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J. Biochem, 2003, Vol. 133, No. 3 325-334
© 2003 Japanese Biochemical Society

BIOCHEMISTRY

Identification of the Carboxyl-Terminal Membrane-Anchoring Region of HPC-1/Syntaxin 1A with the Substituted-Cysteine-Accessibility Method and Monoclonal Antibodies

Kei Suga+,1, Tetsuo Yamamori2 and Kimio Akagawa1

1 Department of Physiology, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611; and 2 Division of Speciation Mechanisms, National Institute for Basic Biology, Okazaki, Aichi 444-8585

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.

+ To whom correspondence should be addressed. Tel: +81-422-47-5511, Fax: +81-422-47-4801, E-mail: ksuga{at}kyorin-u.ac.jp


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