[25] Palmitoylation of G-protein α subunits

doi:10.1016/0076-6879(95)50081-2

Methods in Enzymology

Volume 250, 1995, Pages 314-330

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This chapter describes the procedures used to detect protein palmitoylation. The protocols for metabolic radiolabeling of cells and for analysis of radioactive fatty acids incorporated into proteins are also reviewed. Covalent attachment of palmitate to protein occurs usually through a thioester linkage to cysteine. However, the lipid may also be bound to protein through oxyester linkages to serine or threonine. Fatty acylation of proteins through ester bonds is not specific for palmitate. Longer chain saturated fatty acids, such as stearate, are also detected. Modification of proteins by myristate is primarily by cotranslational addition of the fatty acid through an amide linkage to the amino terminus of the protein. Protein palmitoylation contributes to membrane association of a number of proteins including G-protein α subunits, and the neuronal growth cone protein GAP-43. Palmitoylation of proteins is usually assayed by isolating the protein from cells that have been incubated with radioactive palmitic acid. Thioester linkages are hydrolyzed by treatment with base, and fatty acids cleaved from the protein can be analyzed by reversed phase high-performance liquid chromatography (HPLC) or thin-layer chromatography (TLC).

References (43)

J.R. Hepler et al.
Trends Biochem. Sci.
(1992)
J. Iniguez-Lluhi et al.
Trends Cell Biol.
(1993)
H. Ohguro et al.
EMBO J.
(1991)
S.M. Mumby et al.
T.L.Z. Jones et al.
K. Kokame et al.
Nature (London)
(1992)
T.A. Neubert et al.
J. Biol. Chem.
(1992)
R.S. Johnson et al.
J. Biol. Chem.
(1994)
M.E. Linder et al.
J. Biol. Chem.
(1991)
R. Taussig et al.
Science
(1993)

M.E. Linder et al.

M. Parenti et al.

Biochem. J.

(1993)

A.M. Shenoy-Scaria et al.

J. Cell Biol.

(1994)

M. Degtyarev et al.

Biochemistry

(1993)

M. Veit et al.

FEBS Letters

(1994)

M.J. Schlessinger et al.

L. Muszbek et al.

J. Biol. Chem.

(1993)

H. Hallak et al.

J. Biol. Chem.

(1994)

P.B. Wedegaertner et al.

J. Biol. Chem.

(1993)

S.M. Mumby et al.

J.F. Hancock et al.

Cell (Cambridge, Mass.)

(1989)

Cited by (35)

Palmitoylation controls the catalytic activity and subcellular distribution of phosphatidylinositol 4-kinase IIα
2009, Journal of Biological Chemistry
Citation Excerpt :
Following centrifugation at 210,000 × g for 16 h at 4 °C in an SW40 rotor, 1-ml fractions were collected from the bottom of the tubes, and equal aliquots were subjected to SDS-gel electrophoresis and immunoblotting. Analysis of [3H]Palmitate Incorporation-Sixteen hours after transfection with plasmids encoding wild-type (WT) or mutant PI4KIIα, COS-7 cells were labeled with [3H]palmitate (0.3 mCi/ml) for 3 h as described by Linder et al. (20). Cells were washed with ice-cold PBS and solubilized in RIPA buffer consisting of 0.15 m NaCl, 50 mm Tris-HCl (pH 8.0), 1% Nonidet P40, 0.5% deoxycholate, 0.05% SDS, 2 mm EDTA, 0.2 mm PMSF, and the protease and phosphatase inhibitors described above.
Phosphatidylinositol 4-kinases play essential roles in cell signaling and membrane trafficking. They are divided into type II and III families, which have distinct structural and enzymatic properties and are essentially unrelated in sequence. Mammalian cells express two type II isoforms, phosphatidylinositol 4-kinase IIα (PI4KIIα) and IIβ (PI4KIIβ). Nearly all of PI4KIIα, and about half of PI4KIIβ, associates integrally with membranes, requiring detergent for solubilization. This tight membrane association is because of palmitoylation of a cysteine-rich motif, CCPCC, located within the catalytic domains of both type II isoforms. Deletion of this motif from PI4KIIα converts the kinase from an integral to a tightly bound peripheral membrane protein and abrogates its catalytic activity (Barylko, B., Gerber, S. H., Binns, D. D., Grichine, N., Khvotchev, M., Sudhof, T. C., and Albanesi, J. P. (2001) J. Biol. Chem. 276, 7705-7708). Here we identify the first two cysteines in the CCPCC motif as the principal sites of palmitoylation under basal conditions, and we demonstrate the importance of the central proline for enzymatic activity, although not for membrane binding. We further show that palmitoylation is critical for targeting PI4KIIα to the trans-Golgi network and for enhancement of its association with low buoyant density membrane fractions, commonly termed lipid rafts. Replacement of the four cysteines in CCPCC with a hydrophobic residue, phenylalanine, substantially restores catalytic activity of PI4KIIα in vitro and in cells without restoring integral membrane binding. Although this FFPFF mutant displays a perinuclear distribution, it does not strongly co-localize with wild-type PI4KIIα and associates more weakly with lipid rafts.
The Importance of N-terminal polycysteine and polybasic sequences for G<inf>14</inf>αand G<inf>16</inf>α palmitoylation, plasma membrane localization, and signaling function
2007, Journal of Biological Chemistry
Citation Excerpt :
For experiments with full-length Gα, cells were transfected with 16.8 μg of EE-tagged Gα ± C/S, 4.8 μg of Gβ1, and 2.4 μg of Gγ2. Twenty four hours after transfection, cells were labeled and immunoprecipitated according to a method described in detail elsewhere with minor modifications (27, 28). Specifically, cells for autoradiography were metabolically labeled with 1 mCi/ml [3H]palmitic acid for 2 h at 37°Cin palmitate labeling media (DMEM, 2.5% dialyzed fetal bovine serum, 1× sodium pyruvate, and 1× non-essential amino acids (CellGro)).
Plasma membrane targeting of G protein α (Gα) subunits is essential for competent receptor-to-G protein signaling. Many Gα are tethered to the plasma membrane by covalent lipid modifications at their N terminus. Additionally, it is hypothesized that G_q family members (G_qα,G₁₁α,G₁₄α, and G₁₆α) in particular utilize a polybasic sequence of amino acids in their N terminus to promote membrane attachment and protein palmitoylation. However, this hypothesis has not been tested, and nothing is known about other mechanisms that control subcellular localization and signaling properties of G₁₄α and G₁₆α. Here we report critical biochemical factors that mediate membrane attachment and signaling function of G₁₄α and G₁₆α. We find that G₁₄α and G₁₆α are palmitoylated at distinct polycysteine sequences in their N termini and that the polycysteine sequence along with the adjacent polybasic region are both important for G₁₆α-mediated signaling at the plasma membrane. Surprisingly, the isolated N termini of G₁₄α and G₁₆α expressed as peptides fused to enhanced green fluorescent protein each exhibit differential requirements for palmitoylation and membrane targeting; individual cysteine residues, but not the polybasic regions, determine lipid modification and subcellular localization. However, full-length G₁₆α, more so than G₁₄α, displays a functional dependence on single cysteines for membrane localization and activity, and its full signaling potential depends on the integrity of the polybasic sequence. Together, these findings indicate that G₁₄α and G₁₆α are palmitoylated at distinct polycysteine sequences, and that the adjacent polybasic domain is not required for Gα palmitoylation but is important for localization and functional activity of heterotrimeric G proteins.
G protein βγ complex translocation from plasma membrane to Golgi complex is influenced by receptor γ subunit interaction
2006, Cellular Signalling
On activation of a receptor the G protein βγ complex translocates away from the receptor on the plasma membrane to the Golgi complex. The rate of translocation is influenced by the type of γ subunit associated with the G protein. Complementary approaches — imaging living cells expressing fluorescent protein tagged G proteins and assaying reconstituted receptors and G proteins in vitro — were used to identify mechanisms at the basis of the translocation process. Translocation of Gβγ containing mutant γ subunits with altered prenyl moieties showed that the differences in the prenyl moieties were not sufficient to explain the differential effects of geranylgeranylated γ5 and farnesylated γ11 on the translocation process. The translocation properties of Gβγ were altered dramatically by mutating the C terminal tail region of the γ subunit. The translocation characteristics of these mutants suggest that after receptor activation, Gβγ retains contact with a receptor through the γ subunit C terminal domain and that differential interaction of the activated receptor with this domain controls Gβγ translocation from the plasma membrane.
A G protein γ subunit peptide stabilizes a novel muscarinic receptor state
2006, Biochemical and Biophysical Research Communications
Citation Excerpt :
Purification of G protein subunits. Recombinant Gαi2 and Gαo were purified from Escherichia coli by expressing rat Gαo and Gαi2 with yeast N-myristoyl transferase [16] (kind gift from Dr. M. Linder). Expression and purification were performed based on this previous published procedure with modifications [15].
A prenylated peptide specific to the C terminal tail of a G protein γ subunit type, γ5, inhibits activation of a G protein by the M2 muscarinic receptor. The γ5 peptide was tested for direct effects on the M2 receptor’s properties. The wild type γ5 peptide reduced the affinity of M2 for the agonist, carbachol, more than 5-fold in an antagonist displacement assay. The peptide was inactive when its amino acid sequence was scrambled or when it was unprenylated. Although the wild type peptide reduced the affinity of M2 for the antagonist QNB, it had no effect on the antagonists NMS or atropine. These results suggest that in the presence of the peptide the M2 receptor adopts a novel conformational state that affects the ligand binding surface. The results also suggest that the G protein γ5 subunit tail interacts with a receptor.
Role of the G protein γ subunit in βγ complex modulation of phospholipase Cβ function
2002, Journal of Biological Chemistry
The G protein βγ complex regulates a wide range of effectors, including the phospholipase C isozymes (PLCβs). Different domains on the β subunit are known to contact phospholipase Cβ and affect its regulation. In contrast, the role of the γ subunit in Gβγ modulation of PLCβ function is not known. Results here show that the γ subunit C-terminal domain is involved in mediating Gβγ interactions with phospholipase Cβ. Mutations were introduced to alter the position of the post-translational prenyl modification at the C terminus of the γ subunit with reference to the β subunit. These mutants were appropriately post-translationally modified with the geranylgeranyl moiety. A deletion that shortened the C-terminal domain, insertions that extended this domain, and a point mutation, F59A, that disrupted the interaction of this domain with the β subunit were all affected in their ability to activate PLCβ to varying degrees. All mutants, however, interacted equally effectively with the G_oα subunit. The results indicate that the G protein γ subunit plays a direct role in the modulation of effector function by the βγ complex.
Determining G protein heterotrimer formation
2002, Methods in Enzymology

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[25] Palmitoylation of G-protein α subunits

Publisher Summary

Trends Biochem. Sci.

Trends Cell Biol.

EMBO J.

Nature (London)

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Science

Biochem. J.

J. Cell Biol.

Biochemistry

FEBS Letters

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Cell (Cambridge, Mass.)