Abstract
PROPPINs (β-propellers that bind polyphosphoinositides) are a protein family that binds preferentially phosphatidylinositol 3-phosphate (PtdIns(3)P) and phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) via its FRRG motif. PROPPINs are involved in autophagic functions, but their molecular mechanism is still elusive. To unravel the molecular mechanism of PROPPINs, it is essential to understand the PROPPIN–phosphoinositide binding. Here, we describe a protocol to study the kinetics of the PROPPIN–phosphoinositide binding using a fluorescence resonance energy transfer (FRET) stopped-flow approach. We use FRET between fluorophore-labeled protein and fluorophore-labeled liposomes, monitoring the increase of the acceptor emission in labeled liposomes after the protein–membrane binding. Through this approach, we studied the kinetics of the PROPPIN Atg18 (Autophagy-related protein 18) from Pichia angusta (PaAtg18) and a mutant of its FRRG motif, called FTTG mutant. Stopped-flow experiments demonstrated that the main function of the FRRG motif is to retain, instead of to drive, Atg18 to the membrane, decreasing the Atg18 dissociation rate. Furthermore, this method is suitable for the study of other PI-binding proteins.
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References
Balla T (2013) Phosphoinositides: tiny lipids with Giant impact on cell regulation. Physiol Rev 93:1019–1137
Dickson EJ, Hille B (2019) Understanding phosphoinositides: rare, dynamic, and essential membrane phospholipids. Biochem J 476:1–23
Kutateladze TG (2012) Molecular analysis of protein-phosphoinositide interactions. Curr Top Microbiol 362:111–126
Hammond GRV, Balla T (2015) Polyphosphoinositide binding domains: key to inositol lipid biology. BBA-Mol Cell Biol L 1851:1283–1283
Farre JC, Subramani S (2016) Mechanistic insights into selective autophagy pathways: lessons from yeast. Nat Rev Mol Cell Bio 17:537–552
Busse RA, Scacioc A, Krick R, Perez-Lara A, Thumm M, Kuhnel K (2015) Characterization of PROPPIN-phosphoinositide binding and role of loop 6CD in PROPPIN-membrane binding. Biophys J 108:2223–2234
Krick R, Tolstrup J, Appelles A, Henke S, Thumm M (2006) The relevance of the phosphatidylinositolphosphat-binding motif FRRGT of Atg18 and Atg21 for the Cvt pathway and autophagy. FEBS Lett 580:4632–4638
Baskaran S, Ragusa MJ, Boura E, Hurley JH (2012) Two-site recognition of phosphatidylinositol 3-phosphate by PROPPINs in autophagy. Mol Cell 47:339–348
Krick R, Busse RA, Scacioc A, Stephan M, Janshoff A, Thumm M, Kuhnel K (2012) Structural and functional characterization of the two phosphoinositide binding sites of PROPPINs, a beta-propeller protein family. Proc Natl Acad Sci U S A 109:E2042–E2049
Krick R, Henke S, Tolstrup J, Thumm M (2008) Dissecting the localization and function of Atg18, Atg21 and Ygr223c. Autophagy 4:896–910
Scacioc A, Schmidt C, Hofmann T, Urlaub H, Kuhnel K, Perez-Lara A (2017) Structure based biophysical characterization of the PROPPIN Atg18 shows Atg18 oligomerization upon membrane binding. Sci Rep 7:14008
Metje J (2017) Structural characterization of autophagy related protein complexes. University of Göttingen - Georg-August-Universität Göttingen, Göttingen
Corbin JA, Evans JH, Landgraf KE, Falke JJ (2007) Mechanism of specific membrane targeting by C2 domains: localized pools of target lipids enhance Ca2+ affinity. Biochemistry 46:4322–4336
Nalefski EA, Newton AC (2001) Membrane binding kinetics of protein kinase C beta II mediated by the C2 domain. Biochemistry 40:13216–13229
Perez-Lara A, Thapa A, Nyenhuis SB, Nyenhuis DA, Halder P, Tietzel M, Tittmann K, Cafiso DS, Jahn R (2016) PtdInsP(2) and PtdSer cooperate to trap synaptotagmin-1 to the plasma membrane in the presence of calcium. Elife 5:e15886
Bai JH, Tucker WC, Chapman ER (2004) PIP2 increases the speed of response of synaptotagmin and steers its membrane-penetration activity toward the plasma membrane. Nat Struct Mol Biol 11:36–44
Bai JH, Wang P, Chapman ER (2002) C2A activates a cryptic Ca2+−triggered membrane penetration activity within the C2B domain of synaptotagmin I. Proc Natl Acad Sci U S A 99:1665–1670
Boettcher C, Pries C, Vangent CM (1961) A rapid and sensitive sub-micro phosphorus determination. Anal Chim Acta 24:203–204
Bernasconi CF (1976) Relaxation kinetics. Academic Press, New York
Acknowledgments
We are indebted to Dr. Karin Kühnel for her generous support and advice. We thank Dr. Bruno Ramos-Molina and Dr. Manuel Rosa-Garrido for advice and discussions. This work was supported by a NIH Grant GB10440.155740 and Max Planck Gesellschaft financial support (Grant PSBICH11200) to R.J.
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Pérez-Lara, Á., Jahn, R. (2021). Characterization of PROPPIN–Phosphoinositide Binding by Stopped-Flow Fluorescence Spectroscopy. In: Botelho, R.J. (eds) Phosphoinositides. Methods in Molecular Biology, vol 2251. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1142-5_15
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DOI: https://doi.org/10.1007/978-1-0716-1142-5_15
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