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

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 (hTRi...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Miranda P. Collier, Karen Betancourt Moreira, Kathy H. Li, Yu-Chan Chen, Daniel Itzhak, Rahul Samant, Alexander Leitner, Alma Burlingame, Judith Frydman
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/17df211e25b3430283c572003e4f16b8
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:17df211e25b3430283c572003e4f16b8
record_format	dspace
spelling	oai:doaj.org-article:17df211e25b3430283c572003e4f16b82021-12-02T17:45:17ZNative mass spectrometry analyses of chaperonin complex TRiC/CCT reveal subunit N-terminal processing and re-association patterns10.1038/s41598-021-91086-62045-2322https://doaj.org/article/17df211e25b3430283c572003e4f16b82021-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-91086-6https://doaj.org/toc/2045-2322Abstract 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.Miranda P. CollierKaren Betancourt MoreiraKathy H. LiYu-Chan ChenDaniel ItzhakRahul SamantAlexander LeitnerAlma BurlingameJudith FrydmanNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-15 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Miranda P. Collier Karen Betancourt Moreira Kathy H. Li Yu-Chan Chen Daniel Itzhak Rahul Samant Alexander Leitner Alma Burlingame Judith Frydman Native mass spectrometry analyses of chaperonin complex TRiC/CCT reveal subunit N-terminal processing and re-association patterns
description	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.
format	article
author	Miranda P. Collier Karen Betancourt Moreira Kathy H. Li Yu-Chan Chen Daniel Itzhak Rahul Samant Alexander Leitner Alma Burlingame Judith Frydman
author_facet	Miranda P. Collier Karen Betancourt Moreira Kathy H. Li Yu-Chan Chen Daniel Itzhak Rahul Samant Alexander Leitner Alma Burlingame Judith Frydman
author_sort	Miranda P. Collier
title	Native mass spectrometry analyses of chaperonin complex TRiC/CCT reveal subunit N-terminal processing and re-association patterns
title_short	Native mass spectrometry analyses of chaperonin complex TRiC/CCT reveal subunit N-terminal processing and re-association patterns
title_full	Native mass spectrometry analyses of chaperonin complex TRiC/CCT reveal subunit N-terminal processing and re-association patterns
title_fullStr	Native mass spectrometry analyses of chaperonin complex TRiC/CCT reveal subunit N-terminal processing and re-association patterns
title_full_unstemmed	Native mass spectrometry analyses of chaperonin complex TRiC/CCT reveal subunit N-terminal processing and re-association patterns
title_sort	native mass spectrometry analyses of chaperonin complex tric/cct reveal subunit n-terminal processing and re-association patterns
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/17df211e25b3430283c572003e4f16b8
work_keys_str_mv	AT mirandapcollier nativemassspectrometryanalysesofchaperonincomplextriccctrevealsubunitnterminalprocessingandreassociationpatterns AT karenbetancourtmoreira nativemassspectrometryanalysesofchaperonincomplextriccctrevealsubunitnterminalprocessingandreassociationpatterns AT kathyhli nativemassspectrometryanalysesofchaperonincomplextriccctrevealsubunitnterminalprocessingandreassociationpatterns AT yuchanchen nativemassspectrometryanalysesofchaperonincomplextriccctrevealsubunitnterminalprocessingandreassociationpatterns AT danielitzhak nativemassspectrometryanalysesofchaperonincomplextriccctrevealsubunitnterminalprocessingandreassociationpatterns AT rahulsamant nativemassspectrometryanalysesofchaperonincomplextriccctrevealsubunitnterminalprocessingandreassociationpatterns AT alexanderleitner nativemassspectrometryanalysesofchaperonincomplextriccctrevealsubunitnterminalprocessingandreassociationpatterns AT almaburlingame nativemassspectrometryanalysesofchaperonincomplextriccctrevealsubunitnterminalprocessingandreassociationpatterns AT judithfrydman nativemassspectrometryanalysesofchaperonincomplextriccctrevealsubunitnterminalprocessingandreassociationpatterns
_version_	1718379572447150080

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

Ejemplares similares