Rapid Inactivation of Proteins by Knocksideways

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

The knocksideways system inactivates proteins by using a small molecule to trap them onto mitochondria. It is typically ?3 to 4 orders of magnitude faster than a knockdown. To get the best results out of a knocksideways, five parameters need to be optimized: the bait, the prey, the small molecule, the cell or organism, and the assay. Curr. Protoc. Cell Biol . 61:15.20.1?15.20.7. © 2013 by John Wiley & Sons, Inc.

Keywords: clathrin; AP?1; AP?2; rapamycin; AP21967

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Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1:

Materials

Cells (e.g., HeLa cells) expressing both bait and prey, depleted of the endogenous protein of interest, and growing in a suitable medium at the appropriate temperature
Control cells (i.e., not expressing bait and/or prey)
Small molecule dissolved in ethanol at 2× concentration: either 1 mM rapamycin (Sigma cat. no. R0395‐1MG) or 1 mM AP21967 (Clontech, cat. no. 635057)

Centrifuge
Timer

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Figures

Figure 15.20.1 Schematic diagram of the knocksideways system. The bait and prey constructs both have domains that bind to a small molecule. The bait localizes to the mitochondrial outer membrane; the prey is a protein of interest that cycles between the cytosol and a membrane or other subcellular structure. Addition of the small molecule causes the bait and prey to form heterodimers, thus trapping the prey onto mitochondria.

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Figure 15.20.2 Other Mitotrap‐like constructs. HeLa cells expressing an FKBP‐tagged AP‐2 subunit were transiently transfected with either an HA‐tagged construct consisting of the Tom70 mitochondrial outer membrane sorting signal, an FRB domain and an HA tag (i.e., similar to Mitotrap but without the YFP reporter) (A ), or a modified version of Mitotrap containing a single point mutation in the FRB domain (FRundefined) to enable binding to the rapamycin analog, AP21967 (B ). The cells were treated either with 200 nM rapamycin for 10 min (A) or with 5 µM AP21967 for 10 min, fixed, and labeled for AP‐2‐FKBP. The cells in (A) were double labeled for the HA tag; the cells in (B) were not double labeled because Mitotrap contains a YFP reporter. Scale bar: 20 µm.

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Figure 15.20.3 Knocksideways on Drosophila and mouse cells. (A ) Drosophila (Dmel) cells were stably transfected with Mitotrap and FKBP‐tagged (human) GGA2, incubated either without (A) or with (B) rapamycin for 10 min, and labeled for GGA2. (B ) Embryonic fibroblasts from a mouse strain with an FKBP domain knocked into its AP‐1 gamma gene ( AP1G1 ) were transiently transfected with Mitotrap, incubated with rapamycin for 10 min, and labeled for AP‐1 gamma. Scale bars: 10 µm.

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Literature Cited

Literature Cited
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