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Rapid inactivation of proteins by rapamycin-induced rerouting to mitochondria - PubMed

  • ️Fri Jan 01 2010

Rapid inactivation of proteins by rapamycin-induced rerouting to mitochondria

Margaret S Robinson et al. Dev Cell. 2010.

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.

Copyright (c) 2010 Elsevier Inc. All rights reserved.

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Graphical abstract
Figure 1
Figure 1

FKBP-Tagged AP Complexes (A and B) Schematic diagrams of the strategy that was used. (C–F) Stable cell lines expressing either α-FKBP (C and D) or γ-FKBP (E and F) were mixed with nontransfected cells and double labeled for the construct and either total α or endogenous γ (using a species-specific antibody). The insets show higher magnification views of the areas inside the boxes. Scale bar, 20 μm. (G and H) Homogenates of control HeLa cells and cell lines expressing either α-FKBP (G) or γ-FKBP (H) were depleted of the endogenous subunit, and western blots were probed with antibodies that recognize both the endogenous protein and the tagged construct. (I) Control and α-FKBP-expressing cells were depleted of endogenous α using siRNA, and endocytosis of prebound 125I-labeled transferrin (Tf) was measured. Data are presented as the means (±SE) from three independent experiments.

Figure 2
Figure 2

Localization of Constructs and Effect of Rapamycin (A) Cells expressing Mito-YFP-FRB were labeled with an antibody against a mitochondrial marker, MTC02. (B–E) Stable cell lines coexpressing either α-FKBP (B and C) or γ-FKBP (D and E) together with Mito-YFP-FRB were treated with 200 nM rapamycin for either 0 (B and D) or 10 (C and E) min. Electron micrographs of the same conditions are shown in Figure S1. (F and G) Live-cell imaging of cells coexpressing α-FKBP and Mito-YFP-FRB, and transiently transfected with mCherry-tagged σ2. See Movie S1 for the video link. Rapamycin was added to the cells at time 0 (F), which corresponds to 1 min in the video; the frames in (G) show the same cells 9 s later. The panels on the right of (F) and (G) are higher magnification views of the areas inside the boxes. (H) Localization of clathrin in cells with rerouted AP-2. Mixed populations of cells, expressing either α-FKBP only, or coexpressing α-FKBP and Mito-YFP-FRB, were depleted of endogenous α, treated with 200 nM rapamycin for 10 min, and triple labeled for clathrin. Other triple-labeled images are shown in Figure S2. Scale bars, 20 μm.

Figure 3
Figure 3

Assay for AP-2 Function Two different cell lines that stably express both α-FKBP and Mito-YFP-FRB were depleted of endogenous α and allowed to endocytose fluorescently labeled transferrin for 10 min, with or without first treating with rapamycin for 10 min. As a comparison, control cells, either left untreated or depleted of endogenous α, were also allowed to endocytose fluorescent transferrin for 10 min. After stripping off surface-bound transferrin, internalized transferrin was quantified by flow cytometry.

Figure 4
Figure 4

Assays for AP-1 Function (A and B) Cells coexpressing γ-FKBP and Mito-YFP-FRB were depleted of endogenous γ with siRNA, and incubated with anti-CIMPR for 45 min at 37°C, either without (A) or with (B) rapamycin. See also Figure S3. Scale bar, 20 μm. (C) Cells coexpressing γ-FKBP and Mito-YFP-FRB were depleted of endogenous γ with siRNA. Half of the cells were treated with rapamycin for 10 min before homogenization, then CCVs were isolated and western blots probed with the indicated antibodies. (D) Blots of whole-cell homogenates and CCV preparations from a conventional AP-1 γ knockdown, probed with the same antibodies as in (C).

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