The N-terminal cysteine cluster is essential for membrane targeting of B/K protein.
B/K protein belongs to a family of C-terminal-type (C-type) tandem C2 proteins that contain two C2 Ca(2+)-binding motifs at the C-terminus. Although other C-type tandem C2 proteins have been found to have a unique N-terminal domain that is involved in membrane anchoring (e.g. synaptotagmin) or specific ligand binding (e.g. rabphilin-3A and Doc2), no research has been conducted on the function of the N-terminal domain of B/K protein. In this study we showed that despite lacking a transmembrane domain, both native and recombinant B/K proteins are tightly bound to the membrane fraction, which was completely resistant to 0.1 M Na(2)CO(3), pH 11, or 1 M NaCl treatment. Deletion and mutation analyses indicated that the cysteine cluster at the N-terminal domain (consisting of seven cysteine residues, Cys-19, Cys-23, Cys-26, Cys-27, Cys-30, Cys-35 and Cys-36) is essential for the membrane localization of B/K protein. When wild-type B/K was expressed in PC12 cells, B/K proteins were localized mainly in the perinuclear region (trans-Golgi network), whereas mutant B/K proteins carrying Cys-to-Ala substitutions were present in the cytosol. Based on our findings, we propose that the N-terminal domain of B/K protein contains a novel cysteine-based protein motif that may allow B/K protein to localize in the trans-Golgi network.
Fukuda
M
M
Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. mnfukuda@brain.riken.go.jp
Mikoshiba
K
K
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D019556:COS Cells; D002135:Calcium-Binding Proteins; D002451:Cell Compartmentation / Q000235:genetics; D003545:Cysteine / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D007425:Intracellular Membranes / Q000378:metabolism* / Q000502:physiology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D016716:PC12 Cells; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D017386:Sequence Homology, Amino Acid; D050857:Synaptotagmins; D020080:Tandem Repeat Sequences / Q000235:genetics
D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; C498723:Syt17 protein, mouse; C409229:Syt17 protein, rat; D050857:Synaptotagmins; D003545:Cysteine