Identification of the carboxyl-terminal membrane-anchoring region of HPC-1/syntaxin 1A with the substituted-cysteine-accessibility method and monoclonal antibodies.
HPC-1/syntaxin 1A is a member of the syntaxin family, and functions at the plasma membrane during membrane fusion as the target-soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (t-SNARE). We identified the membrane-anchoring region of HPC-1/syntaxin 1A, and examined its role in anchoring of a protein to the plasma membrane. A series of mutants was created from a cysteine-less mutant of HPC-1/syntaxin 1A by substitution of each residue at the C-terminus with cysteine. The accessibility of the thiol-groups in each mutant was analyzed in vivo. The cysteine (C145) within the N-terminal cytosolic segment was labeled, but not that at C271 or C272, or any of those introduced at the C-terminus. The addition of additional residues to the C-terminal tail of HPC-1/syntaxin 1A allowed labeling by thiol-specific reagents. A monoclonal antibody directed against the C-terminal tail peptide did not react with the protein located at the plasma membrane. In addition, subcellular fractionation and immunocytochemical analyses with various transmembrane mutants showed that the C-terminal tail comprising eight amino acids is essential for anchoring of HPC-1/syntaxin 1A to the plasma membrane. These results indicate that the C-terminal membrane-anchoring region, which comprises 23 amino acids, does not traverse the lipid-bilayer and that the C-terminal tail is essential for anchoring of HPC-1/syntaxin 1A to the plasma membrane.
Suga
Kei
K
Department of Physiology, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan. ksuga@kyorin-u.ac.jp
Yamamori
Tetsuo
T
Akagawa
Kimio
K
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism*; D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism*; D019556:COS Cells; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D050827:Syntaxin 1
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D009419:Nerve Tissue Proteins; C494212:STX1A protein, human; C494214:Stx1a protein, mouse; C494213:Stx1a protein, rat; D050827:Syntaxin 1; D003545:Cysteine