Fig. 1. (A) CLUSTAL W sequence alignment of mouse SBP1 and Vta1p, a putative yeast homologue. (B) CLUSTAL W sequence alignment of mouse and yeast Vps2p. (C) Domain structures of SBP1, Vta1p, mVps2 and Vps2p. Predicted coiled-coil domains are marked with coils. The dotted line through the center of molecules divides the proteins into their positively charged N-terminal and negatively charged C-terminal domains. Molecular masses are given on the right, as calculated from their amino acid composition and determined by SDS-PAGE. Data of Vps2p was described previously (Babst et al., 2002a). N.D. is not determined. (D) Western blotting analysis of SBP1 in mouse tissues. Total extracts from various mouse tissues (50 µg of protein) were run on 10% SDS-PAGE and analyzed by western blotting with affinity purified anti-SBP1 polyclonal antibody. (E) SDS-PAGE analyses of bacterially expressed mVps2. GST-mVps2 was purified with glutathione-beads (lane 1) and subjected to thrombin cleavage on the beads (lane 2). Subsequently, the mVps2 portion was recovered by sedimentation of the beads with centrifugation (lane 3). Coomassie Blue staining revealed that both GST-mVps2 (60 kDa) and thrombin-cleaved mVps2 (35 kDa) migrated slower than expected from their molecular masses (51.1 and 25.1 kDa, respectively). (F) Northern blotting analysis of SBP1. Full-length SBP1 cDNA was labeled with ³²P by random priming and hybridized to multiple tissue northern blots (Clontech) under high stringency conditions. 2 µg of poly(A) + RNA from various mouse tissues was loaded in each lane. (G) Northern blotting analysis of mVps2. As for SBP1, mVps2 mRNA was detected on Multiple Tissue northern blots (Clontech) using full-length mVps2 cDNA as probe.