Amino acid residues 24-31 but not palmitoylation of cysteines 30 and 45 are required for membrane anchoring of glutamic acid decarboxylase, GAD65

Shi, Yongmin; Veit, B; Baekkeskov, Steinunn

doi:10.1083/jcb.124.6.927

Cited by 76 publications

(86 citation statements)

References 47 publications

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“…Taken together, these results indicate that fatty acylation may not be the factor involved in Dcsp's association with cellular membranes. A similar conclusion was reached recently for another membrane-associated protein, glutamic acid decarboxylase GAD65 [19]. GAD65 is palmitoylated on two cysteine residues in the NH2-terminal region and this modification was thought to anchor GAD to membranes [20].…”

Section: Discussionsupporting

confidence: 71%

Association of Drosophila cysteine string proteins with membranes

Goor

Kelly

1996

FEBS Letters

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Section: Discussionsupporting

confidence: 71%

Association of Drosophila cysteine string proteins with membranes

Goor

Kelly

1996

FEBS Letters

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“…The palmitoylated form of glutamic acid decarboxylase (GAD65) is associated with membranes of microvesicles in pancreatic beta cells and synaptic vesicles in ␥-aminobutyric acid-secreting neurons. Membrane localization of GAD65 requires amino acid residues 24-31 of the protein, but not cysteine residues 30 and 45, which are palmitoylated (33). Protein palmitoyltransferase activities are localized in membranes (34)(35)(36), and acylation of RGS4 or GAD65 may require the protein to first bind to membranes before it can be palmitoylated.…”

Section: Discussionmentioning

confidence: 99%

Plasma membrane localization is required for RGS4 function in Saccharomyces cerevisiae

Srinivasa

Bernstein

Blumer

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

139

154

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RGS4, a mammalian GTPase activating protein for G protein ␣ subunits, was identified by its ability to inhibit the pheromone response pathway in Saccharomyces cerevisiae. To define regions of RGS4 necessary for its function in vivo, we assayed mutants for activity in this system. Deletion of the N-terminal 33 aa of RGS4 (⌬1-33) yielded a nonfunctional protein and loss of plasma membrane localization. These functions were restored by addition of a C-terminal membrane-targeting sequence to RGS4 (⌬1-33). Thus, plasma membrane localization is tightly coupled with the ability of RGS4 to inhibit signaling. Fusion of the N-terminal 33 aa of RGS4 to green f luorescent protein was sufficient to localize an otherwise soluble protein to the plasma membrane, defining this N-terminal region as a plasma membrane anchorage domain. RGS4 is palmitoylated, with Cys-2 and Cys-12 the likely sites of palmitoylation. Surprisingly, mutation of the cysteine residues within the N-terminal domain of RGS4 did not affect plasma membrane localization in yeast or the ability to inhibit signaling. Features of the N-terminal domain other than palmitoylation are responsible for the plasma membrane association of RGS4 and its ability to inhibit pheromone response in yeast.Heterotrimeric G proteins couple receptors for hormones, neurotransmitters, and sensory signals to intracellular effector molecules, thereby eliciting cellular responses (1, 2). Guanine nucleotide exchange and hydrolysis on the G protein ␣ subunit drives the cycle of activation and deactivation of these signaling pathways. The duration of G protein-mediated responses is subject to the intrinsic GTPase rate of the G protein ␣ subunit, but is also modulated by extrinsic factors. A recently appreciated form of regulation has come from the discovery that members of a protein family called regulators of G protein signaling, or RGS proteins, stimulate the rate of GTP hydrolysis by G protein ␣ subunits (3-5). RGS proteins are found in species ranging from yeast to mammals and constitute a family of at least 20 mammalian proteins (6-8).All RGS family members share sequence similarity that extends over approximately 130 aa, separated in some cases by insertions of varying length (9-11). This conserved RGS domain is sufficient to stimulate GTPase activity of G protein ␣ subunits in vitro (12)(13)(14). Expression of the RGS homology domain of RET-RGS1 or RGS4 yields a recombinant protein that is a functional GAP (GTPase-activating protein). In the crystal structure of RGS4 bound to AlF 4 Ϫ -activated G i␣1 , only the core domain is visible (15). The RGS homology domain binds to the switch regions of G i␣1 and appears to catalyze GTP hydrolysis by stabilizing the switch regions of the G protein in a conformation that favors the transition state of the reactants (14, 15). Sequences outside the RGS homology domain exhibit considerable diversity among family members.Although rapid progress has been made in dissecting the biochemical mechanism by which RGS proteins regulate G protein a...

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“…Neurons were fixed 24 or 72 hours after transfection. Culture and transfection of COS-7 cells were carried out as described previously (Shi et al, 1994;Namchuk et al, 1997).…”

Section: Cell Culturementioning

confidence: 99%

Palmitoylation controls trafficking of GAD65 from Golgi membranes to axon-specific endosomes and a Rab5a-dependent pathway to presynaptic clusters

Kanaani

Diacovo

El-Husseini

et al. 2004

Journal of Cell Science

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The GABA-synthesizing enzyme GAD65 is synthesized as a soluble cytosolic protein but undergoes post-translational modification(s) to become anchored to the cytosolic face of Golgi membranes before targeting to synaptic vesicle membranes in neuroendocrine cells. Palmitoylation of cysteines 30 and 45 in GAD65 is not required for targeting to Golgi membranes but is crucial for post-Golgi trafficking to presynaptic clusters in neurons. Here, we show that palmitoylated GAD65 colocalizes with the small GTP-binding protein Rab5a in Golgi membranes and in axons but not in dendrites. In the presence of the constitutively positive mutant Rab5(Q79L) palmitoylation resulted in polarized targeting of GAD65 to giant Rab5a-positive axonal endosomes, characterized by the absence of the Rab5a-effector molecule EEA1 and the transferrin receptor. By contrast, Rab5a-positive/EEA1-positive somatodendritic giant endosomes containing the transferrin receptor were devoid of GAD65. Palmitoylation-deficient GAD65 was excluded from endosomal compartments. A dominant negative mutant of Rab5a, Rab5a(S34N), specifically blocked axonal trafficking and presynaptic clustering of palmitoylated GAD65, but did not affect axonal trafficking of mutants of GAD65 that fail to traffic to giant axonal endosomes containing Rab5a(Q79L). Two transmembrane synaptic vesicle proteins, VAMP2 and VGAT also localized to the axonal giant endosomes, and their axonal trafficking and presynaptic clustering was blocked by Rab5a(S34N). The results suggest that palmitoylation of GAD65 regulates the trafficking of the protein from Golgi membranes to an endosomal trafficking pathway in axons that is dependent on Rab5a and is required for the targeting of several synaptic vesicle proteins to presynaptic clusters.

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Amino acid residues 24-31 but not palmitoylation of cysteines 30 and 45 are required for membrane anchoring of glutamic acid decarboxylase, GAD65

Cited by 76 publications

References 47 publications

Association of Drosophila cysteine string proteins with membranes

Association of Drosophila cysteine string proteins with membranes

Plasma membrane localization is required for RGS4 function in Saccharomyces cerevisiae

Palmitoylation controls trafficking of GAD65 from Golgi membranes to axon-specific endosomes and a Rab5a-dependent pathway to presynaptic clusters

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