Abstract
Post-Golgi to apical surface delivery in polarized epithelial cells requires the cytoplasmic dynein motor complex. However, the nature of dynein-cargo interactions and their underlying regulation are largely unknown. Previous studies have shown that the apical surface targeting of rhodopsin requires the dynein light chain, Tctex-1, which binds directly to both dynein intermediate chain (IC) and rhodopsin. In this report, we show that the S82E mutant of Tctex-1, which mimics Tctex-1 phosphorylated at serine 82, has a reduced affinity for dynein IC but not for rhodopsin. Velocity sedimentation experiments further suggest that S82E is not incorporated into the dynein complex. The dominant-negative effect of S82E causes rhodopsin mislocalization in polarized Madin-Darby canine kidney (MDCK) cells. The S82A mutant, which mimics dephosphorylated Tctex-1, can be incorporated into dynein complex but is impaired in its release. Expression of S82A also causes disruption of the apical localization of rhodopsin in MDCK cells. Taken together, these results suggest that the dynein complex disassembles to release cargo due to the specific phosphorylation of Tctex-1 at the S82 residue and that this process is critical for the apical delivery of membrane cargoes.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Cadherins / genetics
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Cadherins / metabolism
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Cell Line
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Contactin 1
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Dogs
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Dyneins / metabolism*
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Humans
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Membrane Glycoproteins / genetics
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Membrane Glycoproteins / metabolism
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Membrane Proteins / genetics
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Membrane Proteins / metabolism
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Microtubule-Associated Proteins / genetics
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Microtubule-Associated Proteins / metabolism*
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Mutation / genetics
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Nerve Tissue Proteins / genetics
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Nerve Tissue Proteins / metabolism
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism*
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Phosphoproteins / genetics
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Phosphoproteins / metabolism
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Phosphorylation / drug effects
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Protein Binding
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Protein Transport / drug effects
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Protein Transport / physiology
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Qb-SNARE Proteins / genetics
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Qb-SNARE Proteins / metabolism
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Recombinant Fusion Proteins / metabolism
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Rhodopsin / genetics
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Rhodopsin / metabolism*
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Sodium-Potassium-Exchanging ATPase / genetics
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Sodium-Potassium-Exchanging ATPase / metabolism
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Tannins / pharmacology
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Tight Junctions / metabolism
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Transfection
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Two-Hybrid System Techniques
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Zonula Occludens-1 Protein
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t-Complex Genome Region
Substances
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Cadherins
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Contactin 1
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GOSR1 protein, human
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Membrane Glycoproteins
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Membrane Proteins
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Microtubule-Associated Proteins
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Nerve Tissue Proteins
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Nuclear Proteins
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Phosphoproteins
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Qb-SNARE Proteins
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Recombinant Fusion Proteins
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TJP1 protein, human
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Tannins
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Zonula Occludens-1 Protein
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Rhodopsin
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Dyneins
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Sodium-Potassium-Exchanging ATPase