Automated Quantification of the Subcellular Localization of Multicompartment Proteins via Q-SCAn

• Type of Material: Article
• Authors: Nicholas C. Bauer, Emory University; Anita Corbett, Emory University; Paul Doetsch, Emory University
• Language: English
• Date: 2013-12-01
• Publisher: Wiley: 12 months
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2013 John Wiley & Sons A/S.
• Final Published Version (URL): http://dx.doi.org/10.1111/tra.12118
• Title of Journal or Parent Work: Traffic
• ISSN: 1398-9219
• Volume: 14
• Issue: 12
• Start Page: 1200
• End Page: 1208
• Grant/Funding Information: This work was supported by the National Institutes of Health [grant numbers P01ES011163 to P.W.D., F31CA168272 to N.C.B.].; We would like to thank the Emory Custom Cloning Core Facility and the Emory University Integrated Cellular Imaging Core of the Winship Cancer Institute (National Cancer Institute Cancer Center Support Grant P30CA138292), especially Debbie Martinson and Dr. Adam Marcus.
• Keywords: FLUORESCENT PROTEINS; fluorescence; Cell Biology; Life Sciences & Biomedicine; image analysis; automated; Saccharomyces cerevisiae; BUDDING YEAST; mitochondria; localization; URACIL-DNA-GLYCOSYLASE; NUCLEAR; quantification; SACCHAROMYCES-CEREVISIAE; SHUTTLE VECTORS; Science & Technology; IMAGE-ANALYSIS; TRANSCRIPTION FACTOR; ANALYSIS SOFTWARE; SORTING SIGNALS; compartmentalization; nuclei
• Research Categories: Chemistry, Biochemistry; Health Sciences, Oncology

Abstract

In eukaryotic cells, proteins can occupy multiple intracellular compartments and move between compartments to fulfill critical biological functions. Unfortunately, no methods have been developed to robustly measure the distribution of a protein among compartments. To address this need, we have developed an automated method termed quantitative subcellular compartmentalization analysis (Q-SCAn). Q-SCAn is a quantitative analytical tool for providing broader and more detailed analysis of the localization of multicompartment proteins as compared to the currently available approaches. In eukaryotic cells, proteins can occupy multiple intracellular compartments and even move between compartments to fulfill critical biological functions or respond to cellular signals. Examples include transcription factors that reside in the cytoplasm but are mobilized to the nucleus as well as dual-purpose DNA repair proteins that are charged with simultaneously maintaining the integrity of both the nuclear and mitochondrial genomes. While numerous methods exist to study protein localization and dynamics, automated methods to quantify the relative amounts of proteins that occupy multiple subcellular compartments have not been extensively developed. To address this need, we present a rapid, automated method termed quantitative subcellular compartmentalization analysis (Q-SCAn). To develop this method, we exploited the facile molecular biology of the budding yeast, Saccharomyces cerevisiae. Individual subcellular compartments are defined by a fluorescent marker protein and the intensity of a target GFP-tagged protein is then quantified within each compartment. To validate Q-SCAn, we analyzed relocalization of the transcription factor Yap1 following oxidative stress and then extended the approach to multicompartment localization by examining two DNA repair proteins critical for the base excision repair pathway, Ntg1 and Ung1. Our findings demonstrate the utility of Q-SCAn for quantitative analysis of the subcellular distribution of multicompartment proteins.