Native mass spectrometry analyses of chaperonin complex TRiC/CCT reveal subunit N-terminal processing and re-association patterns

Miranda P Collier,Rahul Samant,Daniel Itzhak,Alma Burlingame,Karen Betancourt Moreira,Alexander Leitner,Judith Frydman,Yu-Chan Chen,Kathy H Li

doi:10.1038/s41598-021-91086-6

Abstract

The eukaryotic chaperonin TRiC/CCT is a large ATP-dependent complex essential for cellular protein folding. Its subunit arrangement into two stacked eight-membered hetero-oligomeric rings is conserved from yeast to man. A recent breakthrough enables production of functional human TRiC (hTRiC) from insect cells. Here, we apply a suite of mass spectrometry techniques to characterize recombinant hTRiC. We find all subunits CCT1-8 are N-terminally processed by combinations of methionine excision and acetylation observed in native human TRiC. Dissociation by organic solvents yields primarily monomeric subunits with a small population of CCT dimers. Notably, some dimers feature non-canonical inter-subunit contacts absent in the initial hTRiC. This indicates individual CCT monomers can promiscuously re-assemble into dimers, and lack the information to assume the specific interface pairings in the holocomplex. CCT5 is consistently the most stable subunit and engages in the greatest number of non-canonical dimer pairings. These findings confirm physiologically relevant post-translational processing and function of recombinant hTRiC and offer quantitative insight into the relative stabilities of TRiC subunits and interfaces, a key step toward reconstructing its assembly mechanism. Our results also highlight the importance of assigning contacts identified by native mass spectrometry after solution dissociation as canonical or non-canonical when investigating multimeric assemblies.

Highlights

The eukaryotic chaperonin TRiC (TCP1-ring complex, or CCT, cytosolic chaperonin containing TCP1) is an essential protein complex that assists in folding and preventing aggregation of approximately 10% of the proteome, including obligate substrates actin and tubulin[1,2,3]
It is possible that small amounts of assembled T. ni TRiC/CCT co-purify through the steps in our protocol; alternatively, we cannot discount a small amount of co-assembly between insect CCTs and human TRiC, based on sequence conservation of TRiC between these species (Supplementary Fig. 4) and across eukaryotes generally[24]
Proteomic analysis indicated that human TRiC (hTRiC) can contain either α- or β-tubulin remaining bound at the end of a preparation

Summary

Introduction

The eukaryotic chaperonin TRiC (TCP1-ring complex, or CCT, cytosolic chaperonin containing TCP1) is an essential protein complex that assists in folding and preventing aggregation of approximately 10% of the proteome, including obligate substrates actin and tubulin[1,2,3]. The development of a protocol to express and purify correctly assembled and functional recombinant human TRiC ( hTRiC) from insect (Trichoplusia ni) cells has facilitated structural studies and the exploration of new functional a venues[10]. We show that following chemical disruption of the 16-mer, subunits can engage in re-formed canonical and non-canonical contacts; and we discuss potential implications for chaperonin assembly. These findings provide a resource and foundation for future exploration of TRiC assembly and function

Objectives

Methods

Results

Conclusion