Flotillins regulate membrane mobility of the dopamine transporter but are not required for its protein kinase C dependent endocytosis.

Flotillins were proposed to mediate clathrin-independent endocytosis, and recently, flotillin-1 was implicated in the protein kinase C (PKC)-triggered endocytosis of the dopamine transporter (DAT). Since endocytosis of DAT was previously shown to be clathrin-mediated, we re-examined the role of clathrin coat proteins and flotillin in DAT endocytosis using DAT tagged with the hemagglutinin epitope (HA) in the extracellular loop and a quantitative HA antibody uptake assay. Depletion of flotillin-1, flotillin-2 or both flotillins together by small interfering RNAs (siRNAs) did not inhibit PKC-dependent internalization and degradation of HA-DAT. In contrast, siRNAs to clathrin heavy chain and μ2 subunit of clathrin adaptor complex AP-2 as well as a dynamin inhibitor Dyngo-4A significantly decreased PKC-dependent endocytosis of HA-DAT. Similarly, endocytosis and degradation of DAT that is not epitope-tagged were highly sensitive to the clathrin siRNAs and dynamin inhibition but were not affected by flotillin knockdown. Very little co-localization of DAT with flotillins was observed in cells ectopically expressing DAT and in cultured mouse dopaminergic neurons. Depletion of flotillins increased diffusion rates of HA-DAT in the plasma membrane, suggesting that flotillin-organized microdomains may regulate the lateral mobility of DAT. We propose that clathrin-mediated endocytosis is the major pathway of PKC-dependent internalization of DAT, and that flotillins may modulate functional association of DAT with plasma membrane rafts rather than mediate DAT endocytosis.