Rapid inactivation of proteins by rapamycin-induced rerouting to mitochondria

Dev Cell. 2010 Feb 16;18(2):324-31. doi: 10.1016/j.devcel.2009.12.015.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Biological Transport, Active / drug effects
  • Cell Line
  • Clathrin-Coated Vesicles / drug effects
  • Clathrin-Coated Vesicles / metabolism
  • Endocytosis
  • HeLa Cells
  • Humans
  • Kinetics
  • Mice
  • Mitochondria / drug effects*
  • Mitochondria / metabolism*
  • Models, Biological
  • Multiprotein Complexes
  • Proteins / antagonists & inhibitors*
  • Proteins / genetics
  • Proteins / metabolism
  • RNA, Small Interfering / genetics
  • Receptor, IGF Type 2 / metabolism
  • Recombinant Proteins / metabolism
  • Sirolimus / pharmacology*
  • Tacrolimus Binding Proteins / chemistry
  • Tacrolimus Binding Proteins / metabolism
  • Transcription Factor AP-1 / antagonists & inhibitors
  • Transcription Factor AP-1 / genetics
  • Transcription Factor AP-1 / metabolism
  • Transcription Factor AP-2 / antagonists & inhibitors
  • Transcription Factor AP-2 / genetics
  • Transcription Factor AP-2 / metabolism
  • Transfection
  • Transferrin / metabolism

Substances

  • Multiprotein Complexes
  • Proteins
  • RNA, Small Interfering
  • Receptor, IGF Type 2
  • Recombinant Proteins
  • Transcription Factor AP-1
  • Transcription Factor AP-2
  • Transferrin
  • Tacrolimus Binding Proteins
  • Sirolimus