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  Qualitative and quantitative characterization of protein-phosphoinositide interactions with liposome-based methods.

Busse, R. A., Scacioc, A., Hernandez, J. M., Krick, R., Stephan, M., Janshoff, A., et al. (2013). Qualitative and quantitative characterization of protein-phosphoinositide interactions with liposome-based methods. Autophagy, 9(5), 1-8. doi:10.4161/auto.23978.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-000E-EAE9-3 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0027-C842-3
Genre: Journal Article

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1703423.pdf (Publisher version), 2MB
 
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 Creators:
Busse, R. A.1, Author              
Scacioc, A.1, Author              
Hernandez, J. M.1, Author              
Krick, R., Author
Stephan , M., Author
Janshoff, A., Author
Thumm, M., Author
Kühnel, K.1, Author              
Affiliations:
1Department of Neurobiology, MPI for biophysical chemistry, Max Planck Society, ou_578595              

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Free keywords: PROPPIN, isothermal titration calorimetry, liposome flotation assays, multi-angle laser light scattering, small unilamellar vesicle
 Abstract: We characterized phosphoinositide binding of the S. cerevisiae PROPPIN Hsv2 qualitatively with density flotation assays and quantitatively through isothermal titration calorimetry (ITC) measurements using liposomes. We discuss the design of these experiments and show with liposome flotation assays that Hsv2 binds with high specificity to both PtdIns3P and PtdIns(3,5)P2. We propose liposome flotation assays as a more accurate alternative to the commonly used PIP strips for the characterization of phosphoinositide-binding specificities of proteins. We further quantitatively characterized PtdIns3P binding of Hsv2 with ITC measurements and determined a dissociation constant of 0.67 µM and a stoichiometry of 2:1 for PtdIns3P binding to Hsv2. PtdIns3P is crucial for the biogenesis of autophagosomes and their precursors. Besides the PROPPINs there are other PtdIns3P binding proteins with a link to autophagy, which includes the FYVE-domain containing proteins ZFYVE1/DFCP1 and WDFY3/ALFY and the PX-domain containing proteins Atg20 and Snx4/Atg24. The methods described could be useful tools for the characterization of these and other phosphoinositide-binding proteins.

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Language(s): eng - English
 Dates: 2013-032013-05-01
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Method: Peer
 Identifiers: DOI: 10.4161/auto.23978
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Title: Autophagy
Source Genre: Journal
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Pages: - Volume / Issue: 9 (5) Sequence Number: - Start / End Page: 1 - 8 Identifier: ISSN: 1554-8627