The mammalian RIM1, RIM2 and Piccolo proteins appear to be important structural components of the presynaptic active zone; they have 1 or 2 zinc finger domains near the N-terminus, and a PDZ domain followed by 1 or 2 C2 domains at the C-terminus. RIMs are present at all synapses, whereas the much larger Piccolo protein is found in glutamatergic and GABAergic synapses in the brain, but not at cholinergic NMJ's. Although PDZ domains and C2 domains are each relatively common protein motifs, RIM, RIM2 and Piccolo are the only 3 genes/proteins in the mammalian data bases containing the combination of a PDZ domain followed by a C2 domain. The C. elegans genome contains only 2 genes encoding proteins with a PDZ - C2 combination. One of the 2 PDZ - C2 proteins is UNC-10, and Mike Nonet has shown this to be homologous to RIM and RIM2; he has also shown that the UNC-10 protein, like the mammalian RIM and RIM2 proteins, is localized to presynaptic regions. We have been studying the other C. elegans PDZ-C2 gene, which has many of the properties expected of a Piccolo homolog, and we have named it
rip-1 (for RIM-Piccolo related). The
rip-1 locus is extremely large, spanning more than 40 kb of genomic DNA; we have identified 2 alternative transcript start sites and 2 termination sites, and we have demonstrated the presence of all 4 of the possible transcript types. The longest transcript is 26.5 kb, and encodes a predicted 984 kDa protein with an extremely acidic isoelectric point of 4.45. The predicted molecular masses of the other RIP-1 proteins are 939 kDa, 139 kDa, and 94 kDa. With both promoters, we observed GFP expression in neurons; there are approximately 100-150 GFP-expressing neurons (i.e., 1/3 to 1/2 of the nervous system). However, most of the known classes of cholinergic motor neurons do not show any
rip-1 transcription from either promoter. The GFP patterns associated with the two promoters differ from each other in the exact number of positive neurons and their relative intensities. RIP-1 immunostaining of cultured cells shows that RIP-1 protein is present in discrete puncta along neuronal processes, and it colocalizes with general synaptic markers such as UNC-41 and synaptotagmin. In whole worms, RIP-1 immunostaining is seen exclusively in synaptic regions (primarily the nerve ring), but there is little or no overlap with the cholinergic UNC-17 marker.