Abstract
Purpose :
The accumulation of non-resident proteins in the outer segment (OS) of photoreceptors is a hallmark of the genetic disorder Bardet-Biedl Syndrome (BBS) and may underlie retinal degeneration in BBS. The BBSome is a complex of eight BBS proteins that ferries membrane proteins out of cilia, the equivalent of the OS in photoreceptors. Prior work in cultured cells demonstrated that lysine 63-linked ubiquitin (Ub) chains mark dynamic BBSome cargoes for their removal from primary cilia. In this study, we sought to test whether UbK63 chains mark mistargeted proteins for removal from the OS by the BBSome.
Methods :
To determine whether ubiquitin and specific Ub linkages accumulate in OS when BBSome function is compromised, we imaged Ub and Ub linkages in retinal sections of Bbs4-/- mice at developmental stages preceding retinal degeneration. We next isolated UbK63-associated proteins from Bbs4-/- OS using engineered affinity reagents and inventoried the corresponding proteome by quantitative mass spectrometry. To validate the ubiquitination profile of the proteins accumulating in Bbs OS, we biochemically isolated the covalently ubiquitinated proteins from Bbs4-/- OS under denaturating conditions.
Results :
We found that the levels of Ub and UbK63 linkage increase 3- and 2-fold in Bbs4-/- OS respectively compared to controls. The UbK63-associated proteome of Bbs4-/- OS revealed 49 proteins enriched (p < 0.05) by >1.5-fold compared to wildtype. The vast majority of these proteins are membrane proteins that are known to localize to the inner segment, to synaptic vesicles, or to the synaptic terminal. By biochemically isolating ubiquitinated proteins under denaturing conditions, we validated 2 proteins that are differentially ubiquitinated in Bbs4-/- OS compared to wildtype OS. Immunofluorescence imaging further revealed that these validated ubiquitinated non-OS proteins accumulate in Bbs4-/- OS starting at the earliest stages of outer segment morphogenesis.
Conclusions :
We identify several novel cargoes of the BBSome in photoreceptors and demonstrate that UbK63 chains mark these mistargeted cargoes for removal from the OS as they do for removal from cilia for dynamic cargoes. We propose that non-resident proteins are recognized as foreign material by the ubiquitination machinery in the OS before clearance by the BBSome.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.