The Making of Synaptic Ribbons: Carboxy-Terminal Tagging of RIBEYE B-Domain Reduces Steady-State Levels of RIBEYE and Its Efficacy to Build Synaptic Ribbons.

RIBEYE is the central building block of synaptic ribbons, the eponymous presynaptic specializations of ribbon synapses. RIBEYE consists of two major protein regions: a unique amino-terminal, proline-rich A-domain and a carboxy-terminal, NADH-binding B-domain, which is largely identical to tetramer-forming nuclear protein CtBP2. In RIBEYE knockout mice, synaptic ribbons are completely absent, emphasizing the central role of RIBEYE in making synaptic ribbons. In the present study, we used a previously generated transgenic RIBEYE mouse line, in which a large protein tag was fused to the carboxy-terminus of RIBEYE-B-domain (RIBEYE-C-TG). We generated RIBEYE-C-TG-expressing RIBEYE knockout mice to study the impact of carboxy-terminal tagging of RIBEYE B-domain on synaptic ribbon formation. We applied high-resolution confocal immunofluorescence microscopy, conventional and E-PTA transmission electron microscopy to analyze whether carboxy-terminally tagged RIBEYE-C-TG rescues synaptic ribbon deficiency in RIBEYE knockout mice and restores synaptic ribbon in situ. High-resolution immunofluorescence microscopy revealed that transgenic RIBEYE-C-TG expression rescued synaptic ribbon formation in RIBEYE knockout mice. Electron microscopy confirmed that transgenic ribbons consisting of transgenic RIBEYE-C-TG were made, anchored to the active zone, and associated with synaptic vesicles. But transgenic synaptic ribbons formed by RIBEYE-C-TG were considerably smaller in size than synaptic ribbons in control mice. The expression level of RIBEYE-C-TG protein was much lower than untagged RIBEYE protein as demonstrated by qualitative and quantitative Western blot analyses. The decreased expression level of RIBEYE-C-TG protein is not due to decreased activity of the transgenic RIBEYE promoter or alterations of the 5'-UTR as shown by RT-qPCR analyses of RIBEYE transcripts and reporter assays. Our findings suggest that carboxy-terminally tagged RIBEYE-C-TG protein is less stable and less efficient in making synaptic ribbons than the untagged RIBEYE protein. Based on the findings in this study and on recently published structural data from other groups on RIBEYE and RIBEYE B-domain/CtBP2, we predict that carboxy-terminal tagging of RIBEYE B-domain, placed at the interaction interface of RIBEYE B-domain tetramers/RIBEYE B-domain filaments, could interfere with the formation and/or stability of RIBEYE B-domain-dependent oligomeric complexes which are likely essential to build the synaptic ribbon.