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Flotillin-1 is essential for PKC-triggered endocytosis and membrane microdomain localization of DAT

Nature Neuroscience volume 14, pages 469–477 (2011)Cite this article

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A Corrigendum to this article was published on 23 November 2011

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Abstract

Plasmalemmal neurotransmitter transporters (NTTs) regulate the level of neurotransmitters, such as dopamine (DA) and glutamate, after their release at brain synapses. Stimuli including protein kinase C (PKC) activation can lead to the internalization of some NTTs and a reduction in neurotransmitter clearance capacity. We found that the protein Flotillin-1 (Flot1), also known as Reggie-2, was required for PKC-regulated internalization of members of two different NTT families, the DA transporter (DAT) and the glial glutamate transporter EAAT2, and we identified a conserved serine residue in Flot1 that is essential for transporter internalization. Further analysis revealed that Flot1 was also required to localize DAT within plasma membrane microdomains in stable cell lines, and was essential for amphetamine-induced reverse transport of DA in neurons but not for DA uptake. In sum, our findings provide evidence for a critical role of Flot1-enriched membrane microdomains in PKC-triggered DAT endocytosis and the actions of amphetamine.

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Figure 1: PKC triggers endocytosis of heterologously and endogenously expressed DAT.
Figure 2: Flot1 overexpression attenuates Gö6850-mediated inhibition of PKC-triggered endocytosis.
Figure 3: Flot1 is required for the PKC-triggered endocytosis of EM4-YFP-DAT and Hela-eGFP-EAAT2.
Figure 4: Flot1 must be palmitoylated on residue Cys34.
Figure 5: Flot1 is required for the membrane raft localization of DAT but not for transport of DA.
Figure 6: PKC-triggered internalization of DAT requires phosphorylation on Flot1 Ser315.
Figure 7: Flot1 is required for PKC-triggered internalization and AMPH-induced reverse transport of DA in primary DA neurons.

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  • 13 July 2011

    In the version of this article initially published, several images were mirror-reversed or switched during preparation of the artwork and one set of images was switched before analysis. In Figure 2c, left panel, +Gö row, +PMA and –PMA were mirror-reversed in both Surface and Total columns. In Figure 3b, top panel, siFlot1, Total and Surface images were switched and +PMA and –PMA were mirror-reversed within both. In Figure 5a, the TfR blot was mirror-reversed, and its quantification in Figure 5e, right panel (pink line) was also reversed. In Figure 5b, the Flot1 and TfR blots were switched. In Figure 6d, top panel, +PMA and –PMA were mirror-reversed in both rows of the Total column and in the S315A row of the Surface column. Finally, in Figure 4f, the IP and Lysate images in the IB DAT row of the IP Flot1 subpanel were switched before analysis and the P-value for the modulation by PMA of the interaction between wild-type Flot1 and DAT (0.0148) was incorrect. The correct value is 0.0920. All errors have been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank C. Karam, T. Melia, A. Pizzo, H. Stenmark and H. Yano for critical reading of this manuscript, and J. Rothstein (Johns Hopkins) for the Eaat2 cDNA. This work was supported by US National Institute of Neurological Disorders and Stroke research grant RO1 NS050199 and the Parkinson's Disease Foundation (A.Y.) and US National Institute on Drug Abuse research grants PO1 DA12408 (A.G. and J.A.J.), DA13975 (A.G.), K05 DA022413 (J.A.J.) and DA13147 (R.A.V.).

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  1. M Laura Cremona

    Present address: Present address: Department of Endocrinology, Columbia University, College of Physicians and Surgeons, New York, New York, USA.,

Authors and Affiliations

  1. Departments of Neurology, and Pathology and Cell Biology, Columbia University, College of Physicians and Surgeons, New York, New York, USA

    M Laura Cremona, Kelvin Pau, Monique Anderson & Ai Yamamoto

  2. Department of Molecular Physiology and Biophysics, Center for Molecular Neuroscience, Kennedy Center, Vanderbilt University, Nashville, Tennessee, USA

    Heinrich J G Matthies, Erica Bowton, Nicole Speed, Brandon J Lute, Sabrina D Robertson & Aurelio Galli

  3. Center for Molecular Recognition and Departments of Psychiatry and Pharmacology, Columbia University, College of Physicians and Surgeons, New York, New York, USA

    Namita Sen & Jonathan A Javitch

  4. University of North Dakota School of Medicine and Health Science, Grand Forks, North Dakota, USA

    Roxanne A Vaughan

  5. Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, USA

    James E Rothman

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Contributions

M.L.C., K.P., S.D.R., M.A. and A.Y. contributed toward and performed cell surface biotinylation experiments; M.L.C., H.J.G.M., K.P., M.A. and A.Y. contributed toward and performed image-based internalization experiments; M.L.C., N.S., J.A.J. and A.Y. contributed toward and performed DA and tyramine uptake assays; K.P., E.B., N.S., B.J.L. and A.G. contributed toward and performed electrophysiology experiments; R.A.V. contributed critical reagents and critical reading of manuscript; M.L.C., A.Y. and J.E.R. initiated the study and contributed toward and performed the original functional endocytic screen and membrane microdomain localization study; J.E.R. provided direction in endocytosis studies; A.G. and J.A.J. provided direction in DAT and DA studies; A.Y. and J.A.J. wrote the manuscript.

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Correspondence to Ai Yamamoto.

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Supplementary Figures 1–14, Supplementary Table 1 and Supplementary Statistical Analyses (PDF 18436 kb)

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Cremona, M., Matthies, H., Pau, K. et al. Flotillin-1 is essential for PKC-triggered endocytosis and membrane microdomain localization of DAT. Nat Neurosci 14, 469–477 (2011). https://doi.org/10.1038/nn.2781

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