Interactions of subunit CCT3 in the yeast chaperonin CCT/TRiC with Q/N-rich proteins revealed by high-throughput microscopy analysis

Abstract

The eukaryotic chaperonin containing t-complex polypeptide 1 (CCT/TRiC) is an ATP-fueled machine that assists protein folding. It consists of two back-to-back stacked rings formed by eight different subunits that are arranged in a fixed permutation. The different subunits of CCT are believed to possess unique substrate binding specificities that are still mostly unknown. Here, we used highthroughput microscopy analysis of yeast cells to determine changes in protein levels and localization as a result of a Glu to Asp mutation in the ATP binding site of subunits 3 (CCT3) or 6 (CCT6). The mutation in subunit CCT3 was found to induce cytoplasmic foci termed P-bodies where mRNAs, which are not translated, accumulate and can be degraded. Analysis of the changes in protein levels and structural modeling indicate that P-body formation in cells with the mutation in CCT3 is linked to the specific interaction of this subunit with Gln/Asn-rich segments that are enriched in many P-body proteins. An in vitro gel-shift analysis was used to show that the mutation in subunit CCT3 interferes with the ability of CCT to bind a Gln/Asn-rich protein aggregate. More generally, the strategy used in this work can be used to unravel the substrate specificities of other chaperone systems. molecular chaperones | polyQ proteins | protein mis-folding | protein aggregation | high-content analysis C haperonins are ATP-dependent protein-folding machines that are present in all kingdoms of life. They consist of two back-toback stacked oligomeric rings with a cavity at each end, where protein substrate binding and folding take place (for reviews see refs. 1 and 2). The chaperonins can be divided into two groups: group I, found in the bacterial cytoplasm (e.g., GroEL in Escherichia coli), mitochondria, and chloroplasts; and group II found in archaea and the eukaryotic cytosol. Numerous studies have shown that the group I chaperonin GroEL can facilitate the folding of a large number of different proteins in vitro, although its role in the cell is much more limited (for review see ref.3). The group II eukaryotic chaperonin containing t-complex polypeptide 1 (CCT/ TRiC) also seems to have a specialized role in vivo in the folding of actin (4), tubulin (5), and other essential proteins, including regulators of cell division and cytoskeleton formation (6-8), despite seeming to possess broad binding specificity (6). The list of members in the CCT interactome is, however, not yet fully established, and the conditions for entry into this exclusive club remain poorly understood.An important distinction between group I and group II chaperonins is that the former consist of homo-oligomeric rings, whereas the latter usually consist of hetero-oligomeric rings that contain two or three different subunits in the case of many archaeal chaperonins and eight different subunits in the case of CCT. The eight subunits of CCT are arranged in a defined permutation (9), and the orientation of the two rings of CCT with respect to each other is also fixed (10). CCT's heter...

CCT complex restricts neuropathogenic protein aggregation via autophagy

“…CCT1 was previously shown to interact and sequester the N17-huntingtin exon 1 fragments101112 as well as polyQ stretches51. Here we confirmed that CCT2 interacted with huntingtin fragments, polyA stretches, mutant ATXN3 and p62.…”

“…Our data extend beyond the CCT–HTT interaction10111251, as we have seen similar interactions with other autophagy adaptors/cargos such as ATXN3, p62 or polyA tract that indeed accumulate in a similar autophagy-dependency pattern upon CCT depletion. The effects of the CCT depletion on the aggregation and oligomerization of mutant huntingtin fragment, polyalanine expanded proteins, mutant ATXN3 and p62 are primarily autophagy dependent, as opposed to the current assumption that these proteins are direct clients of physiological CCT activity and that this direct chaperone activity on mutant huntingtin is entirely responsible for its anti-aggregation properties (Figs 5 and 6).…”

Novel proteins interacting with peroxisomal protein receptor PEX7 inArabidopsis thaliana

“…Its mammalian and yeast counterparts were originally found to be involved in the correct folding of actin and tubulin, 9 while more recent studies have shown that it actually has a broader range of substrate binding and folding capacity. 10 The potential binding between T-complex and PEX7 is also supported by a previous report in a mammalian system showing that T-complex interacts with the WD40 domain, 11 which largely constitutes PEX7 within the peptides. 12 Because PEX5 also interacts with the WD40 domain of PEX7 for subsequent cargo import, 13 it is tempting to hypothesize that T-complex and PEX5 may competitively bind to PEX7 in the cytosol, in which case T-complex may function as a negative regulator for the import of PTS2-containing proteins.…”

TRiC/CCT chaperonins are essential for maintaining myofibril organization, cardiac physiological rhythm, and lifespan

“…Protein folding is mediated in the eukaryotic cytosol by the TCP-1 ring large multi-subunit complex (TRiC), also called CCT [1][2][3][4][5]. The TCP/ TRiC/CCT1 chaperonin is a highly conserved, macromolecular enzyme comprised of two hetero-oligomeric stacked rings with eight different subunits each [3][4][5][6][7][8]. Both genetic and biochemical evidence reveals that the TRiC complex is required for folding of many essential proteins, including the cytoskeletal proteins myosin, actin and tubulin [9][10][11][12][13][14][15].…”