The secretory carrier membrane protein family: Structure and membrane topology

Secretory carrier membrane proteins (SCAMPs) are integral membrane proteins found in secretory and endocytic carriers implicated to function in membra ne trafficking. Using expressed sequence tag database and library screens a nd DNA sequencing, we have characterized several new SCAMPs spanning the pl ant and animal kingdoms and have defined a broadly conserved protein family . No obvious fungal homologue has been identified, however. We have found t hat SCAMPs share several structural motifs. These include NPF repeats, a le ucine heptad repeat enriched in charged residues, and a proline-rich SH3-li ke and/or WW domain-binding site in the N-terminal domain, which is followe d by a membrane core containing four putative transmembrane spans and three amphiphilic segments that are the most highly conserved structural element s. All SCAMPs are 32-38 kDa except mammalian SCAMP4, which is similar to 25 kDa and lacks most of the N-terminal hydrophilic domain of other SCAMPs. S CAMP4 is authentic as determined by Northern and Western blotting, suggesti ng that this portion of the larger SCAMPs encodes the functional domain. Fo cusing on SCAMP1, we have characterized its structure further by limited pr oteolysis and Western blotting with the use of isolated secretory granules as a uniformly oriented source of antigen and by topology mapping through e xpression of alkaline phosphatase gene fusions in Escherichia coli. Results show that SCAMP1 is degraded sequentially from the N terminus and then the C terminus, yielding an similar to 20-kDa membrane core that contains four transmembrane spans. Using synthetic peptides corresponding to the three c onserved amphiphilic segments of the membrane core, we have demonstrated th eir binding to phospholipid membranes and shown by circular dichroism spect roscopy that the central amphiphilic segment linking transmembrane spans 2 and 3 is alpha-helical. In the intact protein, these segments are likely to reside in the cytoplasm-facing membrane interface. The current model of SC AMP1 suggests that the N and C termini form the cytoplasmic surface of the protein overlying a membrane core, which contains a functional domain locat ed at the cytoplasmic interface with little exposure of the protein on the ectodomain.