Limits...
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.

Show MeSH

Related in: MedlinePlus

Membrane association of novel derivatives of CPY*. Microsomes from wild type cells expressing CPY*, DPY*, or OPY* were treated with Na2CO3 as described under “Experimental Procedures.” Total (T), pellet (P), and supernatant (S) fractions were analyzed by Western blots using antibodies against CPY, Kar2p, or Sec61p as indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2590686&req=5

fig2: Membrane association of novel derivatives of CPY*. Microsomes from wild type cells expressing CPY*, DPY*, or OPY* were treated with Na2CO3 as described under “Experimental Procedures.” Total (T), pellet (P), and supernatant (S) fractions were analyzed by Western blots using antibodies against CPY, Kar2p, or Sec61p as indicated.

Mentions: OPY* was predicted to be similarly cleaved, whereas DPY* was predicted to insert into the bilayer as an integral membrane protein. To test these predictions we used carbonate extraction of microsomes to examine the membrane association of the various proteins. We found that DPY* behaved as an integral membrane protein, whereas both CPY* and OPY* were readily extracted by carbonate (Fig. 2). We therefore conclude that OPY* is soluble, whereas DPY* is membrane associated. DPY* and OPY* thus behave entirely differently from CPY* with regards to their mode of translocation and differently from one another with regards to their solubility/membrane association properties.


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)

Membrane association of novel derivatives of CPY*. Microsomes from wild type cells expressing CPY*, DPY*, or OPY* were treated with Na2CO3 as described under “Experimental Procedures.” Total (T), pellet (P), and supernatant (S) fractions were analyzed by Western blots using antibodies against CPY, Kar2p, or Sec61p as indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Membrane association of novel derivatives of CPY*. Microsomes from wild type cells expressing CPY*, DPY*, or OPY* were treated with Na2CO3 as described under “Experimental Procedures.” Total (T), pellet (P), and supernatant (S) fractions were analyzed by Western blots using antibodies against CPY, Kar2p, or Sec61p as indicated.
Mentions: OPY* was predicted to be similarly cleaved, whereas DPY* was predicted to insert into the bilayer as an integral membrane protein. To test these predictions we used carbonate extraction of microsomes to examine the membrane association of the various proteins. We found that DPY* behaved as an integral membrane protein, whereas both CPY* and OPY* were readily extracted by carbonate (Fig. 2). We therefore conclude that OPY* is soluble, whereas DPY* is membrane associated. DPY* and OPY* thus behave entirely differently from CPY* with regards to their mode of translocation and differently from one another with regards to their solubility/membrane association properties.

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