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First published online May 28, 2004
doi: 10.1242/10.1242/jcs.01131

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LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation

Yukiko Kabeya¹, Noboru Mizushima^1,2,*, Akitsugu Yamamoto³, Satsuki Oshitani-Okamoto¹, Yoshinori Ohsumi¹ and Tamotsu Yoshimori^1,4,5,6,

¹ Department of Cell Biology, National Institute for Basic Biology, Myodaiji 38, Okazaki 444-8585, Japan
² PRESTO, Japan Science and Technology Agency, Honcho 4-1-8, Kawaguchi 332-0012, Japan
³ Department of Bio-Science, Nagahama Institute of Bio-Science and Technology, Tamura 1266, Nagahama 526-0829, Japan
⁴ Department of Cell Genetics, National Institute of Genetics, Yata 1111, Mishima 411-8540, Japan
⁵ Department of Genetics, The Graduate University for Advanced Studies, Yata 1111, Mishima 411-8540, Japan
⁶ CREST, Japan Science and Technology Agency, Honcho 4-1-8, Kawaguchi 332-0012, Japan

View larger version (43K): [in a new window]   Fig. 2. Human Atg4 homologues process from pro-form to form I of LC3, GABARAP and GATE16 at C-termini. (A) Tagged versions of LC3, GABARAP and GATE16. Positions of Myc and HA epitope tags are indicated. The conserved Gly residues are shown in bold; this residue is replaced by Ala in the LC3G120A mutant. Arrow indicates the cleavage site in yeast Atg8 by Aut2/Atg4 (Kirisako et al., 1999). (B) In vivo analysis of C-terminal hydrolase activities of HsAtg4A and HsAtg4B. The double-tagged mammalian Atg8 family proteins in (A) and the indicated human Atg4 family proteins were co-expressed in Atg4-deficient yeast cells. Total lysate of each transformant was analysed by immunoblot analysis using anti-Myc or anti-HA antibodies. Positions of unprocessed substrates and their processed forms are indicated. (C) Cys74 of HsAtg4B is a putative active site. Enzyme activity of wild type HsAtg4B and HsAtg4BC74S mutant was examined as in (B) using Myc-LC3-HA as substrate.

View larger version (27K): [in a new window]   Fig. 1. [14C]-Ethanolamine is incorporated into LC3-II. HeLa cells transfected with Myc-LC3 were labelled with [35S]-methionine/cysteine for 1 hour (lanes 1-2) or [14C]-ethanolamine for 38 hours (lanes 3-4). The cell lysates were immunoprecipitated with anti-Myc antibody and the immunoprecipitates were separated by SDS-PAGE and analysed with a bioimage analyser.

View larger version (27K): [in a new window]   Fig. 3. LC3-II is a substrate of HsAtg4B. (A) LC3-II is cleaved by HsAtg4B in vitro. Lysate from amino-acid-starved ES cells was mixed with lysate from yeast cells expressing FLAG-tagged HsAtg4A, HsAtg4B, HsAtg4BC74S, or vector alone, and incubated for 1 hour at 37°C. After the addition of SDS-PAGE sample buffer to stop the reaction, the products were resolved by SDS-PAGE and subjected to immunoblot using antibody against LC3. The data were shown from the two independent experiments (Exp. 1 and 2). (B) The amount of LC3-II is overestimated by immunoblotting. Total cell lysates (T) were prepared from HeLa cells expressing His-Myc-LC3 after starvation for 1 hour and subjected to immunoprecipitation using anti-Myc antibody (IP). The immunoprecipitates were assayed by immunoblotting (lanes 1-4) or CBB staining (lanes 5 and 6). As controls, mock-transfected cells were used.

View larger version (34K): [in a new window]   Fig. 4. GATE16 and GABARAP exist in two forms after processing at their C-termini. (A) Tagged versions of LC3, GATE16 and GABARAP. Positions of Myc and HA epitope tags are indicated. The conserved Gly residues are shown in bold. LC3C22, G120A lacks the C-terminal fragment and Gly120 is replaced with Ala. GATE16C1, G116A and GABARAPC1, G116A were also produced. The C, GA mutants were tagged only with the Myc epitope at their N-termini. (B) Two forms of mammalian Atg8 family are produced. F9 cells transfected with the tagged proteins shown in (A) were cultured under nutrient-rich or starvation conditions for 2 hours and subjected to immunoblot analysis using an antibody against the Myc epitope. Position of processed forms (I and II) and unprocessed forms (pro) are indicated. An uncharacterized form (*) appeared in GABARAP-transfected cells.

View larger version (21K): [in a new window]   Fig. 5. Two forms of GATE16 and GABARAP differ in subcellular localization. Homogenate of the starved F9 cells expressing Myc-LC3-HA, Myc-GATE16-HA or Myc-GABARAP-HA (T) was centrifuged at 100,000 g for 30 minutes and then supernatant (S) and pellet (P) fractions were collected. These fractions were analysed by immunoblotting using anti-Myc antibody.

View larger version (101K): [in a new window]   Fig. 6. GATE16 and GABARAP localize to the LC3-positive autophagosome under starvation conditions. (A) F9 cells stably transfected with CFP-LC3 (a,d), YFP-GATE16 (b,e), or YFP-GABARAP (c,f) were cultured under nutrient-rich conditions (a-c) or starvation conditions (d-f) for 2 hours at 37°C. Living cells were observed using a Delta Vision microscopic system. Scale bar, 10 µm. (B) F9 cells co-expressing YFP-GATE16 and CFP-LC3 (a,b), YFP-GABARAP and CFP-LC3 (d,e) were starved for 2 hours and subjected to fluorescence microscopic analysis. Merged images (c,f) are shown. Arrowheads indicate the co-localization of both the punctate and ring-shaped structures. Scale bar, 10 µm. (C) The C-terminal glycine is required for autophagosome-targeting of GATE16 and GABARAP. F9 cells expressing YFP-LC3C22, G120A (a), YFP-GATE16C1, G116A (b), or YFP-GABARAPC1, G116A (c) were starved for 2 hours and subjected to fluorescence microscopic analysis. Scale bar, 10 µm.

View larger version (141K): [in a new window]   Fig. 7. YFP-GATE16 is present on autophagosome membranes. F9 cells stably transfected with YFP-GATE16 were cultured in Hanks' solution for 2 hours and fixed. The localization of YFP-GATE16 was examined by silver-enhanced immunogold electron microscopy using anti-GFP antibody. The isolation membrane (arrow), autophagosomes (open arrowheads) and autolysosomes (closed arrowheads) are indicated (A). The typical images of isolation membrane (B), autophagosome (C) and autolysosome (D) are shown at higher magnification. Scale bar, 1 µm.