Live-cell monitoring of protein localization to membrane rafts using protein-fragment complementation

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Merezhko , M , Pakarinen , E , Uronen , R-L & Huttunen , H J 2020 , ' Live-cell monitoring of protein localization to membrane rafts using protein-fragment complementation ' , Bioscience Reports , vol. 40 , no. 1 , 20191290 . https://doi.org/10.1042/BSR20191290

Title: Live-cell monitoring of protein localization to membrane rafts using protein-fragment complementation
Author: Merezhko, Maria; Pakarinen, Emmi; Uronen, Riikka-Liisa; Huttunen, Henri J.
Contributor organization: Neuroscience Center
Institute of Biotechnology
Biosciences
Date: 2020-01-03
Language: eng
Number of pages: 13
Belongs to series: Bioscience Reports
ISSN: 0144-8463
DOI: https://doi.org/10.1042/BSR20191290
URI: http://hdl.handle.net/10138/317254
Abstract: The plasma membrane consists of a variety of discrete domains differing from the surrounding membrane in composition and properties. Selective partitioning of protein to these microdomains is essential for membrane functioning and integrity. Studying the nanoscale size and dynamic nature of the membrane microdomains requires advanced imaging approaches with a high spatiotemporal resolution and, consequently, expensive and specialized equipment, unavailable for most researchers and unsuited for large-scale studies. Thus, understanding of protein partitioning to the membrane microdomains in health and disease is still hampered by the lack of inexpensive live-cell approaches with an appropriate spatial resolution. Here, we have developed a novel approach based on Gaussia princeps luciferase protein-fragment complementation assay to quantitively investigate protein partitioning to cholesterol and sphingomyelin-rich domains, sometimes called 'lipid rafts', in intact living cells with a high-spatial resolution. In the assay, the reporter construct, carrying one half of the luciferase protein, is targeted to lipid microdomains through the fused acetylation motif from Src-family kinase Fyn. A protein of interest carries the second half of the luciferase protein. Together, this serves as a reversible real-time sensor of raft recruitment for the studied protein. We demonstrated that the assay can efficiently detect the dynamic alterations in raft localization of two disease-associated proteins: Akt and APP. Importantly, this method can be used in high-throughput screenings and other large-scale studies in living cells. This inexpensive, and easy to implement raft localization assay will benefit all researchers interested in protein partitioning in rafts.
Subject: AMYLOID PRECURSOR PROTEIN
LIPID RAFTS
GAMMA-SECRETASE
RECEPTOR ACTIVATION
ALPHA-SECRETASE
CHOLESTEROL
PALMITOYLATION
MICRODOMAINS
DOMAIN
OVEREXPRESSION
1182 Biochemistry, cell and molecular biology
Peer reviewed: Yes
Rights: cc_by
Usage restriction: openAccess
Self-archived version: publishedVersion


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