The endoplasmic reticulum (ER) is
responsible for delivering properly folded proteins to their site of
action. This requires a very efficient quality control system to
recognise aberrant proteins and prevent their further transport.
Improperly folded proteins are subjected to rapid proteasomal
degradation after retrograde transport back to the cytosol. (T. Sommer &
D. H. Wolf. Endoplasmic reticulum degradation: reverse protein flow of
no return. FASEB J 11, 1227 (1997)). Here we report that the rapid
proteasomal degradation of a mutated form of the multimembrane-spanning
ABC-transporter Pdr5 (Pdr5*) retained in the ER is initialised at the
lumenal face of the ER-membrane. Using different antibodies directed
against the cytosolic tails or a lumenal loop of the transmembrane
protein, it could be demonstrated that the turnover of Pdr5* demands the
concerted action of both the Sec61 translocon and the ubiquitin-proteasome system. We observed a stabilisation of the entire molecule
within the ER-membrane in yeast mutants characterised by a reduced
translocation capacity or by functionally attenuated proteasomes.
Moreover, no degradation intermediates were detected in any of the
mutants that impede degradation of Pdr5*. Therefore, initial steps are
rate limiting for cleavage and mutations that impede downstream events
prevent initiation of the process. Our data suggest that ER-degradation
is a mechanistically highly integrated process, requiring the combined
operation of components of the degradation system acting at the lumenal
face of the ER-membrane, the Sec61 translocon and the ubiquitin-proteasome system.
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