Abstract
BackgroundThe eukaryotic cytosolic chaperonin CCT is a hetero-oligomeric complex formed by two rings connected back-to-back, each composed of eight distinct subunits (CCTα to CCTζ). CCT complex mediates the folding, of a wide range of newly synthesised proteins including tubulin (α, β and γ) and actin, as quantitatively major substrates.Methodology/Principal FindingsWe disrupted the genes encoding CCTα and CCTδ subunits in the ciliate Tetrahymena. Cells lacking the zygotic expression of either CCTα or CCTδ showed a loss of cell body microtubules, failed to assemble new cilia and died within 2 cell cycles. We also show that loss of CCT subunit activity leads to axoneme shortening and splaying of tips of axonemal microtubules. An epitope-tagged CCTα rescued the gene knockout phenotype and localized primarily to the tips of cilia. A mutation in CCTα, G346E, at a residue also present in the related protein implicated in the Bardet Biedel Syndrome, BBS6, also caused defects in cilia and impaired CCTα localization in cilia.Conclusions/SignificanceOur results demonstrate that the CCT subunits are essential and required for ciliary assembly and maintenance of axoneme structure, especially at the tips of cilia.
Highlights
Cilia are conserved organelles with important sensory and motile functions
The assembly and maintenance of cilia is dependent on bidirectional trafficking of protein complexes between the cell basal body and the cilia tip, the activity known as intraflagellar transport (IFT) [1]
We have investigated the function of CCTa and CCTd subunits of the eukaryotic cytosolic chaperonin in T. thermophila
Summary
Cilia are conserved organelles with important sensory and motile functions. Defects in cilia have been associated with a large number of human diseases, collectively known as ciliopathies. Cilia have a microtubule-based axoneme that is anchored to the basal body. The axoneme is typically composed of 9 doublet-microtubules arranged as a peripheral ring. Motile cilia usually have a pair of singlet microtubules at the center of the axoneme. The assembly and maintenance of cilia is dependent on bidirectional trafficking of protein complexes between the cell basal body and the cilia tip, the activity known as intraflagellar transport (IFT) [1]. Kinesin-2 motors move IFT cargo from the cell body to the tip of cilia, while recycled components are returned to the basal body by cytoplasmic dynein 1b motors [2,3,4,5]. The eukaryotic cytosolic chaperonin CCT is a hetero-oligomeric complex formed by two rings connected backto-back, each composed of eight distinct subunits (CCTa to CCTf). CCT complex mediates the folding, of a wide range of newly synthesised proteins including tubulin (a, b and c) and actin, as quantitatively major substrates
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