Purchase this article with an account.
R. Bhowmick, J. C. Besharse; Identification of a Chaperone Mediated Cargo Complex Association With Intraflagellar Transport Proteins in Photoreceptor. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3799.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Intraflagellar transport (IFT) is a bidirectional motility of multi-subunit protein particles (IFT particles) along axonemal microtubules using kinesin and dynein motors. We have proposed that IFT plays a specific role in the transport of photoreceptor specific proteins into the outer segment, but the specific mechanism by which cargo associates with IFT particles is unknown. We have previously reported that the Dnaj chaperone, MRJ, binds both IFT88/polaris and photoreceptor RetGC1. Since MRJ functions as a co-chaperone with Hsp70, we tested the hypothesis that the MRJ/Hsp70 chaperone pair plays a role in binding of RetGC1 to the IFT complex.
We conducted immunoprecipitation (IP) assays using bovine retinal extracts and antibodies to MRJ, and four IFT proteins (IFT88, 57, 52, and 20) to evaluate IFT protein complexes containing RetGC1, rhodopsin, and the MRJ/HSP70 chaperone pair. Using anti-IFT88 antibodies we carried out IP assays to test the effect of MgATP on cargo and chaperone binding to IFT particles. We also evaluated the role of IFT88 binding to MRJ by measuring its effect on MRJ stimulated HSP70 ATPase activity using in vitro assays.
IP assays using anti-IFT antibodies brought down IFT proteins along with RetGC1, rhodopsin, and HSP70. IPs using anti-MRJ antibodies also brought down the same components, demonstrating the presence of IFT protein complexes containing both cargo proteins and chaperones. Interestingly, binding of cargo proteins and MRJ with IFT proteins was greatly enhanced by addition of MgATP and this effect was totally abolished under conditions preventing ATP hydrolysis. Moreover, in-vitro HSP70 ATPase assays using purified HSP70 along with recombinant MRJ and IFT88 demonstrates that IFT88 greatly stimulates the MRJ dependent ATPase activity of Hsp70.
Our data provide biochemical evidence for a chaperone mediated mediated IFT complex containing RetGC1 and rhodopsin, and suggest that this "IFT-cargo complex" plays a role in trafficking of these cargo proteins into the outer segment. Our data also suggest that the ATPase activity of the MRJ/HSP70 chaperone pair plays a critical role in cargo loading onto the IFT-cargo complex.
This PDF is available to Subscribers Only