SpringerProtocols: Abstract: Reconstitution of Endoplasmic Reticulum-Associated Degradation Using Yeast Membranes and Cytosol

Reconstitution of Endoplasmic Reticulum-Associated Degradation Using Yeast Membranes and Cytosol

By: Robert J. Lee³, Ardythe A. McCracken⁴, Jeffrey L. Brodsky³

Abstract

Full Text

Download PDF (145K)

The first compartment encountered by newly synthesized secreted proteins is the endoplasmic reticulum (ER). Before secreted proteins can traffic beyond the ER they must fold into their final conformations, and components of multiprotein complexes must assemble. Not surprisingly, then, the ER lumen houses a high concentration of molecular chaperones, factors that facilitate protein folding. However, if misfolded secreted proteins arise they may be selected and proteolyzed. This process, which removes potentially toxic proteins from the cell, has been termed ER-associated degradation (ERAD). Surprisingly, ERAD substrates are not degraded within the ER after being selected but are retrotranslocated to the cytoplasm and destroyed by the 26S proteasome. Thus, the ERAD pathway comprises substrate selection, substrate export from the ER, and substrate degradation, and each step in this pathway has been elucidated in part through an in vitro ERAD assay using reagents prepared from a model eukaryote, the yeast Saccharomyces cerevisiae. This chapter describes this in vitro ERAD assay in detail, and special considerations when performing the assay are noted.

Affiliation(s): (3) Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA
(4) Department of Biology, University of Nevada, Reno, NV

Book Title: Ubiquitin-Proteasome Protocols

Series: Methods in Molecular Biology | Volume: 301 | Pub. Date: Apr-01-2005 | Page Range: 175-184 | DOI: 10.1385/1-59259-895-1:175

Subject: Biochemistry

Key Words: Degradation - endoplasmic reticulum - ER-associated degradation - microsome - molecular chaperone - proteasome - translocation - yeast

References

Download References

1.	McCracken, A. A. and Brodsky, J. L. (1996) Assembly of ER-associated protein degradation in vitro: dependence on cytosol, calnexin, and ATP. J. Cell Biol. 132, 291–298.

2.	Ellgaard L. and Helenius, A. (2003) Quality control in the endoplasmic reticulum. Nat. Rev. Mol. Cell Biol. 4, 181–91.

3.	McCracken, A. A. and Brodsky, J. L. (2003) Evolving questions and paradigm shifts in endoplasmic reticulum associated degradation (ERAD). Bioessays 25, 868–877.

4.	Voges, D., Zwickl, P., and Baumeister, W. (1999) The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu. Rev. Biochem. 68, 1015–1068.

5.	Kostova, Z. and Wolf, D. H. (2003). For whom the bell tolls: protein quality control of the endoplasmic reticulum and the ubiquitin-proteasome connection. EMBO J. 22, 2309–2317.

6.	Werner, E. D., Brodsky, J. L., and McCracken, A. A. (1996) Proteasome-dependent endoplasmic reticulum—associated protein degradation: an unconventional route to a familiar fate. Proc. Natl. Acad. Sci. USA 93, 13797–13801.

7.	Pilon, M., Schekman, R., and Römisch, K. (1997) Sec61p mediates export of a misfolded secretory protein from the endoplasmic reticulum to the cytosol for degradation. EMBO J. 16, 4540–4548.

8.	Nishikawa, S., Fewell, S. W., Kato, Y., Brodsky, J. L., and Endo, T. (2001) Molecular chaperones in the yeast endoplasmic reticulum maintain the solubility of proteins from retro-translocation and degradation. J. Cell Biol. 153, 1061–1069.

9.	Zhou, M. and Schekman, R. (1999) The engagement of Sec61p in the ER dislocation process. Mol. Cell 4, 925–34. <Occurrence Type="DOI"><Handle>10.1016/S1097-2765(00)80222-1</Handle></Occurrence>

10.	Brodsky, J. L., Werner, E. D., Dubas, M. E., Goeckeler, J. L., Kruse, K. B., and McCracken, A. A. (1999) The requirement for molecular chaperones during ER-associated protein degradation (ERAD) demonstrates that protein import and export are mechanistically distinct. J. Biol. Chem. 274, 3453–3460.

11.	Gillece, P., Luz, J. M., Lennarz, W. J., de La Cruz, F. J., and Romisch, K. (1999) Export of a cysteine-free misfolded secretory protein from the endoplasmic reticulum for degradation requires interaction with protein disulfide isomerase. J. Cell Biol. 147, 1443–56.

12.	Rothblatt, J. A. and Meyer, D. I. (1986) Secretion in yeast: reconstitution of the translocation and glycosylation of alpha-factor and invertase in a homologous cell-free system. Cell 44, 619–628. <Occurrence Type="DOI"><Handle>10.1016/0092-8674(86)90271-0</Handle></Occurrence>

13.	Deshaies, R. J. and Schekman, R. (1989) SEC62 encodes a putative membrane protein required for protein translocation into the yeast endoplasmic reticulum. J. Cell Biol. 109, 2653–2664.

14.	Brodsky, J. L. and Schekman, R. (1993) A Sec63p-BiP complex from yeast is required for protein translocation in a reconstituted proteoliposome. J. Cell Biol. 123, 1355–63.

15.	Sorger, P. K. and Pelham, H. R. (1987) Purification and characterization of a heat-shock element binding protein from yeast. EMBO J. 6, 3035–3041.

16.	McGuire, M. J., McCullogh, M. L., Croall, D. E., and DeMartino, G. N. (1989) The high molecular weight multicatalytic proteinase, macropain, exists in a latent form in human erythrocytes. Biochim. Biophys. Acta 995, 181–186.

17.	Chu-Ping, M., Vu, J. H., Proske, R. J., Slaughter, C. A., and DeMartino, G. N. (1994) Identification, purification, and characterization of a high molecular weight, ATP-dependent activator (PA700) of the 20S proteasome. J. Biol. Chem. 269, 3539–3547.

18.	DeMartino, G. N., Moomaw, C. R., Zagnitko, O. P., et al. (1994) PA700, an ATP-dependent activator of the 20S proteasome, is an ATPase containing multiple members of a nucleotide-binding protein family. J. Biol. Chem. 269, 20878–20884.

19.	Verma, R., Chen, S., Feldman, R., et al. (2000) Proteasomal proteomics: identification of nucleotide-sensitive proteasome-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes. Mol. Biol. Cell 11, 3425–3439.

20.	Lee, R., Liu, C. W., Harry, C., et al. (2004) Retro-translocation and degradation can be uncoupled during the ER Associated Degradation (ERAD) of a soluble protein. EMBO J. 23, 2206–2215.

21.	Gusarova, V., Caplan, A. J., Brodsky, J. L., and Fisher, E. A. (2001) Apoprotein B degradation is promoted by the molecular chaperones Hsp90 and Hsp70. J. Biol. Chem. 276, 24891–24900.

Comments (Loading...)

Post comment/View all comments