Size, Organization and Dynamics of Soluble SQSTM1 and LC3/SQSTM1 Complexes in Living Cells

Lewis J Kraft,Jacob Dowler,Pallavi Manral,Anne K Kenworthy

doi:10.1016/j.bpj.2015.11.1202

Abstract

Selective autophagy—with the help of molecular adaptors—captures cargo for lysosomal degradation. Among the best studied molecular adaptors is p62/SQSTM1, a homo-oligomeric ubiquitin binding protein, which binds to both cargo and MAP1LC3B (LC3), a protein important for autophagosome biogenesis. Although the mechanisms underlying interaction of LC3 and SQSTM1 have been extensively studied, very little is known about the size or organization of soluble complexes formed between SQSTM1 and LC3 prior to autophagosome binding. To address this question, in the current study we use a combination of FRET and FRAP to study the nanoscale properties of soluble SQSTM1 complexes and SQSTM1/LC3 complexes in living HeLa cells. We show that, independent of puncta, SQSTM1 oligomerizes to form very slowly diffusing complexes that contain multiple copies of SQSTM1 within FRET proximity of one another. Surprisingly, disruption of SQSTM1's PB1 domain, a region of the protein involved in homo-oligomer formation, did not fully dissipate the complexes. Furthermore, wild-type SQSTM1 and PB1 domain mutants were equally capable of binding to LC3. Thus, homo-oligomerization is not required for SQSTM1 to form soluble (puncta independent) complexes or efficiently bind to soluble LC3. Taken together, these findings reveal new insights into the nature of nanometer-sized SQSTM1/LC3 complexes.

Highlights

1079-Pos Board B56 Calibrating Sickle Cell Disease Donna A
Sickle cell disease is fundamentally a kinetic disorder, in which cells containing the mutated hemoglobin (HbS) will cause occlusion if they sickle in the microvasculature, but have minimal consequences if they sickle in the venous return
We have measured the rate of nucleus formation in HbS partially liganded with NO or CO, which we find have equivalent effects in reducing the nucleation rates

Summary

Introduction

1079-Pos Board B56 Calibrating Sickle Cell Disease Donna A. The variable domain of Drp1 limits its interaction with Mff. Removal of this domain allows for formation of a stable Drp1-Mff complex, and results in elevated Drp1 GTPase activity in the presence of Mff. Further characterization of Drp1, Mff, and various mutants of these proteins in MEFs lacking the endogenous protein will build on our current knowledge to refine the role of the variable domain and oligomerization in Drp1 interactions with Mff. 1078-Pos Board B55 Size, Organization and Dynamics of Soluble SQSTM1 and LC3/SQSTM1 Complexes in Living Cells Lewis J. Independent of puncta, SQSTM1 oligomerizes to form very slowly diffusing complexes that contain multiple copies of SQSTM1 within FRET proximity of one another.

Results

Conclusion