Trafficking DAT

Sorkin et al. set out to track endocytosis of the dopamine transporter (DAT) by creating a mutant transporter with a hemagglutinin (HA) epitope-tag in the large extracellular loop. The authors then followed uptake with an HA antibody in a so-called antibody feeding assay. Although DAT undergoes constitutive and PKC-dependent cycling via clathrincoated pits, it lacks conventional internalization sequences. The authors tested whether ubiquitination of DAT could serve as an internalization signal. They screened a library of small interfering RNAs (siRNAs) against endocytic proteins in HeLa cells stably expressing YPF-HA-DAT. Not surprisingly, clathrin heavy chain and dynamin emerged from the screen as molecules critical for endocytosis. Knockdown of the ubiquitin ligase Nedd4-2 (neural precursor cell expressed, developmentally downregulated 4-2) also reduced endocytosis, consistent with the idea that PKC-induced DAT ubiquitination by Nedd4-2 accelerates endocytosis.

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Tatiana Sorkina, Manuel Miranda, Kalen R. Dionne, Brian R. Hoover, Nancy R. Zahniser, and Alexander Sorkin

Source: News tips from the Journal of Neuroscience

Contact: Sara Harris
Society for Neuroscience
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Trafficking DAT