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Retention of prominin in microvilli reveals distinct cholesterol-based lipid micro-domains in the apical plasma membrane

Nature Cell Biology volume 2, pages 582–592 (2000)Cite this article

Abstract

Membrane cholesterol–sphingolipid ‘rafts’, which are characterized by their insolubility in the non-ionic detergent Triton X-100 in the cold, have been implicated in the sorting of certain membrane proteins, such as placental alkaline phosphatase (PLAP), to the apical plasma membrane domain of epithelial cells. Here we show that prominin, an apically sorted pentaspan membrane protein, becomes associated in the trans-Golgi network with a lipid raft that is soluble in Triton X-100 but insoluble in another non-ionic detergent, Lubrol WX. At the cell surface, prominin remains insoluble in Lubrol WX and is selectively associated with microvilli, being largely segregated from the membrane subdomains containing PLAP. Cholesterol depletion results in the loss of prominin's microvillus-specific localization but does not lead to its complete intermixing with PLAP. We propose the coexistence within a membrane domain, such as the apical plasma membrane, of different cholesterol-based lipid rafts, which underlie the generation and maintenance of membrane subdomains.

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Figure 1: Prominin is soluble in Triton X-100 but insoluble in Lubrol WX.
Figure 2: Prominin's insolubility in Lubrol WX is not due to linkage to the actin cytoskeleton or other protein–protein interactions.
Figure 3: Prominin's insolubility in Lubrol WX reflects its association with detergent-resistant complexes.
Figure 4: Cholesterol depletion affects the Lubrol WX-resistant complexes containing prominin.
Figure 5: Clustering of either cholesterol or prominin increases the size of the Lubrol WX-insoluble, prominin-containing complexes.
Figure 6: Prominin becomes insoluble in Lubrol WX within the trans-Golgi/TGN, and the size of the detergent-resistant complexes increases upon exit from the TGN.
Figure 7: The characteristic microvillus-specific pattern of prominin's cell-surface distribution depends on cholesterol.
Figure 8: PLAP does not co-localize with prominin on microvilli and does not redistribute like prominin upon cholesterol depletion.
Figure 9: Formation of the Triton rafts and Lubrol rafts in the TGN and their transport to the planar and the microvillar subdomains of the apical plasma membrane, as revealed after cell lysis in Lubrol WX.

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Acknowledgements

We thank D. Brown for the PLAP-transfected MDCK cells, D. Louvard for the villin-antisense Caco2 cells, D. Buck for the antibody against human prominin (AC133 antigen), C. Thiele for [3H]photo-cholesterol, 10-azistearic acid, cholesterol–mβCD complex and delipidated sera, A. Hellwig for electron microscopy, and M.J. Hannah for help with confocal microscopy. K.R. was the recipient of a fellowship from the Studienstiftung des Deutschen Volkes. W.B.H. was supported by grants from the DFG (SFB 352, C1), the EC (ERB-FMRX-CT96-0023 and ERBBIO4CT960058), the German-Israeli Foundation for Scientific Research and Development, and the Fonds der Chemischen Industrie.

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  1. Katja Röper and Denis Corbeil: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Neurobiology, Interdisciplinary Center of Neuroscience, University of Heidelberg, Im Neuenheimer Feld 364, Heidelberg, D-69120

    Katja Röper, Denis Corbeil & Wieland B. Huttner

  2. Max-Planck-Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 110 , D-01307, Dresden, Germany

    Katja Röper, Denis Corbeil & Wieland B. Huttner

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Correspondence to Wieland B. Huttner.

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Röper, K., Corbeil, D. & Huttner, W. Retention of prominin in microvilli reveals distinct cholesterol-based lipid micro-domains in the apical plasma membrane. Nat Cell Biol 2, 582–592 (2000). https://doi.org/10.1038/35023524

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