title: Autophagic bulk sequestration of cytosolic cargo is independent of LC3, but requires GABARAPs.
abstract: LC3, a mammalian homologue of yeast Atg8, is assumed to play an important part in bulk sequestration and degradation of cytoplasm (macroautophagy), and is widely used as an indicator of this process. To critically examine its role, we followed the autophagic flux of LC3 in rat hepatocytes during conditions of maximal macroautophagic activity (amino acid depletion), combined with analyses of macroautophagic cargo sequestration, measured as transfer of the cytosolic protein lactate dehydrogenase (LDH) to sedimentable organelles. To accurately determine LC3 turnover we developed a quantitative immunoblotting procedure that corrects for differential immunoreactivity of cytosolic and membrane-associated LC3 forms, and we included cycloheximide to block influx of newly synthesized LC3. As expected, LC3 was initially degraded by the autophagic-lysosomal pathway, but, surprisingly, autophagic LC3-flux ceased after ~2h. In contrast, macroautophagic cargo flux was well maintained, and density gradient analysis showed that sequestered LDH partly accumulated in LC3-free autophagic vacuoles. Hepatocytic macroautophagy could thus proceed independently of LC3. Silencing of either of the two mammalian Atg8 subfamilies in LNCaP prostate cancer cells exposed to macroautophagy-inducing conditions (starvation or the mTOR-inhibitor Torin1) confirmed that macroautophagic sequestration did not require the LC3 subfamily, but, intriguingly, we found the GABARAP subfamily to be essential.
authors: Prostate Cancer Research Group, Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo and Oslo University Hospital, N-0318 Oslo, Norway.
authors: Paula
authors: P
authors: Szalai
authors: Cell Biology Section, Institute for Cancer Research, Oslo University Hospital, N-0310 Oslo, Norway.
authors: Linda Korseberg
authors: LK
authors: Hagen
authors: Prostate Cancer Research Group, Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo and Oslo University Hospital, N-0318 Oslo, Norway; Cell Biology Section, Institute for Cancer Research, Oslo University Hospital, N-0310 Oslo, Norway.
authors: Frank
authors: F
authors: Sætre
authors: Prostate Cancer Research Group, Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo and Oslo University Hospital, N-0318 Oslo, Norway.
authors: Morten
authors: M
authors: Luhr
authors: Cell Biology Section, Institute for Cancer Research, Oslo University Hospital, N-0310 Oslo, Norway.
authors: Marianne
authors: M
authors: Sponheim
authors: Cell Biology Section, Institute for Cancer Research, Oslo University Hospital, N-0310 Oslo, Norway.
authors: Anders
authors: A
authors: Øverbye
authors: Prostate Cancer Research Group, Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo and Oslo University Hospital, N-0318 Oslo, Norway; Department of Urology, Oslo University Hospital, N-0310 Oslo, Norway; Department of Cancer Prevention, Institute for Cancer Research, Oslo University Hospital, N-0310 Oslo, Norway.
authors: Ian G
authors: IG
authors: Mills
authors: Prostate Cancer Research Group, Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo and Oslo University Hospital, N-0318 Oslo, Norway; Cell Biology Section, Institute for Cancer Research, Oslo University Hospital, N-0310 Oslo, Norway. Electronic address: p.o.seglen@ncmm.uio.no.
authors: Per O
authors: PO
authors: Seglen
authors: Prostate Cancer Research Group, Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo and Oslo University Hospital, N-0318 Oslo, Norway. Electronic address: k.n.engedal@ncmm.uio.no.
authors: Nikolai
authors: N
authors: Engedal
mesh_terms: D000818:Animals; D001343:Autophagy; D000071187:Autophagy-Related Protein 5; D002478:Cells, Cultured; D008297:Male; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism* / Q000502:physiology*; D011506:Proteins / Q000378:metabolism; D017208:Rats, Wistar; D014617:Vacuoles / Q000378:metabolism
chemical_list: D000071187:Autophagy-Related Protein 5; D008869:Microtubule-Associated Proteins; D011506:Proteins; C545865:Atg5 protein, rat; C502186:GABARAP protein, rat; C526109:Anxa3 protein, rat
keywords: Autophagy; Density gradient; GABARAP; LC3 antibody; Sequestration; Subcellular fractionation