Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 376, Issue 2, Pages 380-392Publisher
ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2007.09.001
Keywords
MP20; tetraspanin; tetraspanin-enriched microdomains; two-dimensional crystal; single particle electron microscopy
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Funding
- NEI NIH HHS [EY015107] Funding Source: Medline
- NIGMS NIH HHS [GM62580] Funding Source: Medline
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Members of the tetraspanin superfamily function as transmembrane scaffold proteins that mediate the assembly of membrane proteins into specific signaling complexes. Tetraspanins also interact with each other and concentrate membrane proteins into tetraspanin-enriched microdomains (TEMs). Here we report that lens-specific tetraspanin MP20 can form multiple types of higher-order assemblies and we present crystalline arrays of MP20. When isolated in the absence of divalent cations, MP20 is solubilized predominantly in tetrameric form, whereas the presence of divalent cations during solubilization promotes the association of MP20 tetramers into higher-order species. This effect only occurs when divalent cations are present during solubilization but not when divalent cations are added to solubilized tetrameric MP20, suggesting that other factors may also be involved. When purified MP20 tetramers are reconstituted with native lens lipids in the presence of magnesium, MP20 forms two-dimensional (2D) crystals. A projection map at 18 angstrom resolution calculated from negatively stained 2D crystals showed that the building block of the crystal is an octamer consisting of two tetramers related to each other by 2-fold symmetry. In addition to 21) crystals, reconstitution of MP20 with native lipids also produced a variety of large protein-lipid complexes, and we present three-dimensional (3D) reconstructions of the four most abundant of these complexes in negative stain. The various complexes formed by MP20 most likely reflect the many ways in which tetraspanins can interact with each other to allow formation of TEMs. (C) 2007 Published by Elsevier Ltd.
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