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

Kanaani, Jamil; Diacovo, Maria Julia; El-Husseini, Alaa; Bredt, David S.; Baekkeskov, Steinunn

doi:10.1242/jcs.01030

Cited by 67 publications

(58 citation statements)

References 35 publications

(45 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Earlier studies have shown that wt GAD65-GFP is predominantly located in Golgi membranes and in presynaptic clusters in neurons, whereas the palmitoylation-deficient GAD65(C30,45A)-GFP fusion protein is detected in a diffuse pattern in the cytosol as well as in Golgi membranes (Kanaani et al, 2002;Kanaani et al, 2004). This difference suggested that a significant fraction of the protein is soluble in the absence of palmitoylation.…”

Section: Resultsmentioning

confidence: 95%

“…A fraction of this membrane-anchored pool of the protein is palmitoylated (Christgau et al, 1992;Shi et al, 1994). However, palmitoylation is not critical for membrane anchoring (Shi et al, 1994;Solimena et al, 1994;Kanaani et al, 2002;Kanaani et al, 2004). Three distinct trafficking signals have been identified in the N-terminal domain of GAD65 (Kanaani et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

“…A Golgi-targeting sequence residing in the first 23 amino acids and a membrane-anchoring signal residing in residues 24-31 are required for targeting to Golgi membranes (Shi et al, 1994;Solimena et al, 1994;Kanaani et al, 2002). The third signal, involving palmitoylation of Cys30 and Cys45 (Shi et al, 1994) is critical for trafficking of the protein from Golgi membranes to presynaptic clusters in axon termini (Kanaani et al, 2002) by a Rab5a-dependent pathway (Kanaani et al, 2004). In addition to the hydrophobic post-translational modifications, GAD65 is phosphorylated on Ser3, Ser6, Ser10 and Ser13 (Namchuk et al, 1997) and acetylated on the penultimate alanine following removal of the N-terminal methionine (Battaglioli et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

A palmitoylation cycle dynamically regulates partitioning of the GABA-synthesizing enzyme GAD65 between ER-Golgi and post-Golgi membranes

Kanaani

Patterson

Schaufele

et al. 2008

Journal of Cell Science

Self Cite

View full text Add to dashboard Cite

GAD65, the smaller isoform of the enzyme glutamic acid decarboxylase, synthesizes GABA for fine-tuning of inhibitory neurotransmission. GAD65 is synthesized as a soluble hydrophilic protein but undergoes a hydrophobic posttranslational modification and becomes anchored to the cytosolic face of Golgi membranes. A second hydrophobic modification, palmitoylation of Cys30 and Cys45 in GAD65, is not required for the initial membrane anchoring but is crucial for post-Golgi trafficking of the protein to presynaptic clusters. The mechanism by which palmitoylation directs targeting of GAD65 through and out of the Golgi complex is unknown. Here, we show that prior to palmitoylation, GAD65 anchors to both ER and Golgi membranes. Palmitoylation, however, clears GAD65 from the ER-Golgi, targets it to the trans-Golgi network and then to a post-Golgi vesicular pathway. FRAP analyses of trafficking of GAD65-GFP reveal a rapid and a slow pool of protein replenishing the Golgi complex. The rapid pool represents non-palmitoylated hydrophobic GAD65-GFP, which exchanges rapidly between the cytosol and ER/Golgi membranes. The slow pool represents palmitoylationcompetent GAD65-GFP, which replenishes the Golgi complex via a non-vesicular pathway and at a rate consistent with a depalmitoylation step. We propose that a depalmitoylationrepalmitoylation cycle serves to cycle GAD65 between Golgi and post-Golgi membranes and dynamically control levels of enzyme directed to the synapse. Supplementary material available online at

show abstract

Section: Resultsmentioning

confidence: 95%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A palmitoylation cycle dynamically regulates partitioning of the GABA-synthesizing enzyme GAD65 between ER-Golgi and post-Golgi membranes

Kanaani

Patterson

Schaufele

et al. 2008

Journal of Cell Science

Self Cite

View full text Add to dashboard Cite

show abstract

“…For example, the palmitoylation motif of GAP-43 broadly targets GFP to the Golgi complex and to the plasma membrane, whereas the fulllength protein is mainly restricted to the axon membrane (Burry et al, 1992). In the case of the proteins GAD-65 and PSD-95, it was shown that other signals in addition to the palmitoylation motif are necessary to achieve the fine targeting of these proteins to presynaptic clusters and postsynaptic densities, respectively (Craven and Bredt, 2000; El-Husseini et al., 2001;Kanaani et al, 2002;Kanaani et al, 2004). For SCG10, we identified region n as a second signal, which participates in the specific sorting of domain A to the Golgi complex, in addition to the palmitoylation motif within region m. Indeed, constructs containing region n (A-GFP, a nm -GFP and SCG10-GFP) were restricted to the Golgi complex and vesicles, whereas the others did not display this limited distribution (a m -GFP, a mc -GFP or ∆n-SCG10-GFP).…”

Section: Journal Of Cell Sciencementioning

confidence: 99%

Two separate motifs cooperate to target stathmin-related proteins to the Golgi complex

Charbaut

Chauvin

Enslen

et al. 2005

Journal of Cell Science

View full text Add to dashboard Cite

newly identified Golgi-specifying sequence. The latter displayed no targeting activity by itself, but retained a Golgi-specifying activity when associated with another membrane-anchoring palmitoylation motif derived from the protein GAP-43. We further identified critical residues for the specific Golgi targeting of domain A. Altogether, our results give new insight into the regulation of the subcellular localization of stathmin family proteins, an important feature of their physiological functions in differentiating and mature neural cells. More generally we provide new information on essential mechanisms of functional protein subcellular targeting. (Stein et al., 1988; Di Paolo et al., 1997a;Gavet et al., 1998;Lutjens et al., 2000;Gavet et al., 2002). SCG10 was also shown to associate with lipid rafts (Maekawa et al., 2001). This specific subcellular distribution has led to proposals that stathmin-related proteins are involved in the local regulation of cellular processes, including microtubule dynamics and possibly others in relation with their other identified partners (Nixon et al., 2002;Liu et al., 2002;Greka et al., 2003). The membrane localization of stathmin-related proteins is achieved by their N-terminal extension composed of one or several specific domains (Fig. 1). Domains A′ and A′′, with unknown functions, are specific to the splice variants RB3, RB3′ and RB3′′. By contrast, domain A, which is the most Nterminal, is conserved in all stathmin-related proteins (60-70% identity) (Stein et al., 1988;Ozon et al., 1997;Ozon et al., 1998) and has been identified as a key element to target SCG10 to the Golgi complex (Di Paolo et al., 1997b). Indeed, it was shown that not only the deletion of domain A in SCG10 redistributed the protein to the cytosol (Di Paolo et al., 1997b), but also that the addition of this same domain to heterologous proteins was able to target the fusion protein to the Golgi complex in COS-7 cells and in neurons (Di Paolo et al., 1997b;Lutjens et al., 2000). Two palmitoylation sites have been identified in domain A of SCG10, but their mutation did not result in the complete solubilization of the protein (Di Paolo et al., 1997b), suggesting that other signals must be involved in the subcellular localization of stathmin-related proteins to the Golgi complex.In this study, we investigated the molecular mechanisms involved in the Golgi targeting of stathmin family proteins. By characterizing the subcellular distribution mediated by various subdomains derived from domain A of SCG10 fused to GFP, we show that it is achieved by the combination of two adjacent motifs, one being responsible for membrane association and the other specifying targeting to the Golgi complex. In addition, we demonstrate that this second motif is able to cooperate with another palmitoylated membrane-anchoring sequence for Golgi targeting and we identified critical residues for its functional integrity. Materials and Methods Plasmid constructsStandard recombinant DNA techniques were carried out as described (Sambroo...

show abstract

“…Angiotensin II can stimulate vesicular redistribution in brain neurons (27). Palmitoylation influences trafficking of the GABA-synthesizing enzyme GAD 65 from Golgi membranes to axon-specific endosomes (28). Circulating factors can reach hypothalamic neurons via fenestrated capillaries.…”

Section: Discussionmentioning

confidence: 99%

Impaired Autofeedback Regulation of Hypothalamic Norepinephrine Release in Experimental Uremia

Klein

Daschner

Vogel

et al. 2005

Journal of the American Society of Nephrology

View full text Add to dashboard Cite

Chronic renal failure (CRF) is associated with multiple hypothalamic dysfunctions, including reduced secretion of gonadotropin-releasing hormone (GnRH). Because GnRH release is tightly controlled by sympathetic neuronal input, a possible alteration of local noradrenergic neurotransmission in experimental CRF was evaluated. Basal, stimulated, and autoinhibited norepinephrine (NE) release was assessed in hypothalamic and hippocampal tissue slices obtained from 5/6-nephrectomized and control rats. Autoinhibition-free NE release from brain slices, prelabeled with [ 3 H]NE and superfused with physiologic buffer, was stimulated by six electrical pulses, 100 Hz (pseudo-one-pulse stimulation). Autoinhibited NE release was induced by 90 pulses at 3 Hz. The release of tritiated NE was measured upon addition of increasing concentrations of unlabeled NE to exogenously activate the inhibitory ␣ 2 -autoreceptor. Although neither basal nor stimulated NE release differed between the groups, significantly lower pIC 50 and I max estimates of the concentration-response curves of exogenous NE on [ 3 H]NE release were observed in CRF rats, suggesting a diminished autoinhibition of hypothalamic noradrenergic terminals in CRF. Western blotting of tissue homogenates disclosed a significantly reduced abundance of ␣ 2 -autoreceptor protein in hypothalamic tissue from CRF rats. These abnormalities were selectively observed in the hypothalamus, whereas noradrenergic autoinhibition seemed unaltered in the hippocampus. The results suggest a diminished autoinhibition of hypothalamic NE release in CRF. Although impaired hypothalamic NE autoinhibition does not explain reduced GnRH secretion in CRF, it may be involved in the pathogenesis of sympathetic hyperactivity associated with this condition. D isorders of the reproductive system, clinically manifesting by impaired libido and fertility in adults and delayed or arrested puberty in adolescents, are common in chronic renal failure (CRF) (1). We and others previously demonstrated defective neuroendocrine activation of the gonadotropic hormone axis both in patients with CRF and in experimental uremia, with evidence for reduced pulsatile release of gonadotropin releasing hormone (GnRH) from the mediobasal hypothalamus (2-5). Experimental findings suggest that uremia may influence the function of hypothalamic neurons by various mechanisms. We observed abnormal extracellular amino acid neurotransmitter concentrations in the mediobasal hypothalamus of uremic rats, compatible with disturbed regulation of hypothalamic neurons by higher neuronal centers (6). The function of brain synaptosomes is altered in experimental uremia (7-10). However, we also demonstrated direct inhibition of GnRH secretion from cultured hypothalamic neurons by a factor circulating in uremic serum (11).Norepinephrinergic axon terminals are located in proximity of GnRH cells in the anterior hypothalamus (12). Norepinephrine (NE) is able to stimulate GnRH release from the hypothalamus in a concentration-dependent manner in vitro ...

show abstract

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

Cited by 67 publications

References 35 publications

A palmitoylation cycle dynamically regulates partitioning of the GABA-synthesizing enzyme GAD65 between ER-Golgi and post-Golgi membranes

A palmitoylation cycle dynamically regulates partitioning of the GABA-synthesizing enzyme GAD65 between ER-Golgi and post-Golgi membranes

Two separate motifs cooperate to target stathmin-related proteins to the Golgi complex

Impaired Autofeedback Regulation of Hypothalamic Norepinephrine Release in Experimental Uremia

Contact Info

Product

Resources

About