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doi: 10.1242/10.1242/jcs.00381

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Mouse Apg16L, a novel WD-repeat protein, targets to the autophagic isolation membrane with the Apg12-Apg5 conjugate

Noboru Mizushima1,2,*, Akiko Kuma2,3, Yoshinori Kobayashi2,3, Akitsugu Yamamoto4, Masami Matsubae5, Toshifumi Takao5, Tohru Natsume6, Yoshinori Ohsumi2,3,* and Tamotsu Yoshimori2,3,7

1 PRESTO, Japan Science and Technology Corporation, Kawaguchi 332-0012, Japan
2 Department of Cell Biology, National Institute for Basic Biology, 38 Nishigonaka, Myodaiji, Okazaki 444-8585, Japan
3 Department of Molecular Biomechanics, School of Life Science, The Graduate University for Advanced Studies, Okazaki 444-8585, Japan
4 Department of Physiology, Kansai Medical University, Moriguchi 570-8506, Japan
5 Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan
6 National Institute of Advanced Industrial Science and Technology, Tokyo 135-0064, Japan
7 Department of Cell Genetics, National Institute of Genetics, Mishima 411-8540, Japan



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  		Fig. 5. Apg16L forms a ~800 kDa protein complex with Apg12-Apg5. (A) Apg16L is present primarily in the cytosol. ES cell homogenate (Total) was fractionated into an initial pellet (P13) and a subsequent pellet (P100) and supernatant (S100) fractions by differential centrifugation. These fractions were analysed by immunoblotting using antibodies against Apg5 and Apg16L. (B) Apg12-Apg5 and Apg16L form a ~800 kDa complex. S100 fractions of tissue homogenates of liver and brain, and cell lysates of wild-type and APG5-/- ES cells were separated by size exclusion chromatography on a Superose 6 column. Each fraction was subjected to immunoblotting using anti-Apg5 and anti-Apg16L antibodies. Positions of the molecular mass standards (in kDa) are shown. V, void fraction.

 


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  		Fig. 1. Identification of proteins interacting with Apg5 in mouse ES cells. (A) Wild-type ES cells stably expressing GFP alone and Apg5-deficient ES cells stably expressing GFP-fused Apg5 (GFP24) were labeled with [35S]methionine/cysteine for 2 hours. Following immunoprecipitation with anti-GFP antibody from cell lysates, the immunoprecipitates were analyzed by SDS-PAGE and a bioimage analyzer. (B) Purification of Apg5-interacting proteins. Total cell lysates were prepared from GFP24 cells and subjected to affinity purification using an anti-GFP antibody-coupled protein-A/Sepharose bead column. Following elution, bound proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. The positions of unconjugated GFP-Apg5, Apg12/GFP-Apg5, and three unknown proteins of 63 kDa, 71 kDa and 144 kDa are indicated. Asterisk indicates the position of immunoglobulin heavy chain partially dissociated from the column.

 


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  		Fig. 2. Structure of Apg16L. (A) Amino acid sequence of Apg16L. Underlined and dotted-underlined residues correspond to peptide sequences obtained by MS analysis of p63 and p71, respectively. Boxes indicate the peptides encoded by exons 8 and 9, which are deleted in spliced isoforms. Sequences outlined in black are WD repeats. (B) Amino acid sequence of mouse Apg16L was aligned with that of S. cerevisiae Apg16 using the BLAST2 program (http://www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html). Identical amino acids are shown with a line between them (|), while similar amino acids are indicated with dots (:). (C) Structural comparison of putative Apg16L homologues. The coiled-coil regions are indicated as gray boxes [analyzed by Multicoil program (Wolf et al., 1997Go), http://multicoil.lcs.mit.edu/cgi-bin/multicoil] and the WD repeats are shown as black boxes (analyzed by PSA Sequence analysis, http://bmerc-www.bu.edu/psa/request.htm). At, Arabidopsis thaliana (AB024031); Ce, Caenorhabditis elegance (U53340, U23449); Dd, Dictyostelium discoideum (AF019236); Dm, Drosophila melanogaster (AY058742); Hs, Homo sapiens (AK027854, AK024453); Mm, Mus musculus; Os, Oryza sativa (AC087852); Pp, Pichia pastoris (Mukaiyama et al., 2002Go) (Y. Sakai, personal communication); Sc, Saccharomyces cerevisiae. The amino acid sequences of Dm Apg16L and Ce Apg16L2, hypothesized from the genomic sequences, might be incomplete, lacking N-terminal sequences.

 


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  		Fig. 3. Spliced isoforms of Apg16L. (A) Alternative splicing of Apg16L mRNA. The 20 exons are indicated by numbers above the line representing Apg16L{gamma}. Alternatively spliced exons in Apg16L{alpha} and Apg16Lß are indicated as broken lines. The positions of the primers (within exons 6 and 10) used in C are indicated by arrows. Corresponding domain structures are shown as in Fig. 2C. (B) Expression of Apg16L in tissues and cell lines. Tissue homogenates were prepared from mouse liver (lane 1), brain (lane 2), the gastrocnemius muscle (lane 3) and kidney (lane 4). Total cell lysates were also prepared from ES cells (lane 5) and HeLa cells transiently transfected with either vector alone (lane 6), Apg16L{alpha} (lane 7) or FLAG-tagged Apg16L{alpha} (lane 8). The mobility of the three isoforms is indicated. (C) Reverse-transcription-PCR analysis of Apg16L mRNA. Total RNA was isolated from mouse liver (lane 1), brain (lane 2), kidney (lane 3), ES cells (lane 4) and HeLa cells (lane 5), and reverse-transcribed into cDNA. A fragment of the Apg16L cDNA corresponding to exons 6-10 was amplified using the primers indicated in A. The cDNA sequences of mouse Apg16L{alpha}, Apg16Lß and Apg16L{gamma} have been deposited in the DDBJ/EMBL/GenBank databases under accession numbers AB087879, AB087880 and AB087881, respectively.

 


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  		Fig. 4. Apg16L interacts with Apg5 and additional Apg16L monomers. (A) Apg16L interacts with Apg5. FLAG-tagged Apg16L and HA-tagged Apg5 were immunoprecipitated from HeLa cells transiently transfected with the indicated plasmids. The interaction of these co-transfected molecules was examined by western-blot analysis using anti-FLAG and anti-HA antibodies. (B) Apg16L forms a homo-oligomer. Wild-type ES cells and Apg5-deficient ES cells were transiently transfected with FLAG-tagged Apg16L and/or GFP-tagged Apg16L. Immunoprecipitates of GFP-Apg16L were examined for the co-immunoprecipitation of FLAG-Apg16L by western-blot analysis using anti-FLAG antibody. (C) Two-hybrid interactions of Apg16L-Apg5 and Apg16L-Apg16L. Interactions between Apg16L, Apg16L deletion constructs and Apg5 within transfected yeast cells were assessed for growth on SC —His —Trp —Leu plates containing 3 mM 3-amino-triazole.

 


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  		Fig. 6. Apg16L co-localizes completely with Apg5 and in part with LC3. ES cells stably co-expressing YFP-LC3 and CFP-Apg5 (A; clone F1-3), CFP-Apg5 and YFP-Apg16L (B; clone Y63D-3), and YFP-LC3 and CFP-Apg16L (C; clone B3-1-5) were cultured in Hanks' solution for 2 hours. Living cells were directly observed with a DeltaVision microscope system. ES cells grow as colonies; 4-7 cells are shown in each panel. Bars, 2 µm.

 


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  		Fig. 7. GFP-Apg16L is present on isolation membranes. ES cells stably expressing GFP-Apg16L were cultured in Hanks' solution for 2 hours and then fixed with 4% paraformaldehyde. The localization of GFP-Apg16L was examined by silver-enhanced immunogold electron microscopy using an anti-GFP antibody. (A,B) Isolation membranes at very early stages. (C) Cup-shaped isolation membrane. (D) Autophagosome. Bar, 1 µm.

 


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  		Fig. 8. Membrane association of Apg16L depends on Apg5 but not on Apg5 conjugation with Apg12. APG5-/- ES cells stably expressing GFP-Apg5 (GFP24) (A) or GFP-Apg5K130R (GKR-1) (B), wild-type ES cells (C) and APG5-/- ES cells (D) were cultured in Hanks' solution for 2 hours. The cells were fixed, permeabilized and subjected to immunofluorescence confocal microscopy using an antiserum against Apg16L (p63C-2) and Cy5-conjugated goat anti-rabbit IgG secondary antibody. GFP-Apg5(KR) labeling, Apg16L staining, merged, and differential interference contrast (DIC) images are shown. Bars, 10 µm.

 




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