Systematic Analysis of Human Cells Lacking ATG8 Proteins Uncovers Roles for GABARAPs and the CCZ1/MON1 Regulator C18orf8/RMC1 in Macroautophagic and Selective Autophagic Flux.

Selective autophagy and macroautophagy sequester specific organelles/substrates or bulk cytoplasm, respectively, inside autophagosomes as cargo for delivery to lysosomes. The mammalian ATG8 orthologues (MAP1LC3A/B/C and GABARAP/L1/L2) are ubiquitin (UB)-like proteins conjugated to the autophagosome membrane and are thought to facilitate cargo receptor recruitment, vesicle maturation, and lysosomal fusion. To elucidate the molecular functions of the ATG8 proteins, we engineered cells lacking genes for each subfamily as well as all six mammalian ATG8s. Loss of GABARAPs alone attenuates autophagic flux basally and in response to macroautophagic or selective autophagic stimuli, including parkin-dependent mitophagy, and cells lacking all ATG8 proteins accumulate cytoplasmic UB aggregates, which are resolved following ectopic expression of individual GABARAPs. Autophagosomes from cells lacking GABARAPs had reduced lysosomal content by quantitative proteomics, consistent with fusion defects, but accumulated regulators of late endosome (LE)/autophagosome maturation. Through interaction proteomics of proteins accumulating in GABARAP/L1/L2-deficient cells, we identified C18orf8/RMC1 as a new subunit of the CCZ1-MON1 RAB7 guanine exchange factor (GEF) that positively regulates RAB7 recruitment to LE/autophagosomes. This work defines unique roles for GABARAP and LC3 subfamilies in macroautophagy and selective autophagy and demonstrates how analysis of autophagic machinery in the absence of flux can identify new regulatory circuits.

Vaites LP ，Paulo JA ，Huttlin EL ，Harper JW ... -  《-》

Atg8 family LC3/GABARAP proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation.

Members of the Atg8 family of proteins are conjugated to autophagosomal membranes, where they have been proposed to drive autophagosome formation and selective sequestration of cargo. In mammals, the Atg8 family consists of six members divided into the LC3 and GABARAP subfamilies. To define Atg8 function, we used genome editing to generate knockouts of the LC3 and GABARAP subfamilies as well as all six Atg8 family members in HeLa cells. We show that Atg8s are dispensable for autophagosome formation and selective engulfment of mitochondria, but essential for autophagosome-lysosome fusion. We find that the GABARAP subfamily promotes PLEKHM1 recruitment and governs autophagosome-lysosome fusion, whereas the LC3 subfamily plays a less prominent role in these processes. Although neither GABARAPs nor LC3s are required for autophagosome biogenesis, loss of all Atg8s yields smaller autophagosomes and a slowed initial rate of autophagosome formation. Our results clarify the essential function of the Atg8 family and identify GABARAP subfamily members as primary contributors to PINK1/Parkin mitophagy and starvation autophagy.

Nguyen TN ，Padman BS ，Usher J ，Oorschot V ，Ramm G ，Lazarou M ... -  《-》

Autophagosome formation and cargo sequestration in the absence of LC3/GABARAPs.

Padman BS ，Nguyen TN ，Lazarou M 《-》

Methods for Studying Interactions Between Atg8/LC3/GABARAP and LIR-Containing Proteins.

LC3/GABARAP proteins (LC3/GABARAPs) are mammalian orthologues of yeast Atg8, small ubiquitin (Ub)-like proteins (UBLs) whose covalent attachment to lipid membranes is crucial for the growth and closure of the double membrane vesicle called the autophagosome. In the past decade, it was demonstrated that Atg8/LC3/GABARAPs are also required for autophagic degradation of cargos in a selective fashion. Cargo selectivity is ensured by receptor proteins, such as p62/SQSTM1, NBR1, Cue5, Atg19, NIX, Atg32, NCOA4, and FAM134B, which simultaneously bind Atg8/LC3/GABARAPs and the cargo together, thereby linking the core autophagic machinery to the target structure: a protein, an organelle, or a pathogen. LC3-interacting regions (LIRs) are short linear motifs within selective autophagy receptors and some other structural and signaling proteins (e.g., ULK1, ATG13, FIP200, and Dvl2), which mediate binding to Atg8/LC3/GABARAPs. Identification and characterization of LIR-containing proteins have provided important insights into the biology of the autophagy pathway, and studying their interactions with the core autophagy machinery represents a growing area of autophagy research. Here, we present protocols for the identification of LIR-containing proteins, i.e., by yeast-two-hybrid screening, glutathione S-transferase (GST) pulldown experiments, and peptide arrays. The use of two-dimensional peptide arrays also represents a powerful method to identify the residues of the LIR motif that are critical for binding. We also describe a biophysical method for studying interactions between Atg8/LC3/GABARAP and LIR-containing proteins and a protocol for preparation and purification of LIR peptides.

Johansen T ，Birgisdottir ÅB ，Huber J ，Kniss A ，Dötsch V ，Kirkin V ，Rogov VV ... -  《-》

Molecular mechanism to target the endosomal Mon1-Ccz1 GEF complex to the pre-autophagosomal structure.

During autophagy, a newly formed double membrane surrounds its cargo to generate the so-called autophagosome, which then fuses with a lysosome after closure. Previous work implicated that endosomal Rab7/Ypt7 associates to autophagosomes prior to their fusion with lysosomes. Here, we unravel how the Mon1-Ccz1 guanosine exchange factor (GEF) acting upstream of Ypt7 is specifically recruited to the pre-autophagosomal structure under starvation conditions. We find that Mon1-Ccz1 directly binds to Atg8, the yeast homolog of the members of the mammalian LC3 protein family. This requires at least one LIR motif in the Ccz1 C-terminus, which is essential for autophagy but not for endosomal transport. In agreement, only wild-type, but not LIR-mutated Mon1-Ccz1 promotes Atg8-dependent activation of Ypt7. Our data reveal how GEF targeting can specify the fate of a newly formed organelle and provide new insights into the regulation of autophagosome-lysosome fusion.

Gao J ，Langemeyer L ，Kümmel D ，Reggiori F ，Ungermann C ... -  《eLife》