title: Size, stoichiometry, and organization of soluble LC3-associated complexes.
abstract: MAP1LC3B, an ortholog of yeast Atg8 and a member of the family of proteins formerly also known as ATG8 in mammals (LC3B henceforth in the text), functions in autophagosome formation and autophagy substrate recruitment. LC3 exists in both a soluble (autophagosome-independent) form as well as a lipid modified form that becomes tightly incorporated into autophagosomal membranes. Although LC3 is known to associate with tens of proteins, relatively little is known about soluble LC3 aside from its interactions with the LC3 lipid conjugation machinery. In previous studies we found autophagosome-independent GFP-LC3B diffuses unusually slowly for a protein of its size, suggesting it may constitutively associate with a high molecular weight complex, form homo-oligomers or aggregates, or reversibly bind microtubules or membranes. To distinguish between these possibilities, we characterized the size, stoichiometry, and organization of autophagosome-independent LC3B in living cells and in cytoplasmic extracts using fluorescence recovery after photobleaching (FRAP) and fluorescence polarization fluctuation analysis (FPFA). We found that the diffusion of LC3B was unaffected by either mutational disruption of its lipid modification or microtubule depolymerization. Brightness and homo-FRET analysis indicate LC3B does not homo-oligomerize. However, mutation of specific residues on LC3B required for binding other proteins and mRNA altered the effective hydrodynamic radius of the protein as well as its stoichiometry. We conclude that when not bound to autophagosomes, LC3B associates with a multicomponent complex with an effective size of ~500 kDa in the cytoplasm. These findings provide new insights into the nature of soluble LC3B and illustrate the power of FRAP and FPFA to investigate the emergent properties of protein complexes in the autophagy pathway.
authors.affiliation: Chemical and Physical Biology Program; Department of Molecular Physiology and Biophysics; Department of Cell and Developmental Biology; Vanderbilt University Medical Center; Nashville, TN USA.
authors.forename: Lewis J
authors.initials: LJ
authors.lastname: Kraft
authors.affiliation: Section on Cellular Biophotonics, Laboratory of Molecular Physiology; National Institute on Alcohol Abuse and Alcoholism; National Institutes of Health; Rockville, MD USA.
authors.forename: Tuan A
authors.initials: TA
authors.lastname: Nguyen
authors.affiliation: Section on Cellular Biophotonics, Laboratory of Molecular Physiology; National Institute on Alcohol Abuse and Alcoholism; National Institutes of Health; Rockville, MD USA.
authors.forename: Steven S
authors.initials: SS
authors.lastname: Vogel
authors.affiliation: Chemical and Physical Biology Program; Department of Molecular Physiology and Biophysics; Department of Cell and Developmental Biology; Vanderbilt University Medical Center; Nashville, TN USA.
authors.forename: Anne K
authors.initials: AK
authors.lastname: Kenworthy
mesh_terms: D001343:Autophagy; D003593:Cytoplasm / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D050356:Lipid Metabolism; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism*; D010588:Phagosomes / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational
chemical_list: C502070:MAP1LC3B protein, human; D008869:Microtubule-Associated Proteins; D046912:Multiprotein Complexes
keywords: ATG8; FCS; FPFA; FRAP; FRET; MAP1LC3B; autophagy; diffusion; fluorescence; hydrodynamic radius