Rapid inactivation of proteins by rapamycin-induced rerouting to mitochondria.
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.
Robinson
Margaret S
MS
University of Cambridge, Cambridge Institute for Medical Research, Cambridge CB2 0XY, UK. msr12@mole.bio.cam.ac.uk
Sahlender
Daniela A
DA
Foster
Samuel D
SD
D000818:Animals; D001483:Base Sequence; D001693:Biological Transport, Active / Q000187:drug effects; D002460:Cell Line; D022163:Clathrin-Coated Vesicles / Q000187:drug effects / Q000378:metabolism; D004705:Endocytosis; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D051379:Mice; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism*; D008954:Models, Biological; D046912:Multiprotein Complexes; D011506:Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology*; D022021:Tacrolimus Binding Proteins / Q000737:chemistry / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D050656:Transcription Factor AP-2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014168:Transferrin / Q000378:metabolism
D046912:Multiprotein Complexes; D011506:Proteins; D034741:RNA, Small Interfering; D017527:Receptor, IGF Type 2; D011994:Recombinant Proteins; D018808:Transcription Factor AP-1; D050656:Transcription Factor AP-2; D014168:Transferrin; D022021:Tacrolimus Binding Proteins; D020123:Sirolimus