Abstract
The phosphodiesterase 6 delta subunit (PDE6δ) shuttles several farnesylated cargos between membranes. The cargo sorting mechanism between cilia and other compartments is not understood. Here we show using the inositol polyphosphate 5'-phosphatase E (INPP5E) and the GTP-binding protein (Rheb) that cargo sorting depends on the affinity towards PDE6δ and the specificity of cargo release. High-affinity cargo is exclusively released by the ciliary transport regulator Arl3, while low-affinity cargo is released by Arl3 and its non-ciliary homologue Arl2. Structures of PDE6δ/cargo complexes reveal the molecular basis of the sorting signal which depends on the residues at the -1 and -3 positions relative to farnesylated cysteine. Structure-guided mutation allows the generation of a low-affinity INPP5E mutant which loses exclusive ciliary localization. We postulate that the affinity to PDE6δ and the release by Arl2/3 in addition to a retention signal are the determinants for cargo sorting and enrichment at its destination.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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ADP-Ribosylation Factors / chemistry
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ADP-Ribosylation Factors / metabolism
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Animals
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Cell Line
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Cilia / metabolism*
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Cyclic Nucleotide Phosphodiesterases, Type 6 / metabolism*
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Fluorescence Polarization
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Green Fluorescent Proteins / metabolism
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Guanosine Triphosphate / metabolism
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Inositol Polyphosphate 5-Phosphatases
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Kinetics
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Mice
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Models, Biological
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Monomeric GTP-Binding Proteins / metabolism
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Mutant Proteins / metabolism
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Neuropeptides / metabolism
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Phosphoric Monoester Hydrolases / metabolism*
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Protein Binding
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Protein Prenylation
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Protein Sorting Signals
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Protein Structure, Secondary
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Protein Transport
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Ras Homolog Enriched in Brain Protein
Substances
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Mutant Proteins
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Neuropeptides
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Protein Sorting Signals
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Ras Homolog Enriched in Brain Protein
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Rheb protein, mouse
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Green Fluorescent Proteins
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Guanosine Triphosphate
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Phosphoric Monoester Hydrolases
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Inositol Polyphosphate 5-Phosphatases
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Cyclic Nucleotide Phosphodiesterases, Type 6
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Arl3 protein, mouse
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ADP-Ribosylation Factors
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Monomeric GTP-Binding Proteins