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J Mol Biol. 2015 Aug 28;427(17):2757-64. doi: 10.1016/j.jmb.2015.06.007. Epub 2015 Jun 21.

Over-Expression Analysis of All Eight Subunits of the Molecular Chaperone CCT in Mammalian Cells Reveals a Novel Function for CCTdelta.

Author information

1
Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, S-106 91 Stockholm, Sweden.
2
Department of Chemistry and Molecular Biology, University of Gothenburg, S-405 30 Gothenburg, Sweden.
3
Department of Chemistry and Molecular Biology, University of Gothenburg, S-405 30 Gothenburg, Sweden. Electronic address: julie.grantham@cmb.gu.se.

Abstract

Chaperonin containing tailless complex polypeptide 1 (CCT) forms a classical chaperonin barrel structure where two rings of subunits surround a central cavity. Each ring consists of eight distinct subunits, creating a complex binding interface that makes CCT unique among the chaperonins. In addition to acting as a multimeric chaperonin, there is increasing evidence indicating that the CCT subunits, when monomeric, possess additional functions. Here we assess the role of the CCT subunits individually, using a GFP (green fluorescent protein) tagging approach to express each of the subunits in their monomeric form in cultured mammalian cells. Over-expression of CCTdelta, but not the other seven CCT subunits, results in the appearance of numerous protrusions at the cell surface. Two point mutations, one in the apical domain and one in the ATP binding pocket of CCTdelta, that abolish protrusion formation have been identified, consistent with the apical domain containing a novel interaction site that is influenced by the ATPase activity in the equatorial domain. Structured illumination microscopy, together with sub-cellular fractionation, reveals that only the wild-type CCTdelta is associated with the plasma membrane, thus connecting spatial organization with surface protrusion formation. Expression of the equivalent subunit in yeast, GFP-Cct4, rescues growth of the temperature-sensitive strain cct4-1 at the non-permissive temperature, indicative of conserved subunit-specific activities for CCTdelta.

KEYWORDS:

ATPase; chaperonin; plasma membrane; spatial organization; structured illumination microscopy

PMID:
26101841
DOI:
10.1016/j.jmb.2015.06.007
[Indexed for MEDLINE]
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