Identification of TbRP2-interacting proteins using proximity-dependent biotinylation (BioID)

The protein RP2 is a tubulin cofactor C-domain containing protein with important roles in ciliogenesis. In humans, mutations in the RP2 gene are associated with 10-15% of cases of X-linked retinitis pigmentosa; a devastating disease characterised by progressive degeneration of retinal photoreceptors. Although XRP2 was initially proposed to function as a GTPase-activating protein (GAP) for tubulin, evidence now suggests that it acts as a GAP for Arl3 (a small GTPase) and together with Arl3 is involved in trafficking proteins to the cilium. I have been studying RP2 function in the flagellated protist Trypanosoma brucei, a tractable model to study eukaryotic flagellum assembly but also a parasite of medical and veterinary importance in subSaharan Africa. Thus, the study of RP2 in trypanosomes has relevance for parasitology, but also the human inherited disease retinitis pigmentosa. However, important differences exist between XRP2 and TbRP2, for instance TbRP2 lacks the consensus sequence specifying N terminal myristoylation (a modification that targets XRP2 to the basal body in mammalian cells) but rather utilises twinned TOF-LisH motifs at the Nterminus of the protein to direct basal body targeting. To further interrogate the targeting and function of TbRP2, I employed proximity-dependent biotin identification (BioID), in combination with quantitative proteomic (SILAC) techniques, to identify putative TbRP2-interacting proteins in vivo. A selected cohort of these proteins were subsequently interrogated by bioinformatics, localised within the cell using a PCR only (pPOT) YFP-tagging strategy and their potential roles in flagellum formation interrogated using inducible RNA interference (RNAi). My studies identified: (i) an Arl3-related protein as the likely molecular client of TbRP2 GAP activity; (ii) the trypanosome mature basal body as a hub for molecular chaperone activity associated with eukaryotic flagellum assembly; and (iii) insight into lineage-specific aspects of basal body biogenesis, as illustrated by the unusual spatial and temporal inheritance of large, trypanosomatid-specific protein of unknown function (TbBBP590).

Name dc:type.qualificationname

Ph.D.

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

Lancaster University

Year dc:date.issued

2017

Authors dc:creator

• Qi, Xin
• McKean, Paul
• Ginger, Michael