Cotranslational ubiquitination of cystic fibrosis transmembrane conductance regulator in vitro.
Ubiquitination is a covalent protein modification that can target proteins in eukaryotic cells for degradation by the 26 S proteasome. Substrates for this degradation pathway include abnormal proteins that arise from misfolding and/or mutation. How and when the ubiquitination machinery recognizes misfolded proteins and targets them for degradation remains largely unknown. We have previously shown that cystic fibrosis transmembrane conductance regulator (CFTR), is rapidly degraded in a ubiquitin-dependent fashion, without any detectable lag following its synthesis (Ward, C. L., and Kopito, R. R. (1994) J. Biol. Chem. 269, 25710-25718), suggesting that ubiquitination and protein synthesis may be temporally linked. In the present study, we have investigated the timing of CFTR ubiquitination relative to its translation in reticulocyte lysates containing 125I-ubiquitin. In synchronized, proteasome-inhibited lysates, translation of full-length CFTR chains was completed in approximately 30 min, whereas modification of CFTR with [125I]ubiquitin was evident by 20 min, indicating that ubiquitination precedes the completion of full-length polypeptide chains. Moreover, ubiquitin was also found to be transferred to nascent CFTR chains while attached to ribosomes. Together, these data establish that ubiquitination, which is widely assumed to be a post-translational event, can occur cotranslationally and suggest a role for ubiquitination early in protein biosynthesis.
Sato
S
S
Department of Biological Sciences, Stanford University, Stanford, California 94305-5020, USA.
Ward
C L
CL
Kopito
R R
RR
D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D010447:Peptide Hydrolases / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D014176:Protein Biosynthesis; D017510:Protein Folding; D012156:Reticulocytes / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D014452:Ubiquitins / Q000378:metabolism*
C505032:CFTR protein, human; D014452:Ubiquitins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease