The hydrophobic cysteine-rich domain of SNAP25 couples with downstream residues to mediate membrane interactions and recognition by DHHC palmitoyl transferases

Jennifer Greaves, Gerald R Prescott, Yuko Fukata, Masaki Fukata, Christine Salaun, Luke H Chamberlain

Research output: Contribution to journalArticle

52 Citations (Scopus)

Abstract

SNAP25 is synthesized as a soluble protein but must associate with the plasma membrane to function in exocytosis; however, this membrane-targeting pathway is poorly defined. SNAP25 contains a palmitoylated cysteine-rich domain with four cysteines, and we show that coexpression of specific DHHC palmitoyl transferases is sufficient to promote SNAP25 membrane association in HEK293 cells. siRNA-mediated knockdown of its SNARE partner, syntaxin 1A, does not affect membrane interaction of SNAP25 in PC12 cells, whereas specific cysteine-to-alanine mutations perturb membrane binding, which is restored by leucine substitutions. These results suggest a role for cysteine hydrophobicity in initial membrane interactions of SNAP25, and indeed other hydrophobic residues in the cysteine-rich domain are also important for membrane binding. In addition to the cysteine-rich domain, proline-117 is also essential for SNAP25 membrane binding, and experiments in HEK293 cells revealed that mutation of this residue inhibits membrane binding induced by coexpression with DHHC17, but not DHHC3 or DHHC7. These results suggest a model whereby SNAP25 interacts autonomously with membranes via its hydrophobic cysteine-rich domain, requiring only sufficient expression of partner DHHC proteins for stable membrane binding. The role of proline-117 in SNAP25 palmitoylation is one of the first descriptions of elements within substrate proteins that modulate DHHC specificity.
LanguageEnglish
Pages1845-1854
Number of pages10
JournalMolecular Biology of the Cell
Volume20
Issue number6
DOIs
Publication statusPublished - 15 Mar 2009

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Transferases
Cysteine
Membranes
HEK293 Cells
Proline
Syntaxin 1
Lipoylation
SNARE Proteins
Mutation
PC12 Cells
Exocytosis
Hydrophobic and Hydrophilic Interactions
Leucine
Alanine
Small Interfering RNA
Membrane Proteins
Proteins
Cell Membrane

Keywords

  • acyltransferases
  • amino acid sequence
  • animals
  • cell line
  • cell membrane
  • cysteine
  • humans
  • hydrophobic and hydrophilic interactions
  • molecular sequence data
  • mutation
  • protein binding
  • rats
  • synaptosomal-associated protein 25

Cite this

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abstract = "SNAP25 is synthesized as a soluble protein but must associate with the plasma membrane to function in exocytosis; however, this membrane-targeting pathway is poorly defined. SNAP25 contains a palmitoylated cysteine-rich domain with four cysteines, and we show that coexpression of specific DHHC palmitoyl transferases is sufficient to promote SNAP25 membrane association in HEK293 cells. siRNA-mediated knockdown of its SNARE partner, syntaxin 1A, does not affect membrane interaction of SNAP25 in PC12 cells, whereas specific cysteine-to-alanine mutations perturb membrane binding, which is restored by leucine substitutions. These results suggest a role for cysteine hydrophobicity in initial membrane interactions of SNAP25, and indeed other hydrophobic residues in the cysteine-rich domain are also important for membrane binding. In addition to the cysteine-rich domain, proline-117 is also essential for SNAP25 membrane binding, and experiments in HEK293 cells revealed that mutation of this residue inhibits membrane binding induced by coexpression with DHHC17, but not DHHC3 or DHHC7. These results suggest a model whereby SNAP25 interacts autonomously with membranes via its hydrophobic cysteine-rich domain, requiring only sufficient expression of partner DHHC proteins for stable membrane binding. The role of proline-117 in SNAP25 palmitoylation is one of the first descriptions of elements within substrate proteins that modulate DHHC specificity.",
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The hydrophobic cysteine-rich domain of SNAP25 couples with downstream residues to mediate membrane interactions and recognition by DHHC palmitoyl transferases. / Greaves, Jennifer; Prescott, Gerald R; Fukata, Yuko; Fukata, Masaki; Salaun, Christine; Chamberlain, Luke H.

In: Molecular Biology of the Cell, Vol. 20, No. 6, 15.03.2009, p. 1845-1854.

Research output: Contribution to journalArticle

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T1 - The hydrophobic cysteine-rich domain of SNAP25 couples with downstream residues to mediate membrane interactions and recognition by DHHC palmitoyl transferases

AU - Greaves, Jennifer

AU - Prescott, Gerald R

AU - Fukata, Yuko

AU - Fukata, Masaki

AU - Salaun, Christine

AU - Chamberlain, Luke H

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N2 - SNAP25 is synthesized as a soluble protein but must associate with the plasma membrane to function in exocytosis; however, this membrane-targeting pathway is poorly defined. SNAP25 contains a palmitoylated cysteine-rich domain with four cysteines, and we show that coexpression of specific DHHC palmitoyl transferases is sufficient to promote SNAP25 membrane association in HEK293 cells. siRNA-mediated knockdown of its SNARE partner, syntaxin 1A, does not affect membrane interaction of SNAP25 in PC12 cells, whereas specific cysteine-to-alanine mutations perturb membrane binding, which is restored by leucine substitutions. These results suggest a role for cysteine hydrophobicity in initial membrane interactions of SNAP25, and indeed other hydrophobic residues in the cysteine-rich domain are also important for membrane binding. In addition to the cysteine-rich domain, proline-117 is also essential for SNAP25 membrane binding, and experiments in HEK293 cells revealed that mutation of this residue inhibits membrane binding induced by coexpression with DHHC17, but not DHHC3 or DHHC7. These results suggest a model whereby SNAP25 interacts autonomously with membranes via its hydrophobic cysteine-rich domain, requiring only sufficient expression of partner DHHC proteins for stable membrane binding. The role of proline-117 in SNAP25 palmitoylation is one of the first descriptions of elements within substrate proteins that modulate DHHC specificity.

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KW - hydrophobic and hydrophilic interactions

KW - molecular sequence data

KW - mutation

KW - protein binding

KW - rats

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