Palmitoylation of the SNAP25 protein family: specificity and regulation by DHHC palmitoyl transferases

Jennifer Greaves, Oforiwa A Gorleku, Christine Salaun, Luke H Chamberlain

Research output: Contribution to journalArticle

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Abstract

SNAP25 plays an essential role in neuronal exocytosis pathways. SNAP25a and SNAP25b are alternatively spliced isoforms differing by only nine amino acids, three of which occur within the palmitoylated cysteine-rich domain. SNAP23 is 60% identical to SNAP25 and has a distinct cysteine-rich domain to both SNAP25a and SNAP25b. Despite the conspicuous differences within the palmitoylated domains of these secretory proteins, there is no information on their comparative interactions with palmitoyl transferases. We report that membrane association of all SNAP25/23 proteins is enhanced by Golgi-localized DHHC3, DHHC7, and DHHC17. In contrast, DHHC15 promoted a statistically significant increase in membrane association of only SNAP25b. To investigate the underlying cause of this differential specificity, we examined a SNAP23 point mutant (C79F) designed to mimic the cysteine-rich domain of SNAP25b. DHHC15 promoted a marked increase in membrane binding and palmitoylation of this SNAP23 mutant, demonstrating that the distinct cysteine-rich domains of SNAP25/23 contribute to differential interactions with DHHC15. The lack of activity of DHHC15 toward wild-type SNAP23 was not overcome by replacing its DHHC domain with that from DHHC3, suggesting that substrate specificity is not determined by the DHHC domain alone. Interestingly, DHHC2, which is closely related to DHHC15, associates with the plasma membrane in PC12 cells and can palmitoylate all SNAP25 isoforms. DHHC2 is, thus, a candidate enzyme to regulate SNAP25/23 palmitoylation dynamics at the plasma membrane. Finally, we demonstrate that overexpression of specific Golgi-localized DHHC proteins active against SNAP25/23 proteins perturbs the normal secretion of human growth hormone from PC12 cells.

Original languageEnglish
Pages (from-to)24629-24638
Number of pages10
JournalJournal of Biological Chemistry
Volume285
Issue number32
Early online date2 Jun 2010
DOIs
Publication statusPublished - 5 Aug 2010
Externally publishedYes

Fingerprint

Synaptosomal-Associated Protein 25
Lipoylation
Transferases
Cysteine
PC12 Cells
Cell membranes
Membranes
Protein Isoforms
Proteins
Cell Membrane
Human Growth Hormone
Exocytosis
Substrate Specificity
Amino Acids
Substrates
Enzymes

Keywords

  • Amino Acid Sequence
  • Animals
  • Cell Membrane
  • Cysteine
  • Gene Expression Regulation, Enzymologic
  • Golgi Apparatus
  • Humans
  • Molecular Sequence Data
  • PC12 Cells
  • Palmitic Acid
  • Protein Isoforms
  • Qb-SNARE Proteins
  • Qc-SNARE Proteins
  • Rats
  • Sequence Homology, Amino Acid
  • Synaptosomal-Associated Protein 25
  • Vesicular Transport Proteins
  • Journal Article
  • Research Support, Non-U.S. Gov't

Cite this

Palmitoylation of the SNAP25 protein family : specificity and regulation by DHHC palmitoyl transferases. / Greaves, Jennifer; Gorleku, Oforiwa A; Salaun, Christine; Chamberlain, Luke H.

In: Journal of Biological Chemistry, Vol. 285, No. 32, 05.08.2010, p. 24629-24638.

Research output: Contribution to journalArticle

Greaves, Jennifer ; Gorleku, Oforiwa A ; Salaun, Christine ; Chamberlain, Luke H. / Palmitoylation of the SNAP25 protein family : specificity and regulation by DHHC palmitoyl transferases. In: Journal of Biological Chemistry. 2010 ; Vol. 285, No. 32. pp. 24629-24638.
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AU - Greaves, Jennifer

AU - Gorleku, Oforiwa A

AU - Salaun, Christine

AU - Chamberlain, Luke H

PY - 2010/8/5

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N2 - SNAP25 plays an essential role in neuronal exocytosis pathways. SNAP25a and SNAP25b are alternatively spliced isoforms differing by only nine amino acids, three of which occur within the palmitoylated cysteine-rich domain. SNAP23 is 60% identical to SNAP25 and has a distinct cysteine-rich domain to both SNAP25a and SNAP25b. Despite the conspicuous differences within the palmitoylated domains of these secretory proteins, there is no information on their comparative interactions with palmitoyl transferases. We report that membrane association of all SNAP25/23 proteins is enhanced by Golgi-localized DHHC3, DHHC7, and DHHC17. In contrast, DHHC15 promoted a statistically significant increase in membrane association of only SNAP25b. To investigate the underlying cause of this differential specificity, we examined a SNAP23 point mutant (C79F) designed to mimic the cysteine-rich domain of SNAP25b. DHHC15 promoted a marked increase in membrane binding and palmitoylation of this SNAP23 mutant, demonstrating that the distinct cysteine-rich domains of SNAP25/23 contribute to differential interactions with DHHC15. The lack of activity of DHHC15 toward wild-type SNAP23 was not overcome by replacing its DHHC domain with that from DHHC3, suggesting that substrate specificity is not determined by the DHHC domain alone. Interestingly, DHHC2, which is closely related to DHHC15, associates with the plasma membrane in PC12 cells and can palmitoylate all SNAP25 isoforms. DHHC2 is, thus, a candidate enzyme to regulate SNAP25/23 palmitoylation dynamics at the plasma membrane. Finally, we demonstrate that overexpression of specific Golgi-localized DHHC proteins active against SNAP25/23 proteins perturbs the normal secretion of human growth hormone from PC12 cells.

AB - SNAP25 plays an essential role in neuronal exocytosis pathways. SNAP25a and SNAP25b are alternatively spliced isoforms differing by only nine amino acids, three of which occur within the palmitoylated cysteine-rich domain. SNAP23 is 60% identical to SNAP25 and has a distinct cysteine-rich domain to both SNAP25a and SNAP25b. Despite the conspicuous differences within the palmitoylated domains of these secretory proteins, there is no information on their comparative interactions with palmitoyl transferases. We report that membrane association of all SNAP25/23 proteins is enhanced by Golgi-localized DHHC3, DHHC7, and DHHC17. In contrast, DHHC15 promoted a statistically significant increase in membrane association of only SNAP25b. To investigate the underlying cause of this differential specificity, we examined a SNAP23 point mutant (C79F) designed to mimic the cysteine-rich domain of SNAP25b. DHHC15 promoted a marked increase in membrane binding and palmitoylation of this SNAP23 mutant, demonstrating that the distinct cysteine-rich domains of SNAP25/23 contribute to differential interactions with DHHC15. The lack of activity of DHHC15 toward wild-type SNAP23 was not overcome by replacing its DHHC domain with that from DHHC3, suggesting that substrate specificity is not determined by the DHHC domain alone. Interestingly, DHHC2, which is closely related to DHHC15, associates with the plasma membrane in PC12 cells and can palmitoylate all SNAP25 isoforms. DHHC2 is, thus, a candidate enzyme to regulate SNAP25/23 palmitoylation dynamics at the plasma membrane. Finally, we demonstrate that overexpression of specific Golgi-localized DHHC proteins active against SNAP25/23 proteins perturbs the normal secretion of human growth hormone from PC12 cells.

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KW - Molecular Sequence Data

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KW - Qc-SNARE Proteins

KW - Rats

KW - Sequence Homology, Amino Acid

KW - Synaptosomal-Associated Protein 25

KW - Vesicular Transport Proteins

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

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