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Fig. 1. CONT represents the single Drosophila homolog of the vertebrate contactin family. CONT is encoded by the transcriptional unit CG1084 (FlyBase), which was predicted from the Drosophila genome sequence. (A) The translation of cont cDNA reveals an ORF of 1390 amino acids. The signal peptide and the GPI anchor signal are indicated in bold. The C-lectin domain is underlined. The predicted N-glycosylation sites are marked in bold and are underlined. These sequence data are available from GenBank under Accession Number AY229991. (B) The modular protein domain structure for the CONT protein is shown in comparison with vertebrate contactin. Except for the N-terminal C-lectin domain in CONT, both proteins display a similar domain organization. (C) The phylogenetic tree obtained after aligning the protein sequences encoding rat contactin, TAG-1, NB-2, NB-3, BIG-1 and BIG-2 with the Drosophila CONT sequence. For this phylogenetic tree analysis, the CONT C-lectin domain was not considered. (D) Immunoblots of lipid rafts and microsomes that were prepared from 18-hour-old embryos. (Left panel) A single protein band with an apparent molecular weight of ~180 kDa can be detected in both fractions when probed with the guinea-pig anti-CONT antiserum, which is directed against the N-terminal C-lectin domain. As a control, Fas1 is highly concentrated in the lipid raft fraction. (Middle panel) The microsomes were incubated with PI-PLC in the presence or absence of ZnCl2 followed by high-speed centrifugation. Solubilized pellet (5 µl) (P) and 25 µl of supernatant (S) were analyzed. As shown in the middle panel, a significant proportion of CONT was released in the supernatant after PI-PLC treatment. This release of CONT from the microsomal pellet was completely inhibited by the presence of ZnCl2. The asterisk shows a degradation band that resulted from incubation at 37°C. As a control, the major fraction of Fas1 was cleaved by PI-PLC and recovered in the supernatant, this release was also inhibited by the presence of ZnCl2. (Right panel) N-glycosidase-F treatment of the microsomal NP-40 extracts reduces the size of CONT to ~155 kDa, close to the size predicted from the cDNA sequence (158 kDa).





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