Targeting of the 67-kDa isoform of glutamic acid decarboxylase to intracellular organelles is mediated by its interaction with the NH2-terminal region of the 65-kDa isoform of glutamic acid decarboxylase.
The two isoforms of glutamic acid decarboxylase (GAD), GAD67 and GAD65, synthesize the neurotransmitter gamma-aminobutyric acid in neurons and pancreatic beta-cells. Previous studies suggest that GAD67 is a soluble cytosolic protein, whereas GAD65 is membrane-associated. Here, we study the intracellular distribution of GAD67 in neurons, pancreatic beta-cells, and fibroblasts transfected either with GAD65 and GAD67 together or with GAD67 alone. Neuronal GAD67 is partially recovered with GAD65 in membrane-containing pellet fractions and Triton X-114 detergent phases. The two proteins co-immunoprecipitate from extracts of brain and GAD65-GAD67 co-transfected fibroblasts, but not when extracts of GAD65 and GAD67 transfected fibroblasts were mixed and used as a starting material for immunoprecipitation. GAD67 is concentrated in the Golgi complex region in GAD65-GAD67 co-transfected fibroblasts, but not in fibroblasts transfected with GAD67 alone. A pool of neuronal GAD67 co-localizes with GAD65 in the Golgi complex region and in many synapses. The two proteins also co-localize in the perinuclear region of some pancreatic beta-cells. GAD67 interacts with the NH2-terminal region of GAD65, even in the absence of palmitoylation of this region of GAD65. Taken together, our results indicate that GAD65-GAD67 association occurs in vivo and is required for the targeting of GAD67 to membranes.
Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.
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Dirkx
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Thomas
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Li
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Lernmark
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Sherwin
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De Camilli
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Solimena
D000818:Animals; D001921:Brain / Q000201:enzymology / Q000648:ultrastructure; D016466:CHO Cells; D002451:Cell Compartmentation; D006224:Cricetinae; D005455:Fluorescent Antibody Technique; D005968:Glutamate Decarboxylase / Q000378:metabolism*; D006056:Golgi Apparatus / Q000201:enzymology; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D007515:Islets of Langerhans / Q000201:enzymology; D007527:Isoenzymes / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins; D013788:Thalamus / Q000201:enzymology
D007527:Isoenzymes; D011994:Recombinant Proteins; D005968:Glutamate Decarboxylase