Fig. 5. Palmitoylation results in targeting of GAD65 to Rab5a(Q79L)-positive/EEA1-negative axonal endosomes. Confocal images of hippocampal neurons co-transfected with plasmids encoding either wt (A-C) or palmitoylation-deficient GAD65-GFP (D) together with HA-tagged constitutively active mutant of Rab5a(Q79L). Neurons were double immunolabeled for GFP, and either HA or endogenous EEA1. (A) wt GAD65-GFP colocalizes with Rab5a(Q79L) in giant axonal vesicles (arrowheads) lining up the axon starting at the base. (B) Enlarged view of more distal axonal vesicles of the neuron shown in A shows the presence of wt GAD65 in axonal endosomes containing HA-Rab5a(Q79L). (C) EEA1, is present in giant endosomal vesicles in the cell body but absent from giant axonal vesicles containing wt GAD65. In the presence of Rab5a(Q79L), EEA1 remains confined to vesicles in the cell body, in contrast to the situation in a non-transfected cell (to the right) where it is also in dendrites. (D) A view of the most distal part of an axon expressing palmitoylation-deficient GAD65 (arrowheads). Axonal puncta containing the palmitoylation-deficient protein do not colocalize with axonal endosomes containing HA-Rab5a(Q79L). (E) The percentage of HA-Rab5a(Q79L)-positive puncta expressing wt GAD65-GFP or palmitoylation-deficient GAD65-GFP was quantified as described in Materials and Methods. The yellow color indicates the percentage of HA-Rab5a(Q79L)-positive puncta expressing wt GAD65-GFP or GAD65(C30,45A)-GFP, respectively. Scale bars, 10 µm.