Flotillins Regulate Membrane Mobility of the Dopamine Transporter but Are Not Required for Its Protein Kinase C Dependent Endocytosis

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