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Stringent requirement for HRD1, SEL1L, and OS-9/XTP3-B for disposal of ERAD-LS substrates.

Bernasconi R, Galli C, Calanca V, Nakajima T, Molinari M - J. Cell Biol. (2010)

Bottom Line: The presence of structural lesions in the luminal, transmembrane, or cytosolic domains determines the classification of misfolded polypeptides as ERAD-L, -M, or -C substrates and results in selection of distinct degradation pathways.In this study, we show that disposal of soluble (nontransmembrane) polypeptides with luminal lesions (ERAD-L(S) substrates) is strictly dependent on the E3 ubiquitin ligase HRD1, the associated cargo receptor SEL1L, and two interchangeable ERAD lectins, OS-9 and XTP3-B.Our data reveal that, in contrast to budding yeast, tethering of mammalian ERAD-L substrates to the membrane changes selection of the degradation pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Research in Biomedicine, 6500 Bellinzona, Switzerland.

ABSTRACT
Sophisticated quality control mechanisms prolong retention of protein-folding intermediates in the endoplasmic reticulum (ER) until maturation while sorting out terminally misfolded polypeptides for ER-associated degradation (ERAD). The presence of structural lesions in the luminal, transmembrane, or cytosolic domains determines the classification of misfolded polypeptides as ERAD-L, -M, or -C substrates and results in selection of distinct degradation pathways. In this study, we show that disposal of soluble (nontransmembrane) polypeptides with luminal lesions (ERAD-L(S) substrates) is strictly dependent on the E3 ubiquitin ligase HRD1, the associated cargo receptor SEL1L, and two interchangeable ERAD lectins, OS-9 and XTP3-B. These ERAD factors become dispensable for degradation of the same polypeptides when membrane tethered (ERAD-L(M) substrates). Our data reveal that, in contrast to budding yeast, tethering of mammalian ERAD-L substrates to the membrane changes selection of the degradation pathway.

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Trapping of BACE476Δ by OS-9.1 upon inactivation of the HRD1 pathway. (A) BACE476Δ was immunoisolated from detergent extracts of cells incubated with a scrambled siRNA, and siRNA targeting HRD1, GP78, GP78, and HRD1 (lanes 1–4) and SEL1L, OS-9, XTP3-B, and XTP3-B + OS-9 (lanes 6–9, respectively). Proteins were separated in SDS polyacrylamide gels and transferred on PVDF. The membranes were blotted with antibodies recognizing endogenous GRP94, BiP, OS-9.1, and OS-9.2 and BACE476Δ as a loading control. (B) Same as described in A for BACE476. Arrowheads indicate the coprecipitated OS-9.1. TCE, total cell extract; IP, immunoprecipitation.
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fig6: Trapping of BACE476Δ by OS-9.1 upon inactivation of the HRD1 pathway. (A) BACE476Δ was immunoisolated from detergent extracts of cells incubated with a scrambled siRNA, and siRNA targeting HRD1, GP78, GP78, and HRD1 (lanes 1–4) and SEL1L, OS-9, XTP3-B, and XTP3-B + OS-9 (lanes 6–9, respectively). Proteins were separated in SDS polyacrylamide gels and transferred on PVDF. The membranes were blotted with antibodies recognizing endogenous GRP94, BiP, OS-9.1, and OS-9.2 and BACE476Δ as a loading control. (B) Same as described in A for BACE476. Arrowheads indicate the coprecipitated OS-9.1. TCE, total cell extract; IP, immunoprecipitation.

Mentions: In an attempt to identify ERAD factors interacting with ERAD-LS versus ERAD-LM substrates, BACE476Δ was tagged with an EFRH tetrapeptide (which is recognized by β1, a monoclonal antibody described in Paganetti et al., 2005), whereas BACE476 was HA tagged. The two proteins were ectopically coexpressed in mock-treated cells (siSCR; Fig. 6, A and B, lane 1; Fig. S4) or in cells with reduced levels of HRD1 (Fig. 6, A and B, lane 2), GP78 (Fig. 6, A and B, lane 3), HRD1 and GP78 (Fig. 6, A and B, lane 4), SEL1L (Fig. 6, A and B, lane 6), OS-9 (Fig. 6, A and B, lane 7), XTP3-B (Fig. 6, A and B, lane 8), and OS-9 and XTP3-B (Fig. 6, A and B, lane 9). Cells were lyzed under conditions that preserve many substrate–chaperone interactions (Molinari et al., 2002). BACE476Δ-β1 (Fig. 6 A) and BACE476-HA (Fig. 6 B) were individually immunoisolated with the appropriate anti-tag antibody together with their interacting partners. The amount of proteins separated in SDS-PAGE and blotted on a PVDF membrane was normalized to ensure equal loading of BACE in each lane (Fig. 6, A and B, bottom). The PVDF membrane was probed with antibodies to GRP94, BiP, and OS-9 to determine whether these ER chaperones, claimed to be involved in ERAD, were found in complexes sufficiently stable to survive the cell lysis and immunoprecipitation protocols. The case for GRP94 is unclear, as variations in the amount of this chaperone coprecipitated with BACE476Δ-β1 (Fig. 6 A) or BACE476-HA (Fig. 6 B) were too small to be considered significant. Variations were also small for BiP. However, a stabilization of BACE476Δ–BiP complexes was observed upon inactivation of HRD1 (Fig. 6 A, lane 2), HRD1 + GP78 (Fig. 6 A, lane 4), SEL1L (Fig. 6 A, lane 6), and upon combined inactivation of OS-9 + XTP3-B (Fig. 6 A, lane 9). These variations were reproducible even when BACE476Δ-β1 was individually expressed in cells subjected to transient interferences (unpublished data). Stabilization of BACE476Δ–BiP complexes upon disassembly of the HRD1 dislocon shows that BiP contributes to delivery of terminally misfolded polypeptides to this machinery.


Stringent requirement for HRD1, SEL1L, and OS-9/XTP3-B for disposal of ERAD-LS substrates.

Bernasconi R, Galli C, Calanca V, Nakajima T, Molinari M - J. Cell Biol. (2010)

Trapping of BACE476Δ by OS-9.1 upon inactivation of the HRD1 pathway. (A) BACE476Δ was immunoisolated from detergent extracts of cells incubated with a scrambled siRNA, and siRNA targeting HRD1, GP78, GP78, and HRD1 (lanes 1–4) and SEL1L, OS-9, XTP3-B, and XTP3-B + OS-9 (lanes 6–9, respectively). Proteins were separated in SDS polyacrylamide gels and transferred on PVDF. The membranes were blotted with antibodies recognizing endogenous GRP94, BiP, OS-9.1, and OS-9.2 and BACE476Δ as a loading control. (B) Same as described in A for BACE476. Arrowheads indicate the coprecipitated OS-9.1. TCE, total cell extract; IP, immunoprecipitation.
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Related In: Results  -  Collection

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fig6: Trapping of BACE476Δ by OS-9.1 upon inactivation of the HRD1 pathway. (A) BACE476Δ was immunoisolated from detergent extracts of cells incubated with a scrambled siRNA, and siRNA targeting HRD1, GP78, GP78, and HRD1 (lanes 1–4) and SEL1L, OS-9, XTP3-B, and XTP3-B + OS-9 (lanes 6–9, respectively). Proteins were separated in SDS polyacrylamide gels and transferred on PVDF. The membranes were blotted with antibodies recognizing endogenous GRP94, BiP, OS-9.1, and OS-9.2 and BACE476Δ as a loading control. (B) Same as described in A for BACE476. Arrowheads indicate the coprecipitated OS-9.1. TCE, total cell extract; IP, immunoprecipitation.
Mentions: In an attempt to identify ERAD factors interacting with ERAD-LS versus ERAD-LM substrates, BACE476Δ was tagged with an EFRH tetrapeptide (which is recognized by β1, a monoclonal antibody described in Paganetti et al., 2005), whereas BACE476 was HA tagged. The two proteins were ectopically coexpressed in mock-treated cells (siSCR; Fig. 6, A and B, lane 1; Fig. S4) or in cells with reduced levels of HRD1 (Fig. 6, A and B, lane 2), GP78 (Fig. 6, A and B, lane 3), HRD1 and GP78 (Fig. 6, A and B, lane 4), SEL1L (Fig. 6, A and B, lane 6), OS-9 (Fig. 6, A and B, lane 7), XTP3-B (Fig. 6, A and B, lane 8), and OS-9 and XTP3-B (Fig. 6, A and B, lane 9). Cells were lyzed under conditions that preserve many substrate–chaperone interactions (Molinari et al., 2002). BACE476Δ-β1 (Fig. 6 A) and BACE476-HA (Fig. 6 B) were individually immunoisolated with the appropriate anti-tag antibody together with their interacting partners. The amount of proteins separated in SDS-PAGE and blotted on a PVDF membrane was normalized to ensure equal loading of BACE in each lane (Fig. 6, A and B, bottom). The PVDF membrane was probed with antibodies to GRP94, BiP, and OS-9 to determine whether these ER chaperones, claimed to be involved in ERAD, were found in complexes sufficiently stable to survive the cell lysis and immunoprecipitation protocols. The case for GRP94 is unclear, as variations in the amount of this chaperone coprecipitated with BACE476Δ-β1 (Fig. 6 A) or BACE476-HA (Fig. 6 B) were too small to be considered significant. Variations were also small for BiP. However, a stabilization of BACE476Δ–BiP complexes was observed upon inactivation of HRD1 (Fig. 6 A, lane 2), HRD1 + GP78 (Fig. 6 A, lane 4), SEL1L (Fig. 6 A, lane 6), and upon combined inactivation of OS-9 + XTP3-B (Fig. 6 A, lane 9). These variations were reproducible even when BACE476Δ-β1 was individually expressed in cells subjected to transient interferences (unpublished data). Stabilization of BACE476Δ–BiP complexes upon disassembly of the HRD1 dislocon shows that BiP contributes to delivery of terminally misfolded polypeptides to this machinery.

Bottom Line: The presence of structural lesions in the luminal, transmembrane, or cytosolic domains determines the classification of misfolded polypeptides as ERAD-L, -M, or -C substrates and results in selection of distinct degradation pathways.In this study, we show that disposal of soluble (nontransmembrane) polypeptides with luminal lesions (ERAD-L(S) substrates) is strictly dependent on the E3 ubiquitin ligase HRD1, the associated cargo receptor SEL1L, and two interchangeable ERAD lectins, OS-9 and XTP3-B.Our data reveal that, in contrast to budding yeast, tethering of mammalian ERAD-L substrates to the membrane changes selection of the degradation pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Research in Biomedicine, 6500 Bellinzona, Switzerland.

ABSTRACT
Sophisticated quality control mechanisms prolong retention of protein-folding intermediates in the endoplasmic reticulum (ER) until maturation while sorting out terminally misfolded polypeptides for ER-associated degradation (ERAD). The presence of structural lesions in the luminal, transmembrane, or cytosolic domains determines the classification of misfolded polypeptides as ERAD-L, -M, or -C substrates and results in selection of distinct degradation pathways. In this study, we show that disposal of soluble (nontransmembrane) polypeptides with luminal lesions (ERAD-L(S) substrates) is strictly dependent on the E3 ubiquitin ligase HRD1, the associated cargo receptor SEL1L, and two interchangeable ERAD lectins, OS-9 and XTP3-B. These ERAD factors become dispensable for degradation of the same polypeptides when membrane tethered (ERAD-L(M) substrates). Our data reveal that, in contrast to budding yeast, tethering of mammalian ERAD-L substrates to the membrane changes selection of the degradation pathway.

Show MeSH