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

Abstract

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

N termini of apPDE4 isoforms are responsible for targeting the isoforms to different cellular membranes

“…Indeed, these results were similar to the results obtained for GAD65 (Kanaani et al 2008), which is a palmitoylationdependent, presynaptic terminal-targeting protein. In the presence of 100 mM 2BR, the GAD65 expression shifted from TGN to both endoplasmic reticulum (ER) and Golgi apparatus (Kanaani et al 2008). It has been believed that palmitoylation of the proteins leads to their incorporation into lipid rafts.…”

Regional Specificity of GABAergic Regulation of Cross-Modal Plasticity in Mouse Visual Cortex after Unilateral Enucleation

“…Although GAD67 produces the majority of GABA throughout interneurons, GAD65 is specialized for the on-demand high production of GABA at axon terminals (Reetz et al, 1991;Wu et al, 2007;Kanaani et al, 2008). In binocular cortex homogenates, the expression of the two GAD protein isoforms remained unchanged (Fig.…”

“…Indeed, long-term ME specifically increased GAD protein expression regardless of age or DE in the medial monocular cortex. Increased GAD levels likely correspond to GABA production above normal levels and are suggestive of an upregulation of the on-demand high production of vesicular GABA, particularly provided by GAD65 at axon terminals (Soghomonian and Martin, 1998;Wu et al, 2007;Kanaani et al, 2008). In the monocular cortex of both normally reared and dark-exposed adult ME mice, the rise in GAD67 could indicate more GABA synthesis throughout interneurons for the purpose of both nonvesicular and vesicular release and for metabolic demands (Belhage et al, 1993;Lau and Murthy, 2012).…”

Comparison of the Pseudorabies Virus Us9 Protein with Homologs from Other Veterinary and Human Alphaherpesviruses

“…␤-1,4-galactosyltransferase and mRFP1 is a monomeric red fluorescent protein) (23), a marker of the TGN, was a kind gift from Jennifer Lippincott-Schwartz (NIH). Plasmids containing the HindIII C fragment of the VZV unique short (Us) region, the EcoRI/BamHI fragment of the PvuII B fragment of the EHV-1 KyA strain, and the HindIII F fragment of the BHV-1 Jura strain were kindly provided by Chuck Grose (University of Iowa), D. J. O'Callaghan (Louisiana State University), and S. I. Chowdhury (Louisiana State University), respectively.…”