Small molecule induced oligomerization, clustering and clathrin-independent endocytosis of the dopamine transporter.

Tatiana Sorkina,Shiqi Ma,Mads Breum Larsen,Simon C Watkins,Alexander Sorkin

doi:10.7554/elife.32293

Abstract

Clathrin-independent endocytosis (CIE) mediates internalization of many transmembrane proteins but the mechanisms of cargo recruitment during CIE are poorly understood. We found that the cell-permeable furopyrimidine AIM-100 promotes dramatic oligomerization, clustering and CIE of human and mouse dopamine transporters (DAT), but not of their close homologues, norepinephrine and serotonin transporters. All effects of AIM-100 on DAT and the occupancy of substrate binding sites in the transporter were mutually exclusive, suggesting that AIM-100 may act by binding to DAT. Surprisingly, AIM-100-induced DAT endocytosis was independent of dynamin, cholesterol-rich microdomains and actin cytoskeleton, implying that a novel endocytic mechanism is involved. AIM-100 stimulated trafficking of internalized DAT was also unusual: DAT accumulated in early endosomes without significant recycling or degradation. We propose that AIM-100 augments DAT oligomerization through an allosteric mechanism associated with the DAT conformational state, and that oligomerization-triggered clustering leads to a coat-independent endocytosis and subsequent endosomal retention of DAT.

Highlights

The activity of plasma membrane receptors, transporters and channels is regulated by endocytosis and post-endocytic trafficking through the endolysosomal system
To gain insight into the mechanism of this novel endocytic regulation of dopamine transporters (DAT), we analyzed AIM-100-induced trafficking of DAT fused to yellow fluorescent protein (YFP) (YFP-DAT) (Sorkina et al, 2003) and YFP-DAT tagged with an extracellular hemagglutinin (HA) epitope (YFP-HA-DAT) (Sorkina et al, 2006) using quantitative fluorescence microscopy
Using the same method of sensitized fluorescence resonance energy transfer (FRET) measuments in cells co-expressing CFP-DAT and YFP-DAT, we found that the intensity of FRET from CFP to YFP was significantly higher in the plasma membrane clusters and intracellular vesicles in AIM-100-treated cells than this intensity in vehicle-treated cells where FRET was measured in various structures and regions of DAT accumulation, such as filopodia, ruffles and cell-cell contacts (Figure 3—figure supplement 1)

Summary

Introduction

The activity of plasma membrane receptors, transporters and channels is regulated by endocytosis and post-endocytic trafficking through the endolysosomal system. Transporters of the SLC6 family share the molecular fold and conformational transition mechanics during substrate transport, some of the trafficking mechanisms discovered for DAT have yet to be demonstrated for other SLC6 transporters (Kristensen et al, 2011; Matthies et al, 2010; Vuorenpaaet al., 2016b; Wu et al, 2015), suggesting there may be cargo-specific mechanisms controlling endocytic trafficking of these transporters. The present findings led to a hypothetical model whereby AIM-100 acts directly on DAT via an allosteric mechanism, and uncovered a novel CIE mechanism driven by the conformation-coupled cargo oligomerization

Results

Discussion

Materials and methods

Funding Funder National Institutes of Health