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First published online 23 March 2004
doi: 10.1242/jcs.01030

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Palmitoylation controls trafficking of GAD65 from Golgi membranes to axon-specific endosomes and a Rab5a-dependent pathway to presynaptic clusters

¹ Departments of Medicine, Microbiology/Immunology, and Diabetes Center, University of California San Francisco, CA 94143-0534, USA
² Department of Physiology, University of California San Francisco, CA 94143-0444, USA

View larger version (40K): [in a new window]   Fig. 1. Palmitoylated wt GAD65-GFP colocalizes with wt HA-Rab5a in Golgi membranes and in axon termini but not in somatodendritic puncta. (A) Confocal images of a hippocampal neuron cotransfected with plasmids encoding wt GAD65-GFP and HA-tagged wt Rab5a. Neurons were double immunolabeled for GFP and HA. GAD65-GFP colocalizes with wt Rab5a in axonal puncta (arrowheads, enlarged frame a) and in Golgi membranes (enlarged frame b) but not in dendritic puncta (enlarged frame c). Scale bars, 10 µm. (B) The percentage of HA-Rab5a(wt)-positive puncta expressing wt GAD65-GFP was analyzed as described in Materials and Methods. The yellow color indicates the percentage of HA-Rab5a(wt)-positive puncta expressing wt GAD65-GFP.

View larger version (59K): [in a new window]   Fig. 2. Palmitoylation-deficient GAD65 colocalizes with Rab5a in Golgi membranes but not in axonal or dendritic puncta. (A) Confocal images of a hippocampal neuron co-transfected with plasmids encoding palmitoylation-deficient GAD65-GFP and HA-tagged wt Rab5a. Neurons were triple immunolabeled for GFP, HA, and the Golgi marker GM130. Arrowheads, axon; arrows, dendrites; scale bars, 10 µm. Palmitoylation-deficient GAD65(C30,45A)-GFP colocalizes with wt Rab5a in Golgi membranes immunostained for GM130 (enlarged frame a, bottom frame in the middle shows overlay of the three colors, frames to the right show overlay of two colors). GAD65(C30,45A) is mostly diffuse in axons and dendrites and does not colocalize with wt Rab5a in axonal (arrowheads) or dendritic (arrows) puncta (enlarged frame b). Frame c shows a view of a distal axon and reveals that the rare axonal puncta containing palmitoylation-deficient GAD65 are devoid of HA-Rab5a. (B) The percentage of HA-Rab5a(wt)-positive puncta expressing palmitoylation-deficient GAD65-GFP was analyzed as described in Materials and Methods. The yellow color indicates the percentage of HA-Rab5a(wt)-positive puncta expressing GAD65(C30,45A)-GFP.

View larger version (83K): [in a new window]   Fig. 3. EEA1-positive somatodendritic early endosomes are devoid of GAD65. Confocal images of hippocampal neurons expressing either GFP-tagged wt Rab5a or GAD65-GFP and immunolabeled for GFP and endogenous EEA1. (A) Wt Rab5a colocalizes with EEA1 in early endosomes in the soma and proximal dendrites (enlarged frame). (B) Enlarged view of a distal axon (arrowheads) and distal dendrites (arrows) of the neuron shown in A, reveals colocalization of Rab5a and EEA1 in dendritic puncta and lack of EEA1 in axonal puncta containing Rab5a. (C) GAD65 is absent from somatodendritic early endosomes containing EEA1. (D) EEA1 is absent from presynaptic clusters (arrowheads) containing GAD65-GFP. Scale bars, 10 µm.

View larger version (24K): [in a new window]   Fig. 4. Lack of colocalization of the GTP-bound Rab5a(Q79L) mutant with either wt or palmitoylation-deficient GAD65 in the soma of primary neurons. Confocal images of the soma of hippocampal neurons co-transfected with plasmids encoding either wt (A) or palmitoylation-deficient GAD65-GFP (B) together with HA-tagged constitutively active mutant of Rab5a(Q79L). Neurons were triple immunolabeled for GFP, HA, and the Golgi marker GM130. (A,B) Neither Wt GAD65-GFP nor palmitoylation-deficient GAD65-GFP are detected in the giant endosomes in the soma containing Rab5a(Q79L). Both Wt GAD65-GFP and palmitoylation-deficient GAD65-GFP are localized in the Golgi compartment, immunostained for GM130 but devoid of Rab5a(Q79L). Scale bar, 10 µm.

View larger version (50K): [in a new window]   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.

View larger version (24K): [in a new window]   Fig. 6. The Golgi localization mutant, 24-60GAD65-GFP, is excluded from Rab5a(Q79L)-positive/EEA1-negative axonal endosomes. Confocal images of hippocampal neurons co-transfected with plasmids encoding HA-Rab5a(Q79L) and the 24-60GAD65-GFP mutant, which targets to vesicles in both dendrites and axons but fails to reach presynaptic clusters. Neurons were double immunolabeled for GFP and HA. The 24-60GAD65-GFP mutant, which is targeted to membranes distinct from Golgi, is absent from giant vesicles containing Rab5a(Q79L) in soma, dendrites (arrow) and axons (arrowhead). Scale bar, 10 µm.

View larger version (46K): [in a new window]   Fig. 7. Accumulation of wt but not palmitoylation-deficient GAD65 in neurite tips in the presence of the Rab5(S34N) mutant. Confocal images of hippocampal neurons 24 hours after transfection with plasmids encoding HA-tagged Rab5a(S34N) (A-D) and wt GAD65-GFP (A,B) or palmitoylation-deficient GAD65-GFP (C,D) and double immunolabeled for GFP and HA. Enlarged frames show (from left to right) immunolabeling for GFP, immunolabeling for HA, and overlay, respectively. (A) Wt GAD65 accumulates with Rab5a(S34N) in the tip of dendrites. (B) View of the distal axon of the neuron shown in A reveals accumulation of wt GAD65 together with Rab5a(S34N) at the tips of the branched axon. (C) Palmitoylation-deficient GAD65 does no accumulate with Rab5a(S34N) at the tip of dendrites. (D) View of the distal axon of the neuron shown in C reveals the absence of palmitoylation-deficient GAD65 in accumulates of Rab5a(S34N) at the tip of axons. Scale bars, 10 µm.

View larger version (72K): [in a new window]   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.

View larger version (83K): [in a new window]   Fig. 9. The Rab5a(S34N) dominant negative mutant inhibits axonal trafficking and presynaptic clustering of VGAT. Confocal images of hippocampal neurons transfected with plasmids encoding VGAT-HA and GFP-tagged Rab5a(S34N). Neurons were double immunolabeled for HA and GFP. (A) A view of the soma reveals that VGAT-HA colocalizes with GFP-Rab5a(S34N) in the Golgi compartment. (B) A view of a neuron expressing VGAT-HA and GFP-Rab5a(S34N). The red color was enhanced to show the axon. VGAT shows a weak diffuse staining in axons and is not detected in presynaptic clusters in the proximal (enlarged frame a) or in the distal axon (enlarged frame b). Scale bars, 10 µm.

View larger version (81K): [in a new window]   Fig. 10. Coexpression of TfR with wt and mutant Rab5a. Confocal images of hippocampal neurons expressing TfR-GFP with or without wt and mutant HA-Rab5a for 72 (A-D) or 24 hours (E-F). Neurons were double immunolabeled for GFP and either HA or endogenous EEA1. (A) A view of a neuron expressing TfR-GFP in somatodendritic endosomes where it colocalizes with endogenous EEA1. High magnification of proximal dendritic puncta (enlarged frame a), however, reveals segregation of TfR-GFP and EEA1 in tubular structures. A view of a distal dendrite (enlarged frame b) reveals puncta that contain TfR-GFP but are devoid of EEA1. (B) TfR-GFP colocalizes with HA-Rab5a(Q79L) in giant endosomal vesicles in the soma (enlarged frame) and in dendrites (arrows) but not in axons (arrowheads). (C) In 72-hour coexpression experiments with Rab5(S34N), TfR traffics to dendritic puncta and its trafficking is similar to conditions without the mutant as shown in D. (E) In 24-hour coexpression experiments, however, with Rab5a(S34N), TfR accumulates in the tips of dendrites (enlarged frames a,b) but not axons (enlarged frame c) and is largely absent from dendritic puncta. (F) For comparison, a view of a neuron coexpressing TfR and wt Rab5a for 24 hours shows trafficking to dendritic puncta. Scale bars, 10 µm.

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