. (a) Hrd3p cross-linked to Hrd1p. 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. 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). (b) Cartoon depicting Hrd1p topology. Top row, linear representation of Hrd1p domains. The start and end of each domain are indicated by the number of the corresponding amino acid residue below. 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). (c) Hrd3p cross-linked to hemi-Hrd1p-GFP, but not RING-Hrd1p-GFP. 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. 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). (d) hemi-Hrd1p expression inhibited Hrd3p cross-linking Hrd1p. The same cross-linking assay in panel a was performed with cells expressing 3HA-Hrd3p with or without the PTDH3-hemi-HRD1 allele. To compensate for lower Hrd1p in the hemi-Hrd1p cells (see Fig. 4 a), four times less lysate was used in the control lanes so that all lanes had identical amounts of immunoprecipitated Hrd1p. (e) Native coimmunoprecipitation of hemi-Hrd1p with Hrd3p. Cells expressing either 1myc-hemi-Hrd1p, 3HA-Hrd3p, or both proteins were lysed under nondenaturing conditions and immunoprecipitated with anti-HA polyclonal antisera. Immunoprecipitates were immunoblotted with the appropriate mAb to detect coimmunoprecipitated 1myc-hemi-Hrd1p (bottom) or immunoprecipitated 3HA-Hrd3p (top). (f) hemi-Hrd1p coimmunoprecipitated with Hrd3p only when expressed in the same cell. 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). Cells expressing each protein individually were mixed in equal quantities and lysed. Cells expressing both proteins were mixed with an equal number of empty cells to ensure an equal protein load and lysed (cartoon). 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).
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Figure 1: Hrd1p and Hrd3p interacted via the Hrd1p NH2-terminal transmembrane domain. (a) Hrd3p cross-linked to Hrd1p. 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. 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). (b) Cartoon depicting Hrd1p topology. Top row, linear representation of Hrd1p domains. The start and end of each domain are indicated by the number of the corresponding amino acid residue below. 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). (c) Hrd3p cross-linked to hemi-Hrd1p-GFP, but not RING-Hrd1p-GFP. 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. 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). (d) hemi-Hrd1p expression inhibited Hrd3p cross-linking Hrd1p. The same cross-linking assay in panel a was performed with cells expressing 3HA-Hrd3p with or without the PTDH3-hemi-HRD1 allele. To compensate for lower Hrd1p in the hemi-Hrd1p cells (see Fig. 4 a), four times less lysate was used in the control lanes so that all lanes had identical amounts of immunoprecipitated Hrd1p. (e) Native coimmunoprecipitation of hemi-Hrd1p with Hrd3p. Cells expressing either 1myc-hemi-Hrd1p, 3HA-Hrd3p, or both proteins were lysed under nondenaturing conditions and immunoprecipitated with anti-HA polyclonal antisera. Immunoprecipitates were immunoblotted with the appropriate mAb to detect coimmunoprecipitated 1myc-hemi-Hrd1p (bottom) or immunoprecipitated 3HA-Hrd3p (top). (f) hemi-Hrd1p coimmunoprecipitated with Hrd3p only when expressed in the same cell. 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). Cells expressing each protein individually were mixed in equal quantities and lysed. Cells expressing both proteins were mixed with an equal number of empty cells to ensure an equal protein load and lysed (cartoon). 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).
Mentions: Physical interactions between Hrd1p and Hrd3p were determined using an in vivo cross-linking assay modified from a procedure for cell lysates (Marcusson et al. 1994). The strains used for the cross-linking assays expressed fully functional, triple HA epitope–tagged versions of Hrd1p and/or Hrd3p from their native promoters. Each modified protein completely complemented the respective alleles for ERAD when expressed in single copy from their native promoters (data not shown). When lysates were derived from cells treated with increasing amounts of cross-linker and Hrd1p was immunoprecipitated using Hrd1p-specific antisera, Hrd3p coimmunoprecipitated in a cross-linker concentration–dependent fashion (Fig. 1 a, top left). In the absence of cellular Hrd1p, no cross-linker–dependent Hrd3p coimmunoprecipitation was observed (Fig. 1 a, top right). The small amount of Hrd3p that coimmunoprecipitated with Hrd1p in the absence of cross-linker was roughly equivalent to that seen in the absence of Hrd1p, indicating that this amount was nonspecific.

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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