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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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Involvement of HRD1 and GP78 in disposal of soluble and membrane-tethered NHK variants. (A) Radiolabeled NHK was immunoisolated after the indicated chase times from cells expressing a scrambled siRNA (siSCR; lanes 1–3), an siRNA targeting HRD1 (siHRD1; lanes 4–6), GP78 (siGP78; lanes 7–9), or both HRD1 and GP78 (siHRD1/siGP78; lanes 10–12). Relevant bands were quantified and plotted. (B and C) Same as described in A for NHKBACE and NHKCD3δ, respectively. Molecular mass markers are shown on the left for all gels (given in kilodaltons).
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fig7: Involvement of HRD1 and GP78 in disposal of soluble and membrane-tethered NHK variants. (A) Radiolabeled NHK was immunoisolated after the indicated chase times from cells expressing a scrambled siRNA (siSCR; lanes 1–3), an siRNA targeting HRD1 (siHRD1; lanes 4–6), GP78 (siGP78; lanes 7–9), or both HRD1 and GP78 (siHRD1/siGP78; lanes 10–12). Relevant bands were quantified and plotted. (B and C) Same as described in A for NHKBACE and NHKCD3δ, respectively. Molecular mass markers are shown on the left for all gels (given in kilodaltons).

Mentions: So far, we showed that deletion of the membrane anchor of ERAD-L substrates confers a strong dependency on the HRD1 machinery independent of the mechanisms regulating disposal of the membrane-tethered version of the proteins under investigation. To challenge our model, we decided to determine requirements for disposal of a soluble ERAD substrate and to verify how requirements would change upon addition of a transmembrane anchor. To this end, we selected a classical ERAD-L substrate, the NHK variant of the secretory protein α1-antitrypsin (Liu et al., 1997). As previously shown in other cell lines (Bernasconi et al., 2008), we confirm that a fraction of NHK escapes ER retention and is secreted extracellularly (∼15% of the labeled protein in HeLa cells; Fig. S2 F). The expectations were that disposal of this ERAD-LS protein should substantially be delayed upon inactivation of the E3 ligase HRD1 and that the strict dependency on HRD1 should be relieved when the folding-defective polypeptide is converted in a membrane-tethered ERAD-LM protein. Reduction of the intralumenal level of HRD1 (Fig. 7 A, lanes 4–6) substantially inhibited NHK degradation without affecting secretion of the protein (Fig. S2 F). The combined inactivation of the GP78 pathway did not further protect the protein from disposal (Fig. 7 A, lanes 10–12). The intervention of HRD1 was not surprising because both SEL1L (Christianson et al., 2008; unpublished data) and OS-9 (Bernasconi et al., 2008; Christianson et al., 2008) have been shown to participate in NHK disposal. Thus, NHK behaves as a bonafide ERAD-LS protein as defined above, which shows strict dependency on the HRD1 pathway for disposal.


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)

Involvement of HRD1 and GP78 in disposal of soluble and membrane-tethered NHK variants. (A) Radiolabeled NHK was immunoisolated after the indicated chase times from cells expressing a scrambled siRNA (siSCR; lanes 1–3), an siRNA targeting HRD1 (siHRD1; lanes 4–6), GP78 (siGP78; lanes 7–9), or both HRD1 and GP78 (siHRD1/siGP78; lanes 10–12). Relevant bands were quantified and plotted. (B and C) Same as described in A for NHKBACE and NHKCD3δ, respectively. Molecular mass markers are shown on the left for all gels (given in kilodaltons).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2812524&req=5

fig7: Involvement of HRD1 and GP78 in disposal of soluble and membrane-tethered NHK variants. (A) Radiolabeled NHK was immunoisolated after the indicated chase times from cells expressing a scrambled siRNA (siSCR; lanes 1–3), an siRNA targeting HRD1 (siHRD1; lanes 4–6), GP78 (siGP78; lanes 7–9), or both HRD1 and GP78 (siHRD1/siGP78; lanes 10–12). Relevant bands were quantified and plotted. (B and C) Same as described in A for NHKBACE and NHKCD3δ, respectively. Molecular mass markers are shown on the left for all gels (given in kilodaltons).
Mentions: So far, we showed that deletion of the membrane anchor of ERAD-L substrates confers a strong dependency on the HRD1 machinery independent of the mechanisms regulating disposal of the membrane-tethered version of the proteins under investigation. To challenge our model, we decided to determine requirements for disposal of a soluble ERAD substrate and to verify how requirements would change upon addition of a transmembrane anchor. To this end, we selected a classical ERAD-L substrate, the NHK variant of the secretory protein α1-antitrypsin (Liu et al., 1997). As previously shown in other cell lines (Bernasconi et al., 2008), we confirm that a fraction of NHK escapes ER retention and is secreted extracellularly (∼15% of the labeled protein in HeLa cells; Fig. S2 F). The expectations were that disposal of this ERAD-LS protein should substantially be delayed upon inactivation of the E3 ligase HRD1 and that the strict dependency on HRD1 should be relieved when the folding-defective polypeptide is converted in a membrane-tethered ERAD-LM protein. Reduction of the intralumenal level of HRD1 (Fig. 7 A, lanes 4–6) substantially inhibited NHK degradation without affecting secretion of the protein (Fig. S2 F). The combined inactivation of the GP78 pathway did not further protect the protein from disposal (Fig. 7 A, lanes 10–12). The intervention of HRD1 was not surprising because both SEL1L (Christianson et al., 2008; unpublished data) and OS-9 (Bernasconi et al., 2008; Christianson et al., 2008) have been shown to participate in NHK disposal. Thus, NHK behaves as a bonafide ERAD-LS protein as defined above, which shows strict dependency on the HRD1 pathway for disposal.

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