T H E J O U R N A L O F C E L L B I O L O G Y
© 2008 Scott and Schekman
The Rockefeller University Press $30.00
J. Cell Biol. Vol. 181 No. 7 1095–1105
Correspondence to Randy Schekman: email@example.com
D.C. Scott ’ s current address is Depts. of Structural Biology and Genetics
and Tumor Cell Biology, St. Jude Children ’ s Research Hospital, Memphis,
Abbreviations used in this paper: ? f, ? -factor; CPY, carboxypeptidase Y; ERAD,
The online version of this paper contains supplemental material.
The ER provides an environment conducive for the folding and
assembly of newly synthesized secretory proteins. To prevent
the premature exit of improperly folded proteins from the ER,
cells have evolved quality control mechanisms to actively moni-
tor the folding state of proteins ( Brodsky and McCracken, 1999 ;
Ellgaard and Helenius, 2003 ). Proteins that irreversibly misfold
are recognized by this quality control system and targeted for
destruction through a process termed ER-associated degradation
(ERAD; Hampton, 2002 ). Although initial studies of ERAD
implied the action of unidentifi ed ER-localized proteases ( Finger
et al., 1993 ), subsequent work clearly defi ned roles for the cyto-
plasmically localized enzymes of the ubiquitin pathway and the
26S proteasome, providing the fi rst indication that misfolded
proteins must be retrotranslocated back across the membrane of
the ER ( Jensen et al., 1995 ; Ward et al., 1995 ). From a mechanistic
standpoint, these results established the need for an ER-localized
protein-conducting channel to direct the fl ow of misfolded pro-
tein export from the ER.
Circumstantial evidence suggested that Sec61p, the main
component of the protein-conducting channel for translocation
into the ER, participates in retrotranslocation from the ER ( Wiertz
et al., 1996 ; Pilon et al., 1997 ; Plemper et al. 1997, 1998 ). In
mammalian cells, Sec61 ? can be coimmunoprecipitated with
class I heavy chains that are targeted for ERAD by the human
cytomegalovirus-encoded glycoprotein US2 ( Wiertz et al., 1996 ).
Subsequently, genetic and biochemical analysis of Sec61p mutants
uncovered alleles more prone to defects in protein retrotrans-
location than translocation ( Pilon et al., 1997 ). Certain ERAD
substrates are stabilized in a partially translocation-defective
mutant, sec61-2 ( Plemper et al., 1997, 1998 ). In an independent
approach, Schmitz et al. (2000) showed that blocking Sec61
channels with translation-arrested ribosomes prevented exit of
cholera toxin from the ER. However, when the crystallographic
structure of SecY/E, an archaeal orthologue of Sec61p, revealed
a strict upper size limit for the pore diameter, it was diffi cult to
conceive how such a small pore could accommodate the larger
retrotranslocation substrates ( Tirosh et al., 2003 ; Van den Berg
et al., 2004 ). Nonetheless, for certain proteins that are subject
to ERAD before the completion of translocation, new evidence
suggests a direct participation of Sec61p in the ERAD process
( Oyadomari et al., 2006 ). For the majority of proteins subject
to ERAD after membrane assembly, the evidence favors one or
more distinct retrotranslocation channels.
Another candidate channel protein, Derlin-1, was identi-
fi ed by virtue of its association with the human cytomegalovirus-
encoded glycoprotein US11 in the process of retrotranslocation
and degradation of class I heavy chains ( Lilley and Ploegh, 2004 ;
com ponent of ER quality control. In ERAD, misfolded pro-
teins are removed from the ER by retrotranslocation into
the cytosol where they are degraded by the ubiquitin –
proteasome system. The identity of the specifi c protein
components responsible for retrotranslocation remains con-
troversial, with the potential candidates being Sec61p, Der1p,
and Doa10. We show that the cytoplasmic N-terminal
isfolded proteins in the endoplasmic reticulum
(ER) are identifi ed and degraded by the ER-
associated degradation pathway (ERAD), a
domain of a short-lived transmembrane ERAD substrate is
exposed to the lumen of the ER during the degradation
process. The addition of N-linked glycan to the N termi-
nus of the substrate is prevented by mutation of a specifi c
cysteine residue of Sec61p, as well as a specifi c cysteine
residue of the substrate protein. We show that the sub-
strate protein forms a disulfi de-linked complex to Sec61p,
suggesting that at least part of the retrotranslocation pro-
cess involves Sec61p.
Role of Sec61p in the ER-associated degradation
of short-lived transmembrane proteins
Daniel C. Scott and Randy Schekman
Department of Molecular and Cell Biology and Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720
JCB • VOLUME 181 • NUMBER 7 • 2008 1096
tion of residue 373 resulted in a slight, but reproducible, stabili-
zation of CPY* ( t 1/2 = 28 – 30 min; Fig. 1, C and D ). In contrast,
CPY* degradation was delayed around twofold in the sec61-2
mutant, which is consistent with previous reports for this mu-
tant ( Plemper et al., 1997 ). In all experiments, we observed a
single electrophoretic species of CPY* unlike the behavior of
Deg1:Sec62 ProtA .
The effect of Sec61p C373I on the production of one of two
electrophoretic species of Deg1:Sec62 ProtA led us to consider the
possibility that a covalent modifi cation accompanies the ERAD
of this substrate. Although the N and C termini of Sec62p are
exposed to the cytosol, the N-terminal Deg signal may infl uence
the disposition of Sec62 during ERAD. Thus, one possibility
was that cryptic N-glycosylation sites in the N terminus of
Sec62p may become exposed to the ER lumen during ERAD.
We found that Deg1:Sec62 ProtA was sensitive to endoglycosidase H,
ProtA is N glycosylated
Ye et al., 2004 ). The interaction of Derlin-1 with glycosylated
class I heavy chains before retrotranslocation and its subsequent
association with deglycosylated heavy chains when cells are
treated with proteasome inhibitors suggest that it is positioned
to interact with substrates before and immediately after they are
retrotranslocated ( Lilley and Ploegh, 2005 ). The role of the yeast
Derlin-1 homologue Der1p in ERAD is poorly defi ned but is
known to be required for the effi cient degradation of misfolded
luminal ER proteins ( Knop et al., 1996 ; Hitt and Wolf, 2004 ).
However, numerous ERAD substrates are degraded independent
of Der1p ( Hill and Cooper, 2000 ; Vashist and Ng, 2004 ). A sub-
set of these Der1p-independent substrates is also independent of
Sec61p but requires Doa10, an ER-localized E3 ubiquitin ligase.
Thus, Doa10 may represent a distinct retrotranslocation channel
in the ER ( Kreft et al., 2006 ).
In this paper, we describe our discovery and characteriza-
tion of a disulfi de-linked intermediate complex formed between a
short-lived transmembrane ERAD substrate and Sec61p. This
complex may accomplish at least an initial stage in the retrotrans-
location of unstable membrane proteins.
Cysteine mutants of Sec61p are protein
retrotranslocation profi cient
To test for defects in the ER export of misfolded membrane pro-
teins, we followed the degradation kinetics of the short-lived
transmembrane substrate Deg1:Sec62 ProtA . The Deg1 sequence
appended to the N terminus of Sec62 ( Deshaies and Schekman,
1989 ) diverts the hybrid protein to the ERAD pathway ( Mayer
et al., 1998 ). In wild-type cells, Deg1:Sec62 ProtA was rapidly de-
graded with t 1/2 of ? 13 min ( Fig. 1, A and B ). Previously, we
reported the effect of a substitution at cysteine 150 of the trans-
location channel ( sec61 – 32 ; C15OY) on the ERAD of an unglyco-
sylated form of ? -factor ( ? f) precursor ( Pilon et al., 1997 ).
To explore the role of each of the cysteine residues of Sec61p in
the ERAD of Deg1:Sec62 ProtA , we created strains in which one or
more of these residues was mutated on a CEN plasmid as the sole
copy of SEC61 . The rate of substrate degradation was accelerated
nearly twofold in a strain harboring Sec61p C373I ( t 1/2 = 7 – 8 min;
Fig. 1, A and B ), whereas Sec61p C121G or C150V had no infl u-
ence on the degradation rate ( t 1/2 = 13 min; Fig. 1, A and B ). As a
control, we also monitored the rate of substrate degradation in a
yeast strain harboring the retrotranslocation (and translocation)-
defective sec61-2 allele ( Plemper et al., 1997 ). In this background
the rate of substrate degradation was not altered, which is consis-
tent with previous reports on the degradation of a misfolded mem-
brane substrate containing a misfolded cytosolic domain (Ste6*;
Huyer et al., 2004 ; see also Discussion). In wild-type and mutant
strains, except C373I, we observed two electrophoretically dis-
tinct species of substrate. The more rapidly migrating species pre-
dominated in C373I ( Fig. 1 A ).
We evaluated the degradation of another unstable ERAD
substrate, CPY * , in the wild-type and Sec61p cysteine mutant
strains. In wild-type cells, CPY* was degraded with a t 1/2 of
? 25 min ( Fig. 1, C and D ). Mutations at position 121 or 150 of
Sec61p retained wild-type degradation kinetics, whereas muta-
Figure 1. Sec61p cysteine mutants are profi cient for ER protein export.
(A) Wild-type and Sec61p mutant cells expressing the transmembrane
ERAD substrate Deg1:Sec62 ProtA were pulse labeled for 5 min and chased
for the indicated times. Deg1:Sec62 ProtA was immunoprecipitated, resolved
by SDS-PAGE, and visualized by audioradiography. (B) Deg1:Sec62 ProtA
decay was quantifi ed by phosphorimager analysis and plotted as the
sum of decay for the two species over time. The data in the plots refl ect
the mean of three independent experiments. Standard deviations for
each time point were typically in the range of 4 – 8% but are not shown
in the plots for ease of viewing. (C) Wild-type and Sec61p mutant cells
expressing CPY* were pulse labeled for 15 min and chased for the
indicated times. CPY* was immunoprecipitated from detergent-solubilized
lysates, resolved by SDS-PAGE, and visualized by audio radiography.
(D) CPY* decay was quantifi ed and plotted as described for Deg1:
Sec62 ProtA in B.
1105 ERAD OF TRANSMEMBRANE PROTEINS BY SEC61P • Scott and Schekman
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