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© The Rockefeller University Press, 0021-9525/2000//591 $5.00
The Journal of Cell Biology, Volume 149, Number 3, , 2000 591-602

The Interaction of the Chaperonin Tailless Complex Polypeptide 1 (Tcp1) Ring Complex (Tric) with Ribosome-Bound Nascent Chains Examined Using Photo-Cross-Linking

^a Department of Medical Biochemistry and Genetics, Texas A&M University, College Station, Texas 77843-1114
^b Department of Chemistry, Texas A&M University, College Station, Texas 77843-1114
^c Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-1114
^d Department of Biological Sciences, Stanford University, Stanford, California 94305-5020
Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020.(650) 723-8475(650) 725-7833

jfrydman{at}leland.stanford.edu

The eukaryotic chaperonin tailless complex polypeptide 1 (TCP1) ring complex (TRiC) (also called chaperonin containing TCP1 [CCT]) is a hetero-oligomeric complex that facilitates the proper folding of many cellular proteins. To better understand the manner in which TRiC interacts with newly translated polypeptides, we examined its association with nascent chains using a photo-cross-linking approach. To this end, a series of ribosome-bound nascent chains of defined lengths was prepared using truncated mRNAs. Photoactivatable probes were incorporated into these ³⁵S- labeled nascent chains during translation. Upon photolysis, TRiC was cross-linked to ribosome-bound polypeptides exposing at least 50–90 amino acids outside the ribosomal exit channel, indicating that the chaperonin associates with much shorter nascent chains than indicated by previous studies. Cross-links were observed for nascent chains of the cytosolic proteins actin, luciferase, and enolase, but not to ribosome-bound preprolactin. The pattern of cross-links became more complex as the nascent chain increased in length. These results suggest a chain length–dependent increase in the number of TRiC subunits involved in the interaction that is consistent with the idea that the substrate participates in subunit-specific contacts with the chaperonin. Both ribosome isolation by centrifugation through sucrose cushions and immunoprecipitation with anti-puromycin antibodies demonstrated that the photoadducts form on ribosome-bound polypeptides. Our results indicate that TRiC/CCT associates with the translating polypeptide shortly after it emerges from the ribosome and suggest a close association between the chaperonin and the translational apparatus.

Key Words: protein folding • actin • luciferase • translation • chaperonin

© 2000 The Rockefeller University Press

Dr. Do's present address is Targeted Therapy, Inc., Oklahoma City, OK 73104.

Abbreviations used in this paper: ANB, N-(5-azido-2-nitrobenzoyl); CCT, chaperonin containing TCP1; GIMc, genes involved in microtubule biogenesis complex; Hsc70, 70-kD heat shock protein cognate; pPL, preprolactin; SRP, signal recognition particle; TCP1, tailless complex polypeptide 1; TRiC, TCP1-ring complex.

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