Abstract
We have developed a method for rapidly inactivating proteins with rapamycin-induced heterodimerization. Cells were stably transfected with siRNA-resistant, FKBP-tagged subunits of the adaptor protein (AP) complexes of clathrin-coated vesicles (CCVs), together with an FKBP and rapamycin-binding domain-containing construct with a mitochondrial targeting signal. Knocking down the endogenous subunit with siRNA, and then adding rapamycin, caused the APs to be rerouted to mitochondria within seconds. Rerouting AP-2 to mitochondria effectively abolished clathrin-mediated endocytosis of transferrin. In cells with rerouted AP-1, endocytosed cation-independent mannose 6-phosphate receptor (CIMPR) accumulated in a peripheral compartment, and isolated CCVs had reduced levels of CIMPR, but normal levels of the lysosomal hydrolase DNase II. Both observations support a role for AP-1 in retrograde trafficking. This type of approach, which we call a "knocksideways," should be widely applicable as a means of inactivating proteins with a time scale of seconds or minutes rather than days.
Copyright (c) 2010 Elsevier Inc. All rights reserved.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Base Sequence
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Biological Transport, Active / drug effects
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Cell Line
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Clathrin-Coated Vesicles / drug effects
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Clathrin-Coated Vesicles / metabolism
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Endocytosis
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HeLa Cells
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Humans
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Kinetics
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Mice
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Mitochondria / drug effects*
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Mitochondria / metabolism*
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Models, Biological
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Multiprotein Complexes
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Proteins / antagonists & inhibitors*
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Proteins / genetics
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Proteins / metabolism
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RNA, Small Interfering / genetics
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Receptor, IGF Type 2 / metabolism
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Recombinant Proteins / metabolism
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Sirolimus / pharmacology*
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Tacrolimus Binding Proteins / chemistry
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Tacrolimus Binding Proteins / metabolism
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Transcription Factor AP-1 / antagonists & inhibitors
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Transcription Factor AP-1 / genetics
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Transcription Factor AP-1 / metabolism
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Transcription Factor AP-2 / antagonists & inhibitors
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Transcription Factor AP-2 / genetics
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Transcription Factor AP-2 / metabolism
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Transfection
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Transferrin / metabolism
Substances
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Multiprotein Complexes
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Proteins
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RNA, Small Interfering
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Receptor, IGF Type 2
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Recombinant Proteins
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Transcription Factor AP-1
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Transcription Factor AP-2
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Transferrin
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Tacrolimus Binding Proteins
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Sirolimus