Polyubiquitin serves as a recognition signal, rather than a ratcheting molecule, during retrotranslocation of proteins across the endoplasmic reticulum membrane.
Polyubiquitination is required for retrotranslocation of proteins from the endoplasmic reticulum back into the cytosol, where they are degraded by the proteasome. We have tested whether the release of a polypeptide chain into the cytosol is caused by a ratcheting mechanism in which the attachment of polyubiquitin prevents the chain from moving back into the endoplasmic reticulum. Using a permeabilized cell system in which major histocompatibility complex class I heavy chains are retrotranslocated under the influence of the human cytomegalovirus protein US11, we demonstrate that polyubiquitination alone is insufficient to provide the driving force for retrotranslocation. Substrate release into the cytosol requires an additional ATP-dependent step. Release requires a lysine 48 linkage of ubiquitin chains. It does not occur when polyubiquitination of the substrate is carried out with glutathione S-transferase (GST)-ubiquitin, and this correlates with poly-GST-ubiquitin not being recognized by a ubiquitin-binding domain in the Ufd1-Npl4 cofactor of the ATPase p97. These data suggest that polyubiquitin does not serve as a ratcheting molecule. Rather, it may serve as a recognition signal for the p97-Ufd1-Npl4 complex, a component implicated in the movement of substrate into the cytosol.
Flierman
Dennis
D
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Ye
Yihong
Y
Dai
Min
M
Chau
Vincent
V
Rapoport
Tom A
TA
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate; D000818:Animals; D001254:Astrocytoma; D002417:Cattle; D002463:Cell Membrane Permeability; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D008099:Liver / Q000502:physiology; D009097:Multienzyme Complexes / Q000378:metabolism; D025821:Polyubiquitin / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D021381:Protein Transport; D014407:Tumor Cells, Cultured
D009097:Multienzyme Complexes; D025821:Polyubiquitin; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex