Abstract
Clarin-1 is the protein product encoded by the gene mutated in Usher syndrome III. Although the molecular function of clarin-1 is unknown, its primary structure predicts four transmembrane domains similar to a large family of membrane proteins that include tetraspanins. Here we investigated the role of clarin-1 by using heterologous expression and in vivo model systems. When expressed in HEK293 cells, clarin-1 localized to the plasma membrane and concentrated in low density compartments distinct from lipid rafts. Clarin-1 reorganized actin filament structures and induced lamellipodia. This actin-reorganizing function was absent in the modified protein encoded by the most prevalent North American Usher syndrome III mutation, the N48K form of clarin-1 deficient in N-linked glycosylation. Proteomics analyses revealed a number of clarin-1-interacting proteins involved in cell-cell adhesion, focal adhesions, cell migration, tight junctions, and regulation of the actin cytoskeleton. Consistent with the hypothesized role of clarin-1 in actin organization, F-actin-enriched stereocilia of auditory hair cells evidenced structural disorganization in Clrn1(-/-) mice. These observations suggest a possible role for clarin-1 in the regulation and homeostasis of actin filaments, and link clarin-1 to the interactive network of Usher syndrome gene products.
Highlights
We found that CLRN1 localized to the plasma membrane when expressed in Human embryonic kidney 293 (HEK293) cells
Because EZ-Link Sulfo-NHS-SS-Biotin does not penetrate the plasma membrane, these biochemical observations prove that CLRN1 localizes on the plasma membrane with its hydrophilic parts exposed to the extracellular side
To determine whether CLRN1 is enriched in specific membrane compartments, we investigated the densities of clarin-1-enriched microdomains by using sucrose density gradient centrifugation, and compared them to the density of membrane microdomains containing the transferrin receptor (Fig. 3A), a membrane protein typically excluded from lipid rafts [27]
Summary
Reagents—Methyl--cyclodextrin (MeCD), puromycin, Brij-58 (catalogue no.: P5884), Brij-96V (catalogue no.: 16011), Brij-98 (catalogue no.: P5641), and Igepal CA-630 (catalogue no.: I8896) were obtained from Sigma-Aldrich. HEK-CLRN cells were sonicated in sample buffer (320 mM sucrose in 50 mM Tris-HCl, pH 7.4), and centrifuged for 10 min at 1000 ϫ g and 4 °C; collected supernatants were centrifuged for 15 min at 22,000 ϫ g and 4 °C. Cells were incubated with Cy3-labeled 10B5 (mouse mAb anti-CLRN1) for 1 h. Affinity Purification of CLRN1—HEK-CLRN cells were lysed in buffer (150 mM NaCl, 50 mM Tris, pH 8.0, and Complete protease inhibitor mixture) containing 1% (v/v) Brij-98 at 4 °C for 1 h, and centrifuged at 22,000 ϫ g and 4 °C for 15 min. Identification of CLRN1 Interacting Proteins—Affinity-purified CLRN1-interacting proteins and proteins non- bound to the resin were separated by SDS-PAGE by using NuPAGE Novex 4 –12% Bis-Tris gel (Invitrogen) Both lanes were cut into five gel bands and subjected to in-gel protein digestion with trypsin as described previously [22]. Specimens from Clrn1Ϫ/Ϫ mice along with age-matched controls were studied by scanning electron microscopy
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