Differential requirements for the EF-hands of human centrin2 in primary ciliogenesis and nucleotide excision repair

Abstract

Centrin2 is a small, conserved calcium-binding protein that localizes to the centriolar distal lumen in human cells. It is required for efficient primary ciliogenesis and nucleotide excision repair (NER). Centrin2 forms part of the xeroderma pigmentosum group C protein complex. To explore how centrin2 contributes to these distinct processes, we mutated the 4 calcium-binding EF-hand domains of human centrin2. Centrin2 in which all EF-hands had been mutated to ablate calcium binding (a ‘4DA’ mutant) was capable of supporting in vitro NER and was as effective as wild-type centrin2 in rescuing the UV sensitivity of centrin2 null cells. However, we found that mutation of any of the EF-hands impaired primary ciliogenesis in hTERT-RPE1 cells to the same extent as deletion of centrin2. Phenotypic analysis of the 4DA mutant revealed defects in centrosome localisation, centriole satellite assembly, ciliary assembly and function and in interactions with POC5 and SFI1. These observations indicate that centrin2 requires calcium-binding capacity for its primary ciliogenesis functions, but not NER, and suggest these functions require that centrin2 be capable of forming complexes with partner proteins.