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Fig. 8. The Rab5a(S34N) dominant negative mutant inhibits axonal trafficking of wt GAD65-GFP but does not affect trafficking of palmitoylation-deficient GAD65 or of the 24-60GAD65-GFP protein to axons and dendrites. Confocal images of hippocampal neurons 72 hours after transfection with plasmids encoding HA-tagged Rab5a(S34N) and either wt GAD65-GFP, palmitoylation-deficient GAD65-GFP, or a targeting mutant, 24-60GAD65-GFP, which targets to vesicles in both dendrites and axons but fails to reach presynaptic clusters. (A) Axonal trafficking and presynaptic clustering of wt GAD65-GFP is almost entirely abolished in the presence of Rab5a(S34N) and both proteins remain predominantly in the soma. (B) The Rab5a mutant does not abolish axonal or dendritic trafficking of palmitoylation-deficient GAD65, which is still present in rare axonal puncta (enlarged frame). (C) Axonal and dendritic targeting of the 24-60GAD65-GFP protein in the absence of Rab5a(S34N). (D) Axonal and dendritic targeting of the 24-60GAD65-GFP protein is not inhibited in the presence of Rab5a(S34N). Scale bars, 10 µm.

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