Caracterização das interações protéicas envolvidas no tráfego celular do transportador de colina de alta afinidade - CHT1

Abstract

The high affinity choline transporter (CHT1) is responsible for choline uptake, which is derived from acetylcholine degradation, in the synaptic cleft of cholinergic neurons. CHT1 activity is very important since cholinergic neurons have very low ability to synthesize acetylcholine de novo. Although CHT1 is mainly a plasma membrane transporter, it is found predominantly recycling between intracellular vesicles and the cell surface. Cellular trafficking seems to play an important role in the transporter activity. In the present experiments, we identified proteins that might interact with CHT1, using yeast two hybrid system. We found that CHT1 interacts with BRI3, Flotillin, SEC14like and MAP1A. These proteins participate in cellular trafficking. To further characterize these interactions we chose SEC14like, a protein that is homologous to yeast SEC14, which is essential in promoting protein transport through the yeast Golgi complex. SEC14like also plays a crucial role in phosphatidylinositol and phosphatidylcholine synthesis and metabolism. SEC14like function in humans is poorly understood. We used co-immunoprecipitation, confocal microscopy and choline uptake to further identify the importance of CHT1-SEC14like interaction. Co-immunoprecipitation assays showed that the two proteins form complexes in mammal cells. Using confocal microscopy, we found that the pattern of distribution of CHT1 was altered in cells that overexpressed SEC14like. Choline uptake analysis suggested that SEC14like overexpression modified transporter activity, decreasing 30% the rate of choline transport. All these data suggest a new possibility of regulation of cholinergic activity by control of the cellular trafficking of CHT1.