Structure based biophysical characterization of the PROPPIN Atg18 shows Atg18 oligomerization upon membrane binding

Andreea Scacioc,Carla Schmidt,Tommy Hofmann,Ángel Pérez-Lara,Henning Urlaub,Karin Kühnel

doi:10.1038/s41598-017-14337-5

Abstract

PROPPINs (β-propellers that bind polyphosphoinositides) are PtdIns3P and PtdIns(3,5)P2 binding autophagy related proteins. They contain two phosphatidylinositolphosphate (PIP) binding sites and a conserved FRRG motif is essential for PIP binding. Here we present the 2.0 Å resolution crystal structure of the PROPPIN Atg18 from Pichia angusta. We designed cysteine mutants for labelling with the fluorescence dyes to probe the distances of the mutants to the membrane. These measurements support a model for PROPPIN-membrane binding, where the PROPPIN sits in a perpendicular or slightly tilted orientation on the membrane. Stopped-flow measurements suggest that initial PROPPIN-membrane binding is driven by non-specific PIP interactions. The FRRG motif then retains the protein in the membrane by binding two PIP molecules as evident by a lower dissociation rate for Atg18 in comparison with its PIP binding deficient FTTG mutant. We demonstrate that the amine-specific cross-linker Bis(sulfosuccinimidyl)suberate (BS3), which is used for protein-protein cross-linking can also be applied for cross-linking proteins and phosphatidylethanolamine (PE). Cross-linking experiments with liposome bound Atg18 yielded several PE cross-linked peptides. We also observed intermolecular cross-linked peptides, which indicated Atg18 oligomerization. FRET-based stopped-flow measurements revealed that Atg18 rapidly oligomerizes upon membrane binding while it is mainly monomeric in solution.

Highlights

During macroautophagy, denoted here as autophagy, an expanding isolation membrane called the phagophore engulfs cytoplasmic content
We previously showed that Hsv[2] binds with an approx. 20 times higher affinity to small unilamellar vesicles (SUVs) containing either PtdIns3P or PtdIns(3,5)P2 than large unilamellar vesicles (LUVs), which had diameters of approx. 40 nm and 100 nm, respectively[16]
We proposed a model for PROPPIN-membrane interactions, where the protein sits perpendicular on the membrane and binds through its two PIP binding sites on the rim of the β-propeller[13]

Summary

Introduction

During macroautophagy, denoted here as autophagy, an expanding isolation membrane called the phagophore engulfs cytoplasmic content. Yeast contains two additional PROPPINs: Atg[21] and Hsv[2] (homologous with swollen vacuole phenotype 2). Loop 6CD protrudes from the β-propeller core and is required for membrane binding in addition to the two PIP binding sites[14,16]. PROPPINs were proposed to bind perpendicular to the membrane through their two PIP binding sites on the rim and the membrane insertion loop 6CD13–16. Atg[18] localizes in a PtdIns3P dependent manner to the preautophagosomal structure (PAS), the site where autophagosome formation is initiated[10,18]. Yeast atg18Δ cells have enlarged vacuoles[9,23] and mutagenesis studies showed that both Atg[18] PIP binding sites are important for maintaining vacuolar homeostasis[13]

Methods

Results

Conclusion