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Sec61p is required for ERAD-L: genetic dissection of the translocation and ERAD-L functions of Sec61P using novel derivatives of CPY.

Willer M, Forte GM, Stirling CJ - J. Biol. Chem. (2008)

Bottom Line: Misfolded proteins in the endoplasmic reticulum (ER) are exported to the cytosol for degradation by the proteasome in a process known as ER-associated degradation (ERAD).CPY* is a well characterized ERAD substrate whose degradation is dependent upon the Hrd1 complex.By changing the translocation properties of a series of novel ERAD substrates, we are able to separate these two events and find that functional Sec61p is essential for the ERAD-L pathway.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom.

ABSTRACT
Misfolded proteins in the endoplasmic reticulum (ER) are exported to the cytosol for degradation by the proteasome in a process known as ER-associated degradation (ERAD). CPY* is a well characterized ERAD substrate whose degradation is dependent upon the Hrd1 complex. However, although the functions of some of the components of this complex are known, the nature of the protein dislocation channel remains obscure. Sec61p has been suggested as an obvious candidate because of its role as a protein-conducting channel through which polypeptides are initially translocated into the ER. However, it has not yet been possible to functionally dissect any role for Sec61p in dislocation from its essential function in translocation. By changing the translocation properties of a series of novel ERAD substrates, we are able to separate these two events and find that functional Sec61p is essential for the ERAD-L pathway.

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ERAD of DPY* and OPY is blocked in sec61-3 cells at 17 °C. A, as in Fig. 5 except cells were incubated at 17 °C after the addition of cold methionine, and samples were collected every 60 min for 4 h. B, quantification of data shown in A. C, wild type and sec62-1 cells expressing DPY* or OPY* were analyzed as described in Fig. 3A. D, wild type cells expressing DPY* were analyzed as described in Fig. 3A with or without the addition of 10 μg/ml cycloheximide (CHX).
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fig6: ERAD of DPY* and OPY is blocked in sec61-3 cells at 17 °C. A, as in Fig. 5 except cells were incubated at 17 °C after the addition of cold methionine, and samples were collected every 60 min for 4 h. B, quantification of data shown in A. C, wild type and sec62-1 cells expressing DPY* or OPY* were analyzed as described in Fig. 3A. D, wild type cells expressing DPY* were analyzed as described in Fig. 3A with or without the addition of 10 μg/ml cycloheximide (CHX).

Mentions: Next we tested for any effect on ERAD following inactivation of the Sec61p-dependent translocase at 17 °C. The ER was preloaded with ERAD-competent substrate by pulse labeling at 30 °C in either wild type or sec61-3 mutant cells. The cells were then shifted to 17 °C and chased in the presence of unlabeled methionine for the times indicated (Fig. 6, A and B). We observed a dramatic increase in the stability of both substrates in sec61-3 cells when compared with wild type controls. Thus functional Sec61p is required for the ER-associated degradation of both DPY* and OPY*.


Sec61p is required for ERAD-L: genetic dissection of the translocation and ERAD-L functions of Sec61P using novel derivatives of CPY.

Willer M, Forte GM, Stirling CJ - J. Biol. Chem. (2008)

ERAD of DPY* and OPY is blocked in sec61-3 cells at 17 °C. A, as in Fig. 5 except cells were incubated at 17 °C after the addition of cold methionine, and samples were collected every 60 min for 4 h. B, quantification of data shown in A. C, wild type and sec62-1 cells expressing DPY* or OPY* were analyzed as described in Fig. 3A. D, wild type cells expressing DPY* were analyzed as described in Fig. 3A with or without the addition of 10 μg/ml cycloheximide (CHX).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2590686&req=5

fig6: ERAD of DPY* and OPY is blocked in sec61-3 cells at 17 °C. A, as in Fig. 5 except cells were incubated at 17 °C after the addition of cold methionine, and samples were collected every 60 min for 4 h. B, quantification of data shown in A. C, wild type and sec62-1 cells expressing DPY* or OPY* were analyzed as described in Fig. 3A. D, wild type cells expressing DPY* were analyzed as described in Fig. 3A with or without the addition of 10 μg/ml cycloheximide (CHX).
Mentions: Next we tested for any effect on ERAD following inactivation of the Sec61p-dependent translocase at 17 °C. The ER was preloaded with ERAD-competent substrate by pulse labeling at 30 °C in either wild type or sec61-3 mutant cells. The cells were then shifted to 17 °C and chased in the presence of unlabeled methionine for the times indicated (Fig. 6, A and B). We observed a dramatic increase in the stability of both substrates in sec61-3 cells when compared with wild type controls. Thus functional Sec61p is required for the ER-associated degradation of both DPY* and OPY*.

Bottom Line: Misfolded proteins in the endoplasmic reticulum (ER) are exported to the cytosol for degradation by the proteasome in a process known as ER-associated degradation (ERAD).CPY* is a well characterized ERAD substrate whose degradation is dependent upon the Hrd1 complex.By changing the translocation properties of a series of novel ERAD substrates, we are able to separate these two events and find that functional Sec61p is essential for the ERAD-L pathway.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom.

ABSTRACT
Misfolded proteins in the endoplasmic reticulum (ER) are exported to the cytosol for degradation by the proteasome in a process known as ER-associated degradation (ERAD). CPY* is a well characterized ERAD substrate whose degradation is dependent upon the Hrd1 complex. However, although the functions of some of the components of this complex are known, the nature of the protein dislocation channel remains obscure. Sec61p has been suggested as an obvious candidate because of its role as a protein-conducting channel through which polypeptides are initially translocated into the ER. However, it has not yet been possible to functionally dissect any role for Sec61p in dislocation from its essential function in translocation. By changing the translocation properties of a series of novel ERAD substrates, we are able to separate these two events and find that functional Sec61p is essential for the ERAD-L pathway.

Show MeSH
Related in: MedlinePlus