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<article doi="10.1038/26506" abstract="Autophagy is a process for the bulk degradation of proteins, in which cytoplasmic components of the cell are enclosed by double-membrane structures known as autophagosomes for delivery to lysosomes or vacuoles for degradation. This process is crucial for survival during starvation and cell differentiation. No molecules have been identified that are involved in autophagy in higher eukaryotes. We have isolated 14 autophagy-defective (apg) mutants of the yeast Saccharomyces cerevisiae and examined the autophagic process at the molecular level. We show here that a unique covalent-modification system is essential for autophagy to occur. The carboxy-terminal glycine residue of Apg12, a 186-amino-acid protein, is conjugated to a lysine at residue 149 of Apg5, a 294-amino-acid protein. Of the apg mutants, we found that apg7 and apg10 were unable to form an Apg5/Apg12 conjugate. By cloning APG7, we discovered that Apg7 is a ubiquitin-E1-like enzyme. This conjugation can be reconstituted in vitro and depends on ATP. To our knowledge, this is the first report of a protein unrelated to ubiquitin that uses a ubiquitination-like conjugation system. Furthermore, Apg5 and Apg12 have mammalian homologues, suggesting that this new modification system is conserved from yeast to mammalian cells."><fig id="2614"><title></title><label>figf1</label><graphic href="https://api.sourcedata.io/file.php?figure_id=2614"/><sd-panel panel_id="9785">Wild-type, <em><sd-tag id="sdTag1" source="sdapi" category="entity" entity_type="gene" role="intervention" text="apg12-1" ext_ids="852518" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG12" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="http://www.ncbi.nlm.nih.gov/gene/"><sd-pretag id="sdPretag14340161636180" parent-tag-id="9" class="component gene">apg12</sd-pretag>-1</sd-tag></em> mutant and Δ<sd-tag id="sdTag2" source="sdapi" category="entity" entity_type="gene" role="intervention" text="apg12" ext_ids="852518" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG12" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="http://www.ncbi.nlm.nih.gov/gene/"><em>apg12</em></sd-tag> <span>cells were cultured in <sd-tag id="sdTag3" source="sdapi" category="entity" entity_type="molecule" role="component" text="nitrogen" ext_ids="CHEBI:25555" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="nitrogen atom" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">nitrogen</sd-tag>-starvation medium containing 1 mM <sd-tag id="sdTag4" source="sdapi" category="entity" entity_type="molecule" role="component" text="PMSF" ext_ids="CHEBI:8102" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="phenylmethanesulfonyl fluoride" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">PMSF</sd-tag>. After incubation for <sd-tag id="sdTag5" source="sdapi" category="time" entity_type="" role="" text="6 h" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">6 h</sd-tag>, cells were observed under a <sd-tag id="sdTag6" source="sdapi" category="assay" entity_type="" role="" text="phase-contrast microscope" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">phase-contrast microscope</sd-tag>. Arrows indicate <sd-tag id="sdTag7" source="sdapi" category="entity" entity_type="subcellular" role="assayed" text="autophagic bodies" ext_ids="GO:0005776" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="autophagic vacuole" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">autophagic bodies</sd-tag>.</span><graphic href="https://api.sourcedata.io/file.php?panel_id=9785"/></sd-panel><sd-panel panel_id="9787"><strong>c</strong>, Wild-type (squares) and Δ<em><sd-tag id="sdTag9" source="sdapi" category="entity" entity_type="gene" role="intervention" text="apg12" ext_ids="852518" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG12" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">apg12</sd-tag></em> (circles) were cultured in <sd-tag id="sdTag11" source="sdapi" category="entity" entity_type="molecule" role="component" text="nitrogen" ext_ids="CHEBI:25555" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="nitrogen atom" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">nitrogen</sd-tag><span>-starvation medium and their viability was determined by <sd-tag id="sdTag10" source="sdapi" category="assay" entity_type="" role="" text="phloxine B staining" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">phloxine B staining</sd-tag>.</span><graphic href="https://api.sourcedata.io/file.php?panel_id=9787"/></sd-panel><sd-panel panel_id="9788"><p><strong>d</strong>, Quantification of autophagic activity of wild-type and Δ<em><sd-tag id="sdTag17" source="sdapi" category="entity" entity_type="gene" role="intervention" text="apg12" ext_ids="852518" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG12" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">apg12</sd-tag></em> cells by <sd-tag id="sdTag15" source="sdapi" category="assay" entity_type="" role="" text="alkaline phosphatase (ALP) assay" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">alkaline phosphatase (ALP) assay</sd-tag> before (black bars) and after (white bars) <sd-tag id="sdTag18" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="nitrogen" ext_ids="CHEBI:25555" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="nitrogen atom" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">nitrogen</sd-tag> starvation for <sd-tag id="sdTag16" source="sdapi" category="time" entity_type="" role="" text="4 h" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">4 h</sd-tag>. Error bars indicate s.d. of three independent experiments.</p><graphic href="https://api.sourcedata.io/file.php?panel_id=9788"/></sd-panel></fig><fig id="2615"><title></title><label>figf2</label><graphic href="https://api.sourcedata.io/file.php?figure_id=2615"/><sd-panel panel_id="9790"><p><strong>a</strong>, <strong>b</strong>, Lysates from Δ<em><sd-tag id="sdTag29" source="sdapi" category="entity" entity_type="gene" role="component" text="apg12" ext_ids="852518" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG12" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">apg12</sd-tag></em> cells carrying only vector or 3 × <sd-tag id="sdTag21" source="sdapi" category="entity" entity_type="protein" role="reporter" text="HA" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">HA</sd-tag>-<sd-tag id="sdTag22" source="sdapi" category="entity" entity_type="gene" role="intervention" text="APG12" ext_ids="852518" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG12" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">APG12</sd-tag> (<strong>a</strong>), and Δ<em><sd-tag id="sdTag23" source="sdapi" category="entity" entity_type="gene" role="intervention" text="apg5" ext_ids="855954" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG5" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">apg5</sd-tag></em>, <em><sd-tag id="sdTag24" source="sdapi" category="entity" entity_type="gene" role="intervention" text="apg7-1" ext_ids="856576" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG7" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">apg7-1</sd-tag></em>, <em><sd-tag id="sdTag25" source="sdapi" category="entity" entity_type="gene" role="intervention" text="apg10-1" ext_ids="850684" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG10" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">apg10-1</sd-tag></em> and Δ<em><sd-tag id="sdTag26" source="sdapi" category="entity" entity_type="gene" role="intervention" text="apg1" ext_ids="852695" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG1" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">apg1</sd-tag></em> cells carrying 3 × <sd-tag id="sdTag31" source="sdapi" category="entity" entity_type="protein" role="reporter" text="HA" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">HA</sd-tag>-<sd-tag id="sdTag27" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Apg12" ext_ids="P38316" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG12" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="https://www.uniprot.org/uniprot/">Apg12</sd-tag> plasmid (<strong>b</strong>) were <sd-tag id="sdTag28" source="sdapi" category="assay" entity_type="" role="" text="immunoblotted" ext_ids="BAO_0002424" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="western blot" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">immunoblotted</sd-tag> using anti-<sd-tag id="sdTag32" source="sdapi" category="entity" entity_type="protein" role="reporter" text="HA" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">HA</sd-tag> antibody. The positions of 3 × <sd-tag id="sdTag125" source="sdapi" category="entity" entity_type="protein" role="reporter" text="HA" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">HA</sd-tag>-<sd-tag id="sdTag33" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Apg12" ext_ids="P38316" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG12" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="https://www.uniprot.org/uniprot/">Apg12</sd-tag> and the larger product (asterisks) are indicated.</p><graphic href="https://api.sourcedata.io/file.php?panel_id=9790"/></sd-panel><sd-panel panel_id="9791"><strong>c</strong><span>, <sd-tag id="sdTag36" source="sdapi" category="assay" entity_type="" role="" text="Immunoblot" ext_ids="BAO_0002424" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="western blot" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">Immunoblot</sd-tag> analysis of wild-type and Δ</span><em><sd-tag id="sdTag37" source="sdapi" category="entity" entity_type="gene" role="intervention" text="apg12" ext_ids="852518" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG12" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">apg12</sd-tag></em> cells harbouring <sd-tag id="sdTag38" source="sdapi" category="entity" entity_type="protein" role="reporter" text="HA" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">HA</sd-tag><span>-<sd-tag id="sdTag35" source="sdapi" category="entity" entity_type="gene" role="intervention" text="APG5" ext_ids="855954" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG5" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">APG5</sd-tag> plasmid.</span><graphic href="https://api.sourcedata.io/file.php?panel_id=9791"/></sd-panel><sd-panel panel_id="9792"><strong>d</strong>, Δ<em><sd-tag id="sdTag44" source="sdapi" category="entity" entity_type="protein" role="assayed" text="apg5" ext_ids="Q12380" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG5" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="https://www.uniprot.org/uniprot/">apg5</sd-tag></em> Δ<em><sd-tag id="sdTag45" source="sdapi" category="entity" entity_type="protein" role="assayed" text="apg12" ext_ids="A6ZLF7///P38316" norm_text="" ext_dbs="Uniprot///Uniprot" in_caption="True" ext_names="ATG12///ATG12" ext_tax_ids="559292///307796" ext_tax_names="Saccharomyces cerevisiae S288C///Saccharomyces cerevisiae YJM789" ext_urls="https://www.uniprot.org/uniprot////https://www.uniprot.org/uniprot/">apg12</sd-tag></em> <span>cells were co-transformed with <sd-tag id="sdTag41" source="sdapi" category="entity" entity_type="protein" role="reporter" text="Myc" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">Myc</sd-tag>-</span><sd-tag id="sdTag46" source="sdapi" category="entity" entity_type="gene" role="component" text="APG12" ext_ids="852518" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG12" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">APG12</sd-tag> and <sd-tag id="sdTag47" source="sdapi" category="entity" entity_type="protein" role="reporter" text="HA" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">HA</sd-tag>-<sd-tag id="sdTag48" source="sdapi" category="entity" entity_type="gene" role="component" text="APG5" ext_ids="855954" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG5" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">APG5</sd-tag><span>. Their lysates were immunoprecipitated with anti-<sd-tag id="sdTag49" source="sdapi" category="entity" entity_type="protein" role="reporter" text="Myc" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">Myc</sd-tag> or anti-</span><sd-tag id="sdTag50" source="sdapi" category="entity" entity_type="protein" role="reporter" text="HA" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">HA</sd-tag> <span>antibodies and detected by <sd-tag id="sdTag42" source="sdapi" category="assay" entity_type="" role="" text="immunoblotting" ext_ids="BAO_0002424" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="western blot" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">immunoblotting</sd-tag> using anti-<sd-tag id="sdTag51" source="sdapi" category="entity" entity_type="protein" role="reporter" text="Myc" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">Myc</sd-tag> antibody. The position of the crossreacting <sd-tag id="sdTag43" source="sdapi" category="entity" entity_type="subcellular" role="component" text="IgG" ext_ids="GO:0071735" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="IgG immunoglobulin complex" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">IgG</sd-tag> heavy chain is indicated</span><graphic href="https://api.sourcedata.io/file.php?panel_id=9792"/></sd-panel></fig><fig id="2616"><title></title><label>figf3</label><graphic href="https://api.sourcedata.io/file.php?figure_id=2616"/><sd-panel panel_id="9794"><strong>b</strong>, Δ<em><sd-tag id="sdTag53" source="sdapi" category="entity" entity_type="gene" role="intervention" text="apg12" ext_ids="852518" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG12" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">apg12</sd-tag></em> cells were transformed with the mutant plasmids and their lysates were <sd-tag id="sdTag54" source="sdapi" category="assay" entity_type="" role="" text="immunoblotted" ext_ids="BAO_0002424" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="western blot" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">immunoblotted</sd-tag> with anti-<sd-tag id="sdTag55" source="sdapi" category="entity" entity_type="protein" role="reporter" text="HA" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">HA</sd-tag> antibody.<graphic href="https://api.sourcedata.io/file.php?panel_id=9794"/></sd-panel><sd-panel panel_id="9795"><p><span>c</span><span>, Autophagic activity was measured as described for Fig. 1d.</span></p><graphic href="https://api.sourcedata.io/file.php?panel_id=9795"/></sd-panel><sd-panel panel_id="9796"><strong>d</strong><span>, Transport of pro-<sd-tag id="sdTag65" source="sdapi" category="entity" entity_type="protein" role="assayed" text="API" ext_ids="P14904" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="APE1" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="https://www.uniprot.org/uniprot/">API</sd-tag> to the <sd-tag id="sdTag66" source="sdapi" category="entity" entity_type="subcellular" role="component" text="vacuole" ext_ids="GO:0005773" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="vacuole" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">vacuole</sd-tag> was examined by</span> <sd-tag id="sdTag67" source="sdapi" category="assay" entity_type="" role="" text="immunoblotting" ext_ids="BAO_0002424" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="western blot" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">immunoblotting</sd-tag> <span>with anti-<sd-tag id="sdTag69" source="sdapi" category="entity" entity_type="protein" role="assayed" text="API" ext_ids="P14904" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="APE1" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="https://www.uniprot.org/uniprot/">API</sd-tag> antiserum. The positions of pro-<sd-tag id="sdTag70" source="sdapi" category="entity" entity_type="protein" role="assayed" text="API" ext_ids="P14904" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="APE1" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="https://www.uniprot.org/uniprot/">API</sd-tag> and mature <sd-tag id="sdTag126" source="sdapi" category="entity" entity_type="protein" role="assayed" text="API" ext_ids="P14904" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="APE1" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="https://www.uniprot.org/uniprot/">API</sd-tag> are indicated.</span><graphic href="https://api.sourcedata.io/file.php?panel_id=9796"/></sd-panel></fig><fig id="2617"><title></title><label>figf4</label><graphic href="https://api.sourcedata.io/file.php?figure_id=2617"/><sd-panel panel_id="9798">Δ<em><sd-tag id="sdTag73" source="sdapi" category="entity" entity_type="gene" role="intervention" text="APG5" ext_ids="855954" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG5" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">apg5</sd-tag></em> cells were transformed with vector alone, wild-type <sd-tag id="sdTag74" source="sdapi" category="entity" entity_type="protein" role="assayed" text="APG5" ext_ids="Q12380" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG5" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="https://www.uniprot.org/uniprot/">APG5</sd-tag> or APG5K149R, and then <sd-tag id="sdTag75" source="sdapi" category="assay" entity_type="" role="" text="immunoblotted" ext_ids="BAO_0002424" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="western blot" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">immunoblotted</sd-tag> with anti-<sd-tag id="sdTag76" source="sdapi" category="entity" entity_type="protein" role="reporter" text="HA" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">HA</sd-tag> (<strong>b</strong>)<graphic href="https://api.sourcedata.io/file.php?panel_id=9798"/></sd-panel><sd-panel panel_id="9799"><span>Autophagic activity was determined by <sd-tag id="sdTag79" source="sdapi" category="assay" entity_type="" role="" text="alkaline phosphatase assay" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">alkaline phosphatase assay</sd-tag> (</span><strong>c</strong>).<graphic href="https://api.sourcedata.io/file.php?panel_id=9799"/></sd-panel><sd-panel panel_id="9800">Δ<em><sd-tag id="sdTag87" source="sdapi" category="entity" entity_type="gene" role="intervention" text="apg5" ext_ids="855954" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG5" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">apg5</sd-tag></em> cells were transformed with vector alone, wild-type APG5 or APG5K149R, and then <sd-tag id="sdTag88" source="sdapi" category="assay" entity_type="" role="" text="immunoblotted" ext_ids="BAO_0002424" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="western blot" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">immunoblotted</sd-tag> anti-<sd-tag id="sdTag89" source="sdapi" category="entity" entity_type="protein" role="assayed" text="API" ext_ids="P14904" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="APE1" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="https://www.uniprot.org/uniprot/">API</sd-tag> (<strong>d</strong>).<graphic href="https://api.sourcedata.io/file.php?panel_id=9800"/></sd-panel></fig><fig id="2618"><title></title><label>figf5</label><graphic href="https://api.sourcedata.io/file.php?figure_id=2618"/><sd-panel panel_id="9802"><strong>c</strong>, <sd-tag id="sdTag91" source="sdapi" category="assay" entity_type="" role="" text="In vitro conjugation" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls=""><em>In vitro</em> conjugation</sd-tag> <span>of <sd-tag id="sdTag92" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Apg5" ext_ids="Q12380" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG5" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="https://www.uniprot.org/uniprot/">Apg5</sd-tag> and</span> <sd-tag id="sdTag94" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Apg12" ext_ids="P38316" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG12" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="https://www.uniprot.org/uniprot/">Apg12</sd-tag>. A cell lysate of Δ<em><sd-tag id="sdTag95" source="sdapi" category="entity" entity_type="gene" role="component" text="apg12" ext_ids="852518" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG12" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">apg12</sd-tag></em> carrying <sd-tag id="sdTag96" source="sdapi" category="entity" entity_type="protein" role="reporter" text="HA" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">HA</sd-tag>-<sd-tag id="sdTag97" source="sdapi" category="entity" entity_type="gene" role="component" text="APG5" ext_ids="855954" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG5" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">APG5</sd-tag> (lane 1) was incubated with an equal amount of lysate from Δ<em><sd-tag id="sdTag98" source="sdapi" category="entity" entity_type="gene" role="component" text="apg5" ext_ids="855954" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG5" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">apg5</sd-tag></em> carrying <sd-tag id="sdTag99" source="sdapi" category="entity" entity_type="protein" role="reporter" text="HA" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">HA</sd-tag>-<sd-tag id="sdTag100" source="sdapi" category="entity" entity_type="gene" role="component" text="apg12" ext_ids="852518" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG12" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">APG12</sd-tag> <span>(2 µ plasmid) (lane 2) at 30 °C with (lane 4-6) or without (lane 7) 5 mM <sd-tag id="sdTag93" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="ATP" ext_ids="CHEBI:15422" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="ATP" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">ATP</sd-tag>. Samples were mixed with SDS-sample buffer at the times indicated.</span><graphic href="https://api.sourcedata.io/file.php?panel_id=9802"/></sd-panel></fig><fig id="2619"><title></title><label>figf6</label><graphic href="https://api.sourcedata.io/file.php?figure_id=2619"/><sd-panel panel_id="9803"><span><sd-tag id="sdTag103" source="sdapi" category="entity" entity_type="cell_type" role="component" text="Spheroplasts" ext_ids="CL:0000524" norm_text="" ext_dbs="CL" in_caption="True" ext_names="spheroplast" ext_tax_ids="" ext_tax_names="" ext_urls=" https://identifiers.org/">Spheroplasts</sd-tag> were generated from cells expressing either</span> <sd-tag id="sdTag116" source="sdapi" category="entity" entity_type="protein" role="reporter" text="HA" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">HA</sd-tag>-<span><sd-tag id="sdTag104" source="sdapi" category="entity" entity_type="gene" role="intervention" text="Apg12" ext_ids="852518" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG12" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">Apg12</sd-tag></span> or <sd-tag id="sdTag127" source="sdapi" category="entity" entity_type="protein" role="reporter" text="HA" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">HA</sd-tag>-<span><sd-tag id="sdTag105" source="sdapi" category="entity" entity_type="gene" role="intervention" text="Apg5" ext_ids="855954" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG5" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">Apg5</sd-tag></span>. Their lysates were mixed and layered on top of a 10-step (18-54 % w/w) sucrose gradient, and centrifuged at 174,000<em>g</em> for 2.5 h (ref. 29). Fifteen fractions were collected and the positions of free <sd-tag id="sdTag117" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Apg5" ext_ids="Q12380" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG5" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="https://www.uniprot.org/uniprot/">Apg5</sd-tag>, free <sd-tag id="sdTag118" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Apg12" ext_ids="P38316" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG12" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="https://www.uniprot.org/uniprot/">Apg12</sd-tag> and <sd-tag id="sdTag119" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Apg5" ext_ids="Q12380" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG5" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="https://www.uniprot.org/uniprot/">Apg5</sd-tag>/<sd-tag id="sdTag120" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Apg12" ext_ids="P38316" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG12" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="https://www.uniprot.org/uniprot/">Apg12</sd-tag> <span>conjugate were examined by <sd-tag id="sdTag106" source="sdapi" category="assay" entity_type="" role="" text="western blotting" ext_ids="BAO_0002424" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="western blot" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">western blotting</sd-tag>. The peak fractions of <sd-tag id="sdTag107" source="sdapi" category="entity" entity_type="protein" role="assayed" text="alcohol dehydrogenase" ext_ids="S5RK20" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ADH1" ext_tax_ids="4932" ext_tax_names="Saccharomyces cerevisiae" ext_urls="https://www.uniprot.org/uniprot/">alcohol dehydrogenase</sd-tag> (ADH)(<sd-tag id="sdTag111" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="cytosol" ext_ids="GO:0005829" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="cytosol" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">cytosol</sd-tag>), <sd-tag id="sdTag108" source="sdapi" category="entity" entity_type="protein" role="assayed" text="ALP" ext_ids="P11491" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="PHO8" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="https://www.uniprot.org/uniprot/">ALP</sd-tag> (</span><sd-tag id="sdTag121" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="vacuole" ext_ids="GO:0005773" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="vacuole" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">vacuole</sd-tag><span>), <sd-tag id="sdTag109" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Kex2" ext_ids="P13134" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="KEX2" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="https://www.uniprot.org/uniprot/">Kex2</sd-tag> (<sd-tag id="sdTag112" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="Golgi" ext_ids="GO:0005794" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="Golgi apparatus" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">Golgi</sd-tag>) and <sd-tag id="sdTag110" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Sec12" ext_ids="P11655" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="SEC12" ext_tax_ids="559292" ext_tax_names="Saccharomyces cerevisiae S288C" ext_urls="https://www.uniprot.org/uniprot/">Sec12</sd-tag> (<sd-tag id="sdTag113" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="endoplasmic reticulum" ext_ids="GO:0005783" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="endoplasmic reticulum" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">endoplasmic reticulum</sd-tag>) are indicated by arrows.</span><graphic href="https://api.sourcedata.io/file.php?panel_id=9803"/></sd-panel></fig></article> |
