Analysis of the palmitoylation and membrane targeting domain of neuromodulin (GAP-43) by site-specific mutagenesis

Liu, Yuechueng; Fisher, Daniel A.C.; Storm, Daniel R.

doi:10.1021/bi00091a023

Cited by 73 publications

(58 citation statements)

References 43 publications

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“…Mutation of Cys-3 and Cys-4 blocks radiolabel incorporation, suggesting that these residues are the sites of modification (27). To study the nature of thioester-linked palmitoylation on a molecular level, we attempted to isolate the N-terminal palmitoylated peptide of GAP-43.…”

Section: Analysis Of Tryptic Digests Of Gap-43-gap-43 Has Beenmentioning

confidence: 99%

“…A previous study showed that the L3 mutant of GAP-43, in which Cys-3 is substituted with Leu, incorporates [ 3 H]palmitate to 75% of the level of the wild type protein (27). When the L3GAP-43 expressed in COS-1 cells was purified, digested with Tev protease, and analyzed by mass spectrometry, a peptide of monoisotopic m/z ϭ 2815.93 was observed, consistent with an acetylated but not palmitoylated N terminus.…”

Section: Analysis Of Tryptic Digests Of Gap-43-gap-43 Has Beenmentioning

confidence: 99%

“…Basic Residues at the N Terminus of GAP-43 Also Contribute to Membrane Binding-The finding that mutation of Cys-3 and Cys-4 leads to decreased palmitate incorporation and decreased membrane binding has led other investigators to conclude that palmitoylation of the N-terminal domain of GAP-43 is necessary for membrane association (27). Here we show that, although GAP-43 is mostly membrane-bound, only one-third of the protein is fatty acylated in vivo at steady state.…”

Section: Figmentioning

confidence: 99%

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Mass Spectrometric Analysis of GAP-43/Neuromodulin Reveals the Presence of a Variety of Fatty Acylated Species

Liang

Neubert

et al. 2002

Journal of Biological Chemistry

101

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GAP-43 (neuromodulin) is a protein kinase C substrate that is abundant in developing and regenerating neurons. Thioester-linked palmitoylation at two cysteines near the GAP-43 N terminus has been implicated in directing membrane binding. Here, we use mass spectrometry to examine the stoichiometry of palmitoylation and the molecular identity of the fatty acid(s) attached to GAP-43 in vivo. GAP-43 expressed in either PC12 or COS-1 cells was acetylated at the N-terminal methionine. Approximately 35% of the N-terminal GAP-43 peptides were also modified by palmitate and/or stearate on Cys residues. Interestingly, a variety of acylated species was detected, in which one of the Cys residues was acylated by either palmitate or stearate, or both Cys residues were acylated by palmitates or stearates or a combination of palmitate and stearate. Depalmitoylation of membrane-bound GAP-43 did not release the protein from the membrane, implying that additional forces function to maintain membrane binding. Indeed, mutation of four basic residues within the N-terminal domain of GAP-43 dramatically reduced membrane localization of GAP-43 without affecting palmitoylation. These data reveal the heterogeneous nature of S-acylation in vivo and illustrate the power of mass spectrometry for identification of key regulatory protein modifications.Covalent modification by acetylation or fatty acylation occurs on a variety of viral and cellular proteins (1). N-terminal acetylation is one of the most common protein modifications, occurring on ϳ85% of eukaryotic proteins (2). The amino acid residue adjacent to the amino-terminal methionine residue determines whether the N-terminal methionine is retained or removed before acetylation (2). Proteins that contain the Nterminal sequence MGXXX(S/T) undergo a different set of modifications. The initiating Met is removed, and myristate is added to the N-terminal glycine. The requirement for glycine at the N terminus is absolute for N-myristoylation to occur.In contrast to N-terminal acetylation and myristoylation, which occur co-translationally, palmitoylation is a post-translational lipid modification. Nearly all palmitoylated proteins are S-acylated by attachment of palmitate via a thioester linkage to the sulfhydryl group of cysteine. Exceptions include adenylate cyclase toxin from Bordetella pertussis, which is modified by amide-linked palmitoylation on the ⑀-amino group of lysine residues (3) and human sonic hedgehog, which is palmitoylated through an amide linkage to the N-terminal cysteine (4). Unlike myristoylation, the enzymology of palmitoylation reactions is poorly understood. Palmitoylacyl transferases have not been thoroughly purified and characterized. Moreover, no study has directly examined the nature of thioester-linked palmitoylation in vivo at a molecular level. All of the studies on thioester-linked palmitoylation are based on incorporation of radioactive palmitate. The stoichiometry of palmitoylation as well as the molecular identity of the attached fatty acid moiety (palmitate a...

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Section: Analysis Of Tryptic Digests Of Gap-43-gap-43 Has Beenmentioning

confidence: 99%

Section: Analysis Of Tryptic Digests Of Gap-43-gap-43 Has Beenmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

See 1 more Smart Citation

Mass Spectrometric Analysis of GAP-43/Neuromodulin Reveals the Presence of a Variety of Fatty Acylated Species

Liang

Neubert

et al. 2002

Journal of Biological Chemistry

101

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“…The first 35 amino acids of SCG-10, which include the palmitoylated cysteines at positions 22 and 24, contain the membrane-targeting information (16). Membrane association of GAP-43 is dependent upon palmitoylated cysteines at positions 3 and 4 (17). Previous studies have demonstrated that the palmitoylated cysteines are necessary for membrane association of SNAP-25 (18,19).…”

mentioning

confidence: 98%

SNAP-25 Is Targeted to the Plasma Membrane through a Novel Membrane-binding Domain

Gonzalo

Greentree

Linder

1999

Journal of Biological Chemistry

109

115

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SNAP-25, syntaxin, and synaptobrevin are SNARE proteins that mediate fusion of synaptic vesicles with the plasma membrane. Membrane attachment of syntaxin and synaptobrevin is achieved through a C-terminal hydrophobic tail, whereas SNAP-25 association with membranes appears to depend upon palmitoylation of cysteine residues located in the center of the molecule. This process requires an intact secretory pathway and is inhibited by brefeldin A. Here we show that the minimal plasma membrane-targeting domain of SNAP-25 maps to residues 85-120. This sequence is both necessary and sufficient to target a heterologous protein to the plasma membrane. Palmitoylation of this domain is sensitive to brefeldin A, suggesting that it uses the same membranetargeting mechanism as the full-length protein. As expected, the palmitoylated cysteine cluster is present within this domain, but surprisingly, membrane anchoring requires an additional five-amino acid sequence that is highly conserved among SNAP-25 family members. Significantly, the membrane-targeting module coincides with the protease-sensitive stretch (residues 83-120) that connects the two ␣-helices that SNAP-25 contributes to the four-helix bundle of the synaptic SNARE complex. Our results demonstrate that residues 85-120 of SNAP-25 represent a protein module that is physically and functionally separable from the SNARE complexforming domains.Intracellular membrane trafficking depends on specific interactions between vesicles and target membranes. Biochemical and genetic studies demonstrate that integral membrane proteins referred to as SNAREs (soluble N-ethylmaleimidesensitive factor attachment protein receptors) are important elements in this process (1, 2). The best characterized SNARE proteins are those that mediate synaptic vesicle exocytosis in nerve terminals (reviewed in Ref.3). SNAP-25 (synaptosomeassociated protein of 25 kDa) and syntaxin are plasma membrane proteins that bind to the synaptic vesicle protein, synaptobrevin/vesicle-associated membrane protein. The critical role that these proteins play in neurotransmission is underscored by the fact that all three are targets of tetanus or botulinum neurotoxins that block neurotransmitter release.SNAP-25, syntaxin, and synaptobrevin bind to each other to form a stable heterotrimeric complex that consists of a fourhelix bundle (4, 5). SNAP-25 contributes two helices from its Nand C-terminal domains; syntaxin and synaptobrevin each provide one. Syntaxin and synaptobrevin are anchored in opposing membranes through transmembrane domains at their C termini. The parallel orientation of these membrane-anchored helices within the complex has led to a model in which the zippering action of complex formation brings the membranes together, with the energy of complex formation driving membrane fusion (6 -8). However, whether SNARE proteins directly mediate fusion remains uncertain (9). Unlike synaptobrevin and syntaxin, SNAP-25 is associated with membranes through palmitoylated cysteines found near the center of the molecul...

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“…B-50/GAP-43 is conveyed by fast anterograde axonal transportS,6,27,31,48,49,54 attached to vesicles by palmitoylation of its N-terminal domain. 36,47 In the rat sciatic nerve, B-50/GAP-4335'54 and synaptophysin ~3 are also translocated retrogradely.…”

mentioning

confidence: 99%

Ultrastructural localization of B-50/growth-associated protein-43 to anterogradely transported synaptophysin-positive and calcitonin gene-related peptide-negative vesicles in the regenerating rat sciatic nerve

et al. 1996

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Abstract--The growth-associated protein-43/B-50 (B-50/GAP-43) is conveyed from the neuronal soma into the axon by fast axonal transport and moved to the nerve terminal. To visualize and determine the type of vesicles by which B-50/GAP-43 is anterogradely transported in the regenerating rat sciatic nerve, we have investigated Lowicryl HM20 embedded nerve pieces dissected from the proximal side of a collection ligature. Ultrastructurally, numerous vesicular profiles of various sizes, tubules and mitochondria were seen to accumulate proximal to the collection ligature. Both, in unmyelinated and myelinated axons, B-50/GAP-43 immunoreactivity was associated with vesicular profiles which had a diameter of 50 nm. A fraction of the B-50/GAP-43 label co-localized with the small vesicle marker synaptophysin. Co-localization of B-50/GAP-43 was not detected with the large dense-core vesicle marker calcitonin gene-related peptide.These results indicate that, in rat sciatic nerve axons, B-50/GAP-43 is anterogradely transported in small 50 nm vesicles of the constitutive pathway. These transport vesicles were distinguished in two types. We suggest that one type carrying, both, B-50 GAP-43 and synaptophysin has as destination the nerve terminal, whereas the second type, which only contains B-50/GAP-43 and no synaptophysin, may be primarily targeted to the axolemma for local membrane fusion.

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Analysis of the palmitoylation and membrane targeting domain of neuromodulin (GAP-43) by site-specific mutagenesis

Cited by 73 publications

References 43 publications

Mass Spectrometric Analysis of GAP-43/Neuromodulin Reveals the Presence of a Variety of Fatty Acylated Species

Mass Spectrometric Analysis of GAP-43/Neuromodulin Reveals the Presence of a Variety of Fatty Acylated Species

SNAP-25 Is Targeted to the Plasma Membrane through a Novel Membrane-binding Domain

Ultrastructural localization of B-50/growth-associated protein-43 to anterogradely transported synaptophysin-positive and calcitonin gene-related peptide-negative vesicles in the regenerating rat sciatic nerve

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