Sinais de tráfego envolvidos no envolvidos no endereçamento do transportador vesicular de acetilcolina (VAChT)

Abstract

The vesicular acetylcholine transporter (VAChT) is the protein responsible for packaging cytoplasmic acetylcholine (ACh) into the synaptic vesicle of cholinergic neurons. The cytosolic C-terminal region of VAChT is important in intracellular trafficking events and targeting to secretory organelles. In this study, we evaluate the existence of trafficking signals in this region. Using confocal microscopy and GFP (green fluorescent protein) as a molecular label, the intracellular trafficking of several VAChT constructs was observed. Our results suggest that amino acids 471-490 contain important signals for endocytosis, trafficking, endoplasmic reticulum (ER) export and the targeting of VAChT to synaptic like microvesicles (SLMVs). In the region comprehended by residues 481-485 seems to exist a signal for endoplasmic reticulum export since the mutation of these amino acids retains the transporter in the ER. Mutation of the di-leucine motif (L485 L486) relocates the transporter to the soma plasma membrane. Protein-protein interaction experiments demonstrated that the C-terminal region of VAChT interacts with subunit of clathrin adaptor complex AP2 in a di-leucine motif dependent manner. To evaluate the role of clathrin in the trafficking and endocytosis of VAChT, we used the AP180-C construct. When AP180-C is overexpressed the clathrin-mediated endocytosis is blockade. These experiments demonstrated that clathrin mediated endocytosis is an essential event for VAChT trafficking. In addition, our results showed that the amino acids involved in the interaction with the clathrin endocytic machinery are present in the sequence between residues 471-490 of the C-terminal region of VAChT. In our next step, we tried to identify proteins that interact with VAChT and contribute in its trafficking and sorting to synaptic vesicles. For this reason, we performed a screening in a human brain cDNA library. The interaction between VAChT and SEC14like was detected in this system and was confirmed by co-immunoprecipitation. SEC14 is implicated in vesicle trafficking and in phosphatidylinositol and phosphatidylcholine transfer between membranes. Co-localization experiments suggest not only that VAChT and SEC14like co-localize but also that the subcellular distribution of VAChT appears to be altered in the SEC14 presence. Further studies must be performed to verify the implications of SEC14like overexpression on VAChT trafficking. Our results strongly suggest that: 1- there is an ER export signal between amino acids 481-485; 2- the di-leucine motif is responsible for VAChT interaction with AP2; 3- the clathrin dependent endocytosis has a fundamental role in endocytosis and trafficking of the transporter; and 4- the overexpression of SEC14like seems to interfere with VAChT trafficking.