Characterization of PROPPIN-Phosphoinositide Binding and Role of Loop 6CD in PROPPIN-Membrane Binding

Abstract

PROPPINs (β-propellers that bind polyphosphoinositides) are a family of PtdIns3P- and PtdIns(3,5)P2-binding proteins that play an important role in autophagy. We analyzed PROPPIN-membrane binding through isothermal titration calorimetry (ITC), stopped-flow measurements, mutagenesis studies, and molecular dynamics (MD) simulations. ITC measurements showed that the yeast PROPPIN family members Atg18, Atg21, and Hsv2 bind PtdIns3P and PtdIns(3,5)P2 with high affinities in the nanomolar to low-micromolar range and have two phosphoinositide (PIP)-binding sites. Single PIP-binding site mutants have a 15- to 30-fold reduced affinity, which explains the requirement of two PIP-binding sites in PROPPINs. Hsv2 bound small unilamellar vesicles with a higher affinity than it bound large unilamellar vesicles in stopped-flow measurements. Thus, we conclude that PROPPIN membrane binding is curvature dependent. MD simulations revealed that loop 6CD is an anchor for membrane binding, as it is the region of the protein that inserts most deeply into the lipid bilayer. Mutagenesis studies showed that both hydrophobic and electrostatic interactions are required for membrane insertion of loop 6CD. We propose a model for PROPPIN-membrane binding in which PROPPINs are initially targeted to membranes through nonspecific electrostatic interactions and are then retained at the membrane through PIP binding.

Membrane scission driven by the PROPPIN Atg18

“…In line with previous binding studies Baskaran et al, 2012;Busse et al, 2015), purified Atg18 wt bound to PI3P as well as to PI(3,5)P 2 -containing liposomes (Fig 5A and E). First, we tested membrane binding of the various Atg18 mutant forms in a liposome centrifugation assay.…”

“…In line with previous binding studies Baskaran et al, 2012;Busse et al, 2015), purified Atg18 wt bound to PI3P as well as to PI(3,5)P 2 -containing liposomes (Fig 5A and E). Since lipid binding involves two lipid binding sites and two blades of Atg18 (Baskaran et al, 2012;Krick et al, 2012;Watanabe et al, 2012;Busse et al, 2015), its preservation suggests that the protein can reach its native b-propeller conformation independently of the CD loop. Atg18 FGGG bound PI(3,5)P 2 and PI3P liposomes very poorly (Fig 5B and E).…”

“…First, we tested membrane binding of the various Atg18 mutant forms in a liposome centrifugation assay. In line with previous binding studies Baskaran et al, 2012;Busse et al, 2015), purified Atg18 wt bound to PI3P as well as to PI(3,5)P 2 -containing liposomes ( Fig 5A and E). Atg18 wt bound better to 5% PI(3,5)P 2 than to 5% PI3P.…”

“…Key features shared by PROPPINs are their two lipid interaction sites with a capacity to bind PI3P, PI(3,5)P 2 , and PI(5)P, phosphoinositides that are particularly enriched on endosomes, lysosomes, and autophagosomes (Schink et al, 2013;McCartney et al, 2014;Vicinanza et al, 2015). It must face the membrane surface when the protein binds PI3P and PI(3,5)P 2 (Baskaran et al, 2012;Krick et al, 2012;Watanabe et al, 2012;Busse et al, 2015). It must face the membrane surface when the protein binds PI3P and PI(3,5)P 2 (Baskaran et al, 2012;Krick et al, 2012;Watanabe et al, 2012;Busse et al, 2015).…”

Sensing Membrane Curvature in Macroautophagy

“…Each has been shown to bind membranes via two phosphoinositide binding sites plus other sequences that insert in the membrane 88 . Hsv2 has also been examined on liposomes of different size and revealed to have a more than 10-fold increase in apparent affinity when the membranes become highly curved 89 . The mechanism of curvature recognition for both RavZ and Hsv2 remains uncertain.…”