Over-Expression Analysis of All Eight Subunits of the Molecular Chaperone CCT in Mammalian Cells Reveals a Novel Function for CCTdelta. Issue 17 (28th August 2015)

• Record Type: Journal Article
• Authors: Spiess, Matthias; Echbarthi, Meriem; Svanström, Andreas; Karlsson, Roger; Grantham, Julie
• Is Part Of: Journal of molecular biology. Volume 427:Issue 17(2015:Sep. 01)
• Page Start: 2757
• Page End: 2764
• Publication Date: 2015-08-28
• Subjects: CCT Chaperonin containing tailless complex polypeptide 1 -- SIM structured illumination microscopy -- wt wild type; chaperonin -- structured illumination microscopy -- spatial organization -- plasma membrane -- ATPase; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2015.06.007
• Languages: English
• ISSNs: 0022-2836

Abstract

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 ( g reen f luorescent p rotein) 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. Graphical abstract: Highlights: CCT is an essential chaperonin formed from eight protein subunits. The monomeric CCTdelta subunit induces cell surface protrusions in mammalian cells. This requires an interaction surface in the apical domain and ATPase activity. Cct4 monomer activity affects the growth of yeast. CCT function extends beyond oligomeric chaperonin-mediated protein folding.