Abstract
Purpose :
Biological processes are imperfect and errors in trafficking to the outer segment (OS) of photoreceptors can result in the entry of inner segment proteins into the OS. The correction of such errors has been illuminated by studies of Bardet-Biedl Syndrome (BBS), a mendelian disorder whose cardinal features include retinal degeneration. The BBSome, a complex of BBS proteins, ferries membrane proteins out of cilia and retinal degeneration in BBS is likely caused by defects in the removal of mistargeted proteins from the OS, which is a specialized cilium. To better understand the pathological imbalances that leads to photoreceptor death in BBS, we sought to define the characteristics of proteins that are removed from the OS by the BBSome.
Methods :
Past work in cultured cells has shown that ubiquitin (Ub) chains linked at lysine 63 (K63) mark cargoes for BBSome-mediated exit. We stained retinal sections of Bbs4 knockout mice at P15 with pan-Ub and linkage-specific probes. We next biochemically isolated the ubiquitinated proteins that accumulate in Bbs4-/- OSs using Tandem Ubiquitin Binding Entities (TUBEs). Experimental replicates and quantitative mass spectrometry enabled a statistically robust identification of BBSome cargoes in photoreceptors.
Results :
Ubiquitin levels in the OS of Bbs4-/- mice are considerably elevated compared to control mice. Specifically, K63Ub chains accumulate in Bbs4-/- OS. Mass spectrometry analyses of K63Ub-associated proteins found 49 proteins statistically enriched (p < 0.05) by 1.5-fold in Bbs4-/- OS compared to wildtype OS while only 10 proteins were depleted. The vast majority of proteins linked to K63Ub that accumulate in Bbs4-/- OS are membrane proteins that are known to localize to the inner segment, to synaptic vesicles, or to the synaptic terminal. The identification of Syntaxin 3, a synaptic protein known to mislocalize to the OS in Bbs knockout mice, in this dataset directly validates our approach. The preliminary characterization of hits from our data set suggests the existence of a novel class of BBSome cargoes that may be subject to quality control.
Conclusions :
The analysis of our mass spectrometry data set shows that K63Ub chains mark unwanted proteins for clearance from the OS by the BBSome. Our study generalizes the function of BBSome in the clearance of unwanted proteins from the OS and suggests the existence of quality control mechanisms that maintain the proteome of photoreceptor OS.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.