Rapid Inactivation of Proteins by Rapamycin-Induced Rerouting to Mitochondria

Summary 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 year

  2010

• Venue

  Developmental Cell

• Publication date

  2010-02-16

• Fields of study

  Biology, Medicine

• Identifiers
  DOI 10.1016/j.devcel.2009.12.015PMID 20159602PMCID 2845799
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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