The Cotranslational Contacts between Ribosome-bound Nascent Polypeptides and the Subunits of the Hetero-oligomeric Chaperonin TRiC Probed by Photocross-linking

Yuanlong Shao,Anne S. Meyer,William R. Skach,Stephanie A. Etchells,Anne Roobol,Arthur E. Johnson,Martin J. Carden,Judith Frydman,Alice Y. Yam,Yiwei Miao

doi:10.1074/jbc.m504110200

Abstract

The hetero-oligomeric eukaryotic chaperonin TRiC (TCP-1-ring complex, also called CCT) interacts cotranslationally with a diverse subset of newly synthesized proteins, including actin, tubulin, and luciferase, and facilitates their correct folding. A photocross-linking approach has been used to map the contacts between individual chaperonin subunits and ribosome-bound nascent chains of increasing length. Whereas a cryo-EM study suggests that chemically denatured actin interacts with only two TRiC subunits (delta and either beta or epsilon), actin and luciferase chains photocross-link to at least six TRiC subunits (alpha, beta, delta, epsilon, xi, and theta) at different stages of translation. Furthermore, the photocross-linking of actin, but not luciferase, nascent chains to TRiC subunits zeta and theta was length-dependent. In addition, a single photoreactive probe incorporated at a unique site in actin nascent chains of different lengths reacted covalently with multiple TRiC subunits, thereby indicating that the nascent chain samples the polypeptide binding sites of different subunits. We conclude that elongating actin and luciferase nascent chains contact multiple TRiC subunits upon emerging from the ribosome, and that the TRiC subunits contacted by nascent actin change as it elongates and starts to fold.

Highlights

The hetero-oligomeric eukaryotic chaperonin TRiC (TCP-1-ring complex, called CCT) interacts cotranslationally with a diverse subset of newly synthesized proteins, including actin, tubulin, and luciferase, and facilitates their correct folding
The nascent chain remains bound to the ribosome as a peptidyl-tRNA in a ribosome-nascent chain complex (RNC)
Because the length of the nascent polypeptide is determined by the length of the truncated mRNA, one can examine nascent chain length dependence by preparing intermediates using truncated mRNAs of different lengths

Summary

EXPERIMENTAL PROCEDURES

MRNA, and tRNA—Amber codons were introduced into plasmids coding for mouse ␤-actin and luciferase [14] using the QuikChange protocol (Stratagene), and the primary sequence of each construct was confirmed by DNA sequencing. Fractions enriched in TRiC were isolated by gel filtration on Superose 6 and immunoprecipitated with various subunit-specific antibodies as described below. Supernatants (110 ␮l) were added to 650 ␮l of buffer A (20 mM HEPES (pH 7.5), 140 mM KCl, 0.1% (w/v) SDS, 1% (v/v) Nonidet P-40, 1% (w/v) sodium deoxycholate) to dissociate the TRiC complex [24]. After sedimentation to remove aggregates, each supernatant (750 ␮l) received 20 ␮l of protein A-Sepharose beads (Sigma) in buffer A containing 10% (w/v) delipidated bovine serum albumin (Sigma) before being rocked for at least 2 h at 4 °C. Beads were pelleted, washed 3 times with 750 ␮l of buffer A, and resuspended in 40 ␮l of sample buffer (65 °C, 30 min). After SDS-PAGE, radioactive species were detected and quantified using a Bio-Rad FX phosphorimager

RESULTS

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DISCUSSION