Co-expression of CCT subunits hints at TRiC assembly
The eukaryotic cytosolic chaperonin, t-complex polypeptide 1 (TCP-1) ring complex or TRiC, is responsible for folding a tenth of the proteins in the cell. TRiC is a double-ringed barrel with each ring composed of eight different CCT (chaperonin containing TCP-1) subunits. In order for the subunits to assemble together into mature TRiC, which is believed to contain one and only one of each of these subunits per ring, they must be translated from different chromosomes, correctly folded and assembled. When expressed alone in Escherichia coli, the subunits CCT4 and CCT5, interestingly, form TRiC-like homo-oligomeric rings. To explore potential subunit-subunit interactions, we co-expressed these homo-oligomerizing CCT4 and CCT5 subunits or the archaeal chaperonin Mm-Cpn (Methanococcus maripaludis chaperonin) with CCT1-8, one at a time. We found that CCT5 shifted all of the CCT subunits, with the exception of CCT6, into double-barrel TRiC-like complexes, while CCT4 only interacted with CCT5 and CCT8 to form chaperonin rings. We hypothesize that these specific interactions may be due to the formation of hetero-oligomers in E. coli, although more work is needed for validation. We also observed the interaction of CCT5 and Mm-Cpn with smaller fragments of the CCT subunits, confirming their intrinsic chaperone activity. Based on this hetero-oligomer data, we propose that TRiC assembly relies on subunit exchange with some stable homo-oligomers, possibly CCT5, as base assembly units. Eventually, analysis of CCT arrangement in various tissues and at different developmental times is anticipated to provide additional insight on TRiC assembly and CCT subunit composition.
KeywordsChaperonin TRiC CCT Assembly Hetero-oligomer
We would like to thank the members of the Jonathan King lab for helpful discussions on this topic and the members of Gisou van der Goot lab for critical reading of this manuscript.
- Archibald JM, Logsdon JM, Doolittle WF (2000) Origin and evolution of eukaryotic chaperonins: phylogenetic evidence for ancient duplications in CCT genes. Mol Biol Evol 17:1456–1466. https://doi.org/10.1093/oxfordjournals.molbev.a026246 CrossRefGoogle Scholar
- Cong Y, Baker ML, Jakana J, Woolford D, Miller EJ, Reissmann S, Kumar RN, Redding-Johanson AM, Batth TS, Mukhopadhyay A, Ludtke SJ, Frydman J, Chiu W (2010) 4.0-A resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement. Proc Natl Acad Sci 107:4967–4972. https://doi.org/10.1073/pnas.0913774107 CrossRefGoogle Scholar
- Hein MY, Hubner NC, Poser I, Cox J, Nagaraj N, Toyoda Y, Gak IA, Weisswange I, Mansfeld J, Buchholz F, Hyman AA, Mann M (2015) A human interactome in three quantitative dimensions organized by stoichiometries and abundances. Cell 163:712–723. https://doi.org/10.1016/j.cell.2015.09.053 CrossRefGoogle Scholar
- Horwich AL, Fenton WA, Chapman E, Farr GW (2007) Two families of chaperonin: physiology and mechanism. Annu Rev Cell Dev Biol 23:115–145. https://doi.org/10.1146/annurev.cellbio.23.090506.123555 CrossRefGoogle Scholar
- Leitner A, Joachimiak LA, Bracher A, Mönkemeyer L, Walzthoeni T, Chen B, Pechmann S, Holmes S, Cong Y, Ma B, Ludtke S, Chiu W, Hartl FU, Aebersold R, Frydman J (2012) The molecular architecture of the eukaryotic chaperonin TRiC/CCT. Struct 20:814–825. https://doi.org/10.1016/j.str.2012.03.007 CrossRefGoogle Scholar
- Roobol A, Holmes FE, Hayes NVL et al (1995) Cytoplasmic chaperonin complexes enter neurites developing in vitro and differ in subunit composition within single cells. J Cell Sci 108:1477–1488Google Scholar
- Skouri-Panet F, Michiel M, Ferard C et al (2012) Structural and functional specificity of small heat shock protein HspB1 and HspB4, two cellular partners of HspB5: role of the in vitro hetero-complex formation in chaperone activity. Biochimie 94:975–984. https://doi.org/10.1016/j.biochi.2011.12.018 CrossRefGoogle Scholar
- Yokota S, Yanagi H, Yura T, Kubota H (2001b) Cytosolic chaperonin-containing t-complex polypeptide 1 changes the content of a particular subunit species concomitant with substrate binding and folding activities during the cell cycle. Eur J Biochem 268:4664–4673. https://doi.org/10.1046/j.1432-1327.2001.02393.x CrossRefGoogle Scholar