Developmental Cell
Volume 18, Issue 2, 16 February 2010, Pages 324-331
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Rapid Inactivation of Proteins by Rapamycin-Induced Rerouting to Mitochondria

https://doi.org/10.1016/j.devcel.2009.12.015Get rights and content
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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.

Highlights

► We have developed a way of getting rid of proteins quickly (i.e., minutes not days) ► This approach provides new insights into the function of the coat protein AP-1

CELLBIO
PROTEINS

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