Abstract
Purpose:
Photoreceptors (PR) are highly polarized and compartmentalized cells with large amounts of proteins synthesized in the inner segment (IS) and delivered to the outer segment (OS) and synaptic terminal. The photoreceptor-specific protein, Tulp1, is localized to the IS and synapse and is hypothesized to be involved in protein movement. To better understand the molecular processes that regulate protein trafficking in photoreceptors, we aim to identify compartment-specific Tulp1 binding partners.
Methods:
Serial tangential sectioning of Long Evans rat retinas was utilized to isolate the IS and synaptic PR compartments. The ganglion cell layer was collected as a Tulp1 negative control tissue. Lysates isolated from these compartments were immunoprecipitated using a polyclonal Tulp1 antibody. Retinal lysates from the tulp1-/- mouse were also immunoprecipitated to determine non-specific binding. The resultant Tulp1-bound proteins were separated by SDS-PAGE, protein bands excised, digested with trypsin and identified by liquid chromatography tandem mass spectromety (LC-MS/MS) on an LTQ-Orbitrap Elite hybrid mass spectrometer. Relative quantitation was performed by comparing the normalized spectral counts for the identified proteins across these samples.
Results:
Potential Tulp1 binding partners were identified from the specific PR compartments. In the IS, the primary proteins included members of the phototransduction cascade and constituents of the cytoskeleton involved in membrane dynamics. In the synaptic region, the primary proteins included members of the ribbon active zone. A separate subset of proteins were identified in both the IS and synapse. These included members of the GTPase activating family of proteins. None of these proteins were identified in the negative control samples.
Conclusions:
Tulp1 has two distinct compartment-specific interactomes. Our results support the hypothesis that Tulp1 is involved in the trafficking of proteins from the IS to the OS and the continuous membrane remodeling and vesicle cycling at the synaptic terminal. These findings may offer insight into possible mechanisms underlying photoreceptor degeneration caused by mutations in TULP1.
Keywords: 648 photoreceptors •
695 retinal degenerations: cell biology •
663 proteomics