Intracellular Context Affects Levels of a Chemically Dependent Destabilizing Domain

Thomas J. Wandless,Maria Gasset,Ling-chun Chen,Emily L. Egeler,Mark A. Sellmyer,Rishi Rakhit

doi:10.1371/journal.pone.0043297

Abstract

The ability to regulate protein levels in live cells is crucial to understanding protein function. In the interest of advancing the tool set for protein perturbation, we developed a protein destabilizing domain (DD) that can confer its instability to a fused protein of interest. This destabilization and consequent degradation can be rescued in a reversible and dose-dependent manner with the addition of a small molecule that is specific for the DD, Shield-1. Proteins encounter different local protein quality control (QC) machinery when targeted to cellular compartments such as the mitochondrial matrix or endoplasmic reticulum (ER). These varied environments could have profound effects on the levels and regulation of the cytoplasmically derived DD. Here we show that DD fusions in the cytoplasm or nucleus can be efficiently degraded in mammalian cells; however, targeting fusions to the mitochondrial matrix or ER lumen leads to accumulation even in the absence of Shield-1. Additionally, we characterize the behavior of the DD with perturbants that modulate protein production, degradation, and local protein QC machinery. Chemical induction of the unfolded protein response in the ER results in decreased levels of an ER-targeted DD indicating the sensitivity of the DD to the degradation environment. These data reinforce that DD is an effective tool for protein perturbation, show that the local QC machinery affects levels of the DD, and suggest that the DD may be a useful probe for monitoring protein quality control machinery.

Highlights

Proteins are important for almost every cellular process
These data reinforce that destabilizing domain (DD) is an effective tool for protein perturbation, show that the local QC machinery affects levels of the DD, and suggest that the DD may be a useful probe for monitoring protein quality control machinery
To support the above findings and since it is difficult to quantify fluorescent proteins extracellularly, we investigated the effects of Shield-1, CHX, and MG132 on the flux of an endoplasmic reticulum (ER)-targeted DD fused to a luminescent reporter protein

Summary

Introduction

Proteins are important for almost every cellular process. a significant portion of modern biology is devoted to studying the production and interactions of proteins. We developed a small, inherently unstable protein domain based on the FK506- and rapamycinbinding protein (FKBP12), termed a destabilizing domain (DD) [2] This instability can be conferred to a genetically fused protein of interest, and the resulting fusion protein is rapidly degraded in the absence of stabilizing ligand. The addition of a specific small molecule ligand, Shield-1, can rescue the fusion protein from degradation in a rapid, dose-dependent, and reversible manner. This system has been widely applied in variety of cell types and organisms [3,4,5,6,7,8,9,10,11]

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Conclusion