DEVCEL
1838
S1534-5807(10)00013-4
10.1016/j.devcel.2009.12.015
Elsevier Inc.
Resource
Rapid Inactivation of Proteins by Rapamycin-Induced Rerouting to Mitochondria
Margaret S.
Robinson
1
∗
msr12@mole.bio.cam.ac.uk
Daniela A.
Sahlender
1
Samuel D.
Foster
1
1
University of Cambridge, Cambridge Institute for Medical Research, Cambridge CB2 0XY, UK
∗
Corresponding author
Published: February 15, 2010
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.
Graphical Abstract
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
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10.1016/j.devcel.2009.12.015
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On the cover: The cover image of Arabidopsis thaliana in flower was processed using Photoshop's glowing edges filter (credit: Alexander Simpson). In this issue, a regulator of flowering time, FPA, is shown to control RNA cleavage and alternative polyadenylation. This function affects antisense RNAs at the FLC locus; at other sites, defective RNA 3′ end formation in the absence of FPA can result in readthrough transcription of silenced loci. For details, see Hornyik et al., pp. 203–213, and the Preview by Rosonina and Manley, pp. 172–174.
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