Biochemical characterization of native usher protein complexes from a vesicular subfraction of tracheal epithelial cells

Marisa Zallocchi, Joseph H. Sisson, Dominic Cosgrove

Research output: Contribution to journalArticlepeer-review

12 Scopus citations


Usher syndrome is the major cause of deaf/blindness in the world. It is a genetic heterogeneous disorder, with nine genes already identified as causative for the disease. We noted expression of all known Usher proteins in bovine tracheal epithelial cells and exploited this system for large-scale biochemical analysis of Usher protein complexes. The dissected epithelia were homogenized in nondetergent buffer and sedimented on sucrose gradients. At least two complexes were evident after the first gradient: one formed by specific isoforms of CDH23, PCDH15, and VLGR-1 and a different one at the top of the gradient that included all of the Usher proteins and rab5, a transport vesicle marker. TEM analysis of these top fractions found them enriched in 100-200 nm vesicles, confirming a vesicular association of the Usher complex(es). Immunoisolation of these vesicles confirmed some of the associations already predicted and identified novel interactions. When the vesicles are lysed in the presence of phenylbutyrate, most of the Usher proteins cosediment into the gradient at a sedimentation coefficient of approximately 50 S, correlating with a predicted molecular mass of 2 × 106 Da. Although it is still unclear whether there is only one complex or several independent complexes that are trafficked within distinct vesicular pools, this work shows for the first time that native Usher protein complexes occur in vivo. This complex(es) is present primarily in transport vesicles at the apical pole of tracheal epithelial cells, predicting that Usher proteins may be directionally transported as complexes in hair cells and photoreceptors.

Original languageEnglish (US)
Pages (from-to)1236-1247
Number of pages12
Issue number6
StatePublished - Feb 16 2010

ASJC Scopus subject areas

  • Biochemistry


Dive into the research topics of 'Biochemical characterization of native usher protein complexes from a vesicular subfraction of tracheal epithelial cells'. Together they form a unique fingerprint.

Cite this