Abstract
The adaptor complex 3 (AP-3) targets membrane proteins from endosomes to lysosomes, lysosome-related organelles and synaptic vesicles. Phosphatidylinositol-4-kinase type II alpha (PI4KIIalpha) is one of several proteins possessing catalytic domains that regulate AP-3-dependent sorting. Here we present evidence that PI4KIIalpha uniquely behaves both as a membrane protein cargo as well as an enzymatic regulator of adaptor function. In fact, AP-3 and PI4KIIalpha form a complex that requires a dileucine-sorting motif present in PI4KIIalpha. Mutagenesis of either the PI4KIIalpha-sorting motif or its kinase-active site indicates that both are necessary to interact with AP-3 and properly localize PI4KIIalpha to LAMP-1-positive endosomes. Similarly, both the kinase activity and the sorting signal present in PI4KIIalpha are necessary to rescue endosomal PI4KIIalpha siRNA-induced mutant phenotypes. We propose a mechanism whereby adaptors use canonical sorting motifs to selectively recruit a regulatory enzymatic activity to restricted membrane domains.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Amino Acid Motifs
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Animals
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Base Sequence
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Binding Sites / genetics
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Catalytic Domain / genetics
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Cell Line
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DNA-Binding Proteins / chemistry
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism*
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Endosomes / metabolism*
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Humans
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Lysosomal Membrane Proteins / metabolism
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Minor Histocompatibility Antigens
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Multiprotein Complexes
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Mutagenesis, Site-Directed
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PC12 Cells
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Phosphotransferases (Alcohol Group Acceptor) / antagonists & inhibitors
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Phosphotransferases (Alcohol Group Acceptor) / chemistry*
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Phosphotransferases (Alcohol Group Acceptor) / genetics
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Phosphotransferases (Alcohol Group Acceptor) / metabolism*
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Protein Structure, Tertiary
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RNA, Small Interfering / genetics
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Rats
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Transcription Factors / chemistry
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Transcription Factors / genetics
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Transcription Factors / metabolism*
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Transfection
Substances
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DNA-Binding Proteins
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LAMP1 protein, human
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Lysosomal Membrane Proteins
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Minor Histocompatibility Antigens
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Multiprotein Complexes
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RNA, Small Interfering
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Recombinant Fusion Proteins
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Transcription Factors
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enhancer-binding protein AP-3
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Phosphotransferases (Alcohol Group Acceptor)
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phosphatidylinositol phosphate 4-kinase