Abstract
The GABA-synthesizing enzyme GAD65 is synthesized as a soluble cytosolic protein but undergoes post-translational modification(s) to become anchored to the cytosolic face of Golgi membranes before targeting to synaptic vesicle membranes in neuroendocrine cells. Palmitoylation of cysteines 30 and 45 in GAD65 is not required for targeting to Golgi membranes but is crucial for post-Golgi trafficking to presynaptic clusters in neurons. Here, we show that palmitoylated GAD65 colocalizes with the small GTP-binding protein Rab5a in Golgi membranes and in axons but not in dendrites. In the presence of the constitutively positive mutant Rab5(Q79L) palmitoylation resulted in polarized targeting of GAD65 to giant Rab5a-positive axonal endosomes, characterized by the absence of the Rab5a-effector molecule EEA1 and the transferrin receptor. By contrast, Rab5a-positive/EEA1-positive somatodendritic giant endosomes containing the transferrin receptor were devoid of GAD65. Palmitoylation-deficient GAD65 was excluded from endosomal compartments. A dominant negative mutant of Rab5a, Rab5a(S34N), specifically blocked axonal trafficking and presynaptic clustering of palmitoylated GAD65, but did not affect axonal trafficking of mutants of GAD65 that fail to traffic to giant axonal endosomes containing Rab5a(Q79L). Two transmembrane synaptic vesicle proteins, VAMP2 and VGAT also localized to the axonal giant endosomes, and their axonal trafficking and presynaptic clustering was blocked by Rab5a(S34N). The results suggest that palmitoylation of GAD65 regulates the trafficking of the protein from Golgi membranes to an endosomal trafficking pathway in axons that is dependent on Rab5a and is required for the targeting of several synaptic vesicle proteins to presynaptic clusters.
Highlights
The sorting of newly synthesized membrane proteins to distinct domains in polarized cells appears to begin in the Golgi/trans Golgi network (TGN), where proteins can segregate and exit in separate transport vesicles (Keller et al, 2001)
Wt GAD65 colocalizes with Rab5a in axons but not in dendrites and palmitoylation is required for this colocalization
We addressed the question whether endogenous GAD65 colocalizes with green fluorescent protein (GFP)-Rab5a(wt)
Summary
The sorting of newly synthesized membrane proteins to distinct domains in polarized cells appears to begin in the Golgi/trans Golgi network (TGN), where proteins can segregate and exit in separate transport vesicles (Keller et al, 2001). While targeting of proteins to apical vs basolateral surfaces in epithelial cells sometimes occurs directly from the TGN, the trafficking pathways are often indirect They can involve additional sorting steps at the plasma membrane and/or in early endosomes and transcytosis from one domain to another before a final destination is reached (reviewed in Keller and Simons, 1997). It was suggested that basolateral and apical domains of epithelial cells correspond to the somatodendritic and axonal surfaces of neurons, respectively (Dotti and Simons, 1990). The pathways involved in targeting of proteins to presynaptic termini in axons remain obscure It is unclear whether axonal proteins are sorted in axonally directed transport vesicles directly at the TGN or whether they undergo additional sorting steps. Neurons appear to utilize a variety of membrane transport pathways to achieve the precise localization of newly synthesized proteins in axons
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