Abstract
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 x 10(6) 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.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Animals
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Cadherin Related Proteins
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Cadherins / chemistry
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Cadherins / genetics
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Carrier Proteins / chemistry
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Carrier Proteins / genetics
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Cattle
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Cell Cycle Proteins
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Cell Fractionation / methods
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Cytoskeletal Proteins
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Extracellular Matrix Proteins / chemistry
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Extracellular Matrix Proteins / genetics
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Humans
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Membrane Proteins / chemistry
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Membrane Proteins / genetics
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Mice
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Multiprotein Complexes / biosynthesis
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Multiprotein Complexes / chemistry*
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Multiprotein Complexes / genetics
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Nerve Tissue Proteins / chemistry
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Nerve Tissue Proteins / genetics
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Protein Isoforms / biosynthesis
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Protein Isoforms / chemistry
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Protein Isoforms / genetics
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Protein Precursors / chemistry
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Protein Precursors / genetics
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Protein Structure, Tertiary / genetics
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Protein Transport / genetics
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Receptors, G-Protein-Coupled / chemistry
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Receptors, G-Protein-Coupled / genetics
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Respiratory Mucosa / chemistry*
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Respiratory Mucosa / cytology*
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Respiratory Mucosa / ultrastructure
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Trachea / chemistry*
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Trachea / cytology*
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Trachea / ultrastructure
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Transport Vesicles / chemistry*
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Transport Vesicles / genetics
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Transport Vesicles / ultrastructure
Substances
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CDHR15 protein, mouse
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Cadherin Related Proteins
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Cadherins
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Carrier Proteins
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Cdh23 protein, mouse
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Cell Cycle Proteins
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Clrn1 protein, mouse
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Cytoskeletal Proteins
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Extracellular Matrix Proteins
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Mass1 protein, mouse
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Membrane Proteins
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Multiprotein Complexes
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Nerve Tissue Proteins
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Protein Isoforms
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Protein Precursors
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Receptors, G-Protein-Coupled
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Recombinant Proteins
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Sans protein, mouse
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Ush1c protein, mouse
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Ush2a protein, mouse