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

The smaller isoform of the GABA synthesizing enzyme glutamic acid decarboxylase, GAD₆₅, is synthesized as a soluble protein that undergoes posttranslational modification(s) in the NH₂-terminal region to become anchored to the membrane of small synaptic-like microvesicles in pancreatic β cells, and synaptic vesicles in GABA-ergic neurons. A soluble hydrophilic form, a soluble hydrophobic form, and a hydrophobic firmly membrane-anchored form have been detected in β cells. A reversible and hydroxylamine sensitive palmitoylation has been shown to distinguish the firmly membrane-anchored form from the soluble yet hydrophobic form, suggesting that palmitoylation of cysteines in the NH₂-terminal region is involved in membrane anchoring. In this study we use site-directed mutagenesis to identify the first two cysteines in the NH₂-terminal region, Cys 30 and Cys 45, as the sites of palmitoylation of the GAD₆₅ molecule. Mutation of Cys 30 and Cys 45 to Ala results in a loss of palmitoylation but does not significantly alter membrane association of GAD₆₅ in COS-7 cells. Deletion of the first 23 amino acids at the NH₂ terminus of the GAD₆₅30/45A mutant also does not affect the hydrophobicity and membrane anchoring of the GAD₆₅ protein. However, deletion of an additional eight amino acids at the NH₂ terminus results in a protein which is hydrophilic and cytosolic. The results suggest that amino acids 24-31 are required for hydrophobic modification and/or targeting of GAD₆₅ to membrane compartments, whereas palmitoylation of Cys 30 and Cys 45 may rather serve to orient or fold the protein at synaptic vesicle membranes.