Abstract
Purpose: :
Photoreceptors are specialized neurons, consisting of several discrete compartments organized in a linear array. These compartments include the photosensitive outer segment, the biosynthetic inner segment, the nuclear region, and the synaptic terminal from which glutamate is tonically released in the dark. The signals and mechanisms that ensure the proper sorting, targeting and trafficking of proteins to these various compartments are poorly understood, particularly in the case of the synaptic terminal. Synaptophysin spans the membrane four times and is found in oligomers within the synaptic vesicle membrane, where it is believed to contribute to the regulation of various aspects of the synaptic vesicle cycle. The objective was to determine the minimal sequence within synaptophysin that signals the protein to be targeted to the synaptic terminal.
Methods: :
Our approach was to generate constructs consisting of a membrane-associated YFP as a reporter fused to various regions of synaptophysin, and express those constructs in the rod photoreceptors of transgenic Xenopus laevis. The subcellular localization of these proteins in tadpole photoreceptors was assessed by confocal microscopy.
Results: :
The YFP reporter was found throughout the cell but strongly accumulated in the outer segment, as we have previously described for membrane proteins lacking targeting information in this cell type. We found that the cytoplasmic C-terminus of synaptophysin, but not the N-terminus or the intracellular loop region, fused to the reporter was restricted to the inner segment plasma membrane and synaptic terminal. Several deletion constructs of the C-terminus revealed that the last 10 residues of synaptophysin’s C-terminus were sufficient to target the reporter to the synaptic terminal.
Conclusions: :
We conclude that the C-terminal 10 amino acids of synaptophysin contain a targeting signal that directs the subcellular localization of this protein in photoreceptors.
Keywords: photoreceptors • synapse • cell membrane/membrane specializations