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Novel SCAMPs lacking NPF repeats: ubiquitous and synaptic vesicle-specific forms implicate SCAMPs in multiple
R Fernández-Chacón1, T C Südhof
1Center for Basic Neuroscience, Department of Molecular Genetics, and Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, Dallas, Texas 75390-9111, USA.
Abstract:
In vertebrates, secretory carrier membrane proteins (SCAMPs) 1-3 constitute a family of putative membrane-trafficking proteins composed of cytoplasmic N-terminal sequences with NPF repeats, four central transmembrane regions (TMRs), and a cytoplasmic tail. SCAMPs probably function in endocytosis by recruiting EH-domain proteins to the N-terminal NPF repeats but may have additional functions mediated by their other sequences. We now demonstrate that SCAMPs form a much larger and more heterogeneous protein family than envisioned previously, with an evolutionary conservation extending to invertebrates and plants. Two novel vertebrate SCAMPs (SCAMPs 4 and 5), single SCAMP genes in Caenorhabditis elegans and Drosophila melanogaster, and multiple SCAMPs in Arabidopsis thaliana were identified. Interestingly, the novel SCAMPs 4 and 5 lack the N-terminal NPF repeats that are highly conserved in all other SCAMPs. RNA and Western blotting experiments showed that SCAMPs 1-4 are ubiquitously coexpressed, whereas SCAMP 5 is only detectable in brain where it is expressed late in development coincident with the elaboration of mature synapses. Immunocytochemistry revealed that SCAMP 5 exhibits a synaptic localization, and subcellular fractionations demonstrated that SCAMP 5 is highly enriched in synaptic vesicles. Our studies characterize SCAMPs as a heterogeneous family of putative trafficking proteins composed of three isoforms that are primarily synthesized outside of neurons (SCAMPs 2-4), one isoform that is ubiquitously expressed but highly concentrated on synaptic vesicles (SCAMP 1), and one brain-specific isoform primarily localized to synaptic vesicles (SCAMP 5). The conservation of the TMRs in all SCAMPs with the variable presence of N-terminal NPF repeats suggests that in addition to the role of some SCAMPs in endocytosis mediated by their NPF repeats, all SCAMPs perform a "core" function in membrane traffic mediated by their TMRs.