Identification of a novel protein complex containing annexin A4, rabphilin and synaptotagmin

Willshaw, Angela; Grant, Karen; Yan, Jun; Rockliffe, Nichola; Ambavarapu, Sailaja; Burdyga, Galina; Varro, Andrea; Fukuoka, Shin-Ichi; Gawler, Debra

doi:10.1016/S0014-5793(03)01513-8

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Volume 559, Issue 1 , Pages 13-21, 13 February 2004

Identification of a novel protein complex containing annexin A4, rabphilin and synaptotagmin

Edited by Gianni Cesareni

Angela Willshaw
- The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, UK
- Present address: Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Karen Grant
- The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, UK
Jun Yan
- Amersham Biosciences, Little Chalfont HP7 9LL, UK
Nichola Rockliffe
- The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, UK
Sailaja Ambavarapu
- The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, UK
Galina Burdyga
- The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, UK
Andrea Varro
- The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, UK
Shin-Ichi Fukuoka
- Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan
Debra Gawler
- The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, UK
- Corresponding author. Fax: (44)-151-7945327

Received 16 September 2003; received in revised form 14 November 2003; accepted 24 November 2003.

Abstract

Rabphilin is a synaptic vesicle-associated protein proposed to play a role in regulating neurotransmitter release. Here we report the isolation and identification of a novel protein complex containing rabphilin, annexin A4 and synaptotagmin 1. We show that the rabphilin C2B domain interacts directly with the N-terminus of annexin A4 and mediates the co-complexing of these two proteins in PC12 cells. Analyzing the cellular localisation of these co-complexing proteins we find that annexin A4 is located on synaptic membranes and co-localises with rabphilin at the plasma membrane in PC12 cells. Given that rabphilin and synaptotagmin are synaptic vesicle proteins involved in neurotransmitter release, the identification of this complex suggests that annexin A4 may play a role in synaptic exocytosis.

Keywords: Annexin, Rabphilin, C2 domain, Ca²⁺-regulated secretion

Abbreviations: PBS, phosphate-buffered saline, GST, glutathione S-transferase, ECL, enhanced chemiluminescence, TFA, trifluoroacetic acid, GFAP, glial fibrillary acidic protein