Phosphorylation of the LIR Domain of SCOC Modulates ATG8 Binding Affinity and Specificity

Martina Wirth,Dhira Joshi,Harold B.J. Jefferies,Ashish Jain,Minoo Razi,Jack-Ansgar Bruun,Terje Johansen,Nicola O'Reilly,Rebecca Lee,Yakubu Princely Abudu,Sharon A. Tooze,Wenxin Zhang,Stephane Mouilleron,Eva Sjøttem,Hallvard Lauritz Olsvik

doi:10.1016/j.jmb.2021.166987

Abstract

Autophagy is a highly conserved degradative pathway, essential for cellular homeostasis and implicated in diseases including cancer and neurodegeneration. Autophagy-related 8 (ATG8) proteins play a central role in autophagosome formation and selective delivery of cytoplasmic cargo to lysosomes by recruiting autophagy adaptors and receptors. The LC3-interacting region (LIR) docking site (LDS) of ATG8 proteins binds to LIR motifs present in autophagy adaptors and receptors. LIR-ATG8 interactions can be highly selective for specific mammalian ATG8 family members (LC3A-C, GABARAP, and GABARAPL1-2) and how this specificity is generated and regulated is incompletely understood.We have identified a LIR motif in the Golgi protein SCOC (short coiled-coil protein) exhibiting strong binding to GABARAP, GABARAPL1, LC3A and LC3C. The residues within and surrounding the core LIR motif of the SCOC LIR domain were phosphorylated by autophagy-related kinases (ULK1-3, TBK1) increasing specifically LC3 family binding. More distant flanking residues also contributed to ATG8 binding. Loss of these residues was compensated by phosphorylation of serine residues immediately adjacent to the core LIR motif, indicating that the interactions of the flanking LIR regions with the LDS are important and highly dynamic.Our comprehensive structural, biophysical and biochemical analyses support and provide novel mechanistic insights into how phosphorylation of LIR domain residues regulates the affinity and binding specificity of ATG8 proteins towards autophagy adaptors and receptors.

Highlights

Autophagy is a highly conserved recycling and stress survival pathway ensuring cellular health and homeostasis.[1]
Trans-Golgi network and GABARAP-positive puncta and this localization might occur through binding other proteins, such as Arl[1] and fasciculation and elongation protein zeta 1 (FEZ1),39,40 or dimerization with endogenous wild type short coiled-coil protein (SCOC).[50]
Phosphorylation of LC3-interacting region (LIR) motifs of autophagy receptors and adaptors plays an important role in regulating selective autophagy pathways, e.g. mitophagy[33,34,35,36,37,38] and xenophagy.[32]

Summary

Introduction

Autophagy is a highly conserved recycling and stress survival pathway ensuring cellular health and homeostasis.[1]. Many LIR-containing proteins interact with ATG8 proteins in a highly selective manner, suggesting functional differences between LC3 and GABARAP subfamily proteins.[5,13,14,15,28] Rogov et al defined a GABARAP interaction motif (GIM), H0-[V/I]1-X2-C3, and showed that valine or isoleucine in position X1 promotes GABARAP binding.[29] We recently showed that the binding preference of LIR motifs to GABARAPs is regulated by the X2 residue within the core LIR motif as well as residues in the adjacent C-terminal region (positions X4-X10).[30] ATG8-subfamily binding specificity is determined by the residues of the LIRdocking site (LDS). In GST-pull down experiwhich are up to eight amino acid residues distant ments SCOC isoform 3 interacted strongly with from the SCOC core LIR motif, to ATG8 protein LC3A, LC3C, GABARAP and GABARAPL1, much binding. (Supplementary Figure 1B) suggesting that the LIR motif is not essential for SCOC localization to the

Results

Discussion

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