AMINO-ACID-RESIDUES-24-31 BUT NOT PALMITOYLATION OF CYSTEINE-30 AND CYSTEINE-45 ARE REQUIRED FOR MEMBRANE ANCHORING OF GLUTAMIC-ACID DECARBOXYLASE, GAD(65)

The smaller isoform of the GABA synthesizing enzyme glutamic acid deca rboxylase, GAD(65), is synthesized as a soluble protein that undergoes posttranslational modification(s) in the NH2-terminal region to becom e anchored to the membrane of small synaptic-like microvesicles in pan creatic beta cells, and synaptic vesicles in GABA-ergic neurons. A sol uble hydrophilic form, a soluble hydrophobic form, and a hydrophobic f irmly membrane-anchored form have been detected in beta cells. A rever sible and hydroxylamine sensitive palmitoylation has been shown to dis tinguish the firmly membrane-anchored form from the soluble yet hydrop hobic form, suggesting that palmitoylation of cysteines in the NH2-ter minal region is involved in membrane anchoring. In this study we use s ite-directed mutagenesis to identify the first two cysteines in the NH 2-terminal region, Cys 30 and Cys 45, as the sites of palmitoylation o f the GAD(65) molecule. Mutation of Cys 30 and Cys 45 to Ala results i n a loss of palmitoylation but does not significantly alter membrane a ssociation of GAD(65) in COS-7 cells. Deletion of the first 23 amino a cids at the NH2 terminus of the GAD(65)30/45A mutant also does not aff ect the hydrophobicity and membrane anchoring of the GAD(65) protein. However, deletion of an additional eight amino acids at the NH2 termin us results in a protein which is hydrophilic and cytosolic. The result s suggest that amino acids 24-31 are required for hydrophobic modifica tion and/or targeting of GAD(65) to membrane compartments, whereas pal mitoylation of Cys 30 and Cys 45 may rather serve to orient or fold th e protein at synaptic vesicle membranes.