Autophagy is a unique membrane trafficking pathway describing the formation and targeting of double membrane autophagosomes to the vacuole/lysosome. The biogenesis of autophagosomes and their delivery to the vacuole/lysosome depend on multiple membrane fusion events. Using a cell-free system, we have investigated the ability of LC3 and GATE-16, two mammalian Atg8 orthologs, to mediate membrane fusion. We found that both proteins promote tethering and membrane fusion, mediated by the proteins' N-terminal α helices. We further show that short, 10 amino acid long synthetic peptides derived from the N terminus of LC3 or GATE-16 are sufficient to promote membrane fusion. Our data indicate that the fusion activity of LC3 is mediated by positively charged amino acids, whereas the activity of GATE-16 is mediated by hydrophobic interactions. Finally, we demonstrate that LC3 and GATE-16 N termini in general and specific residues needed for the fusion activity are essential for the proteins role in autophagosome biogenesis.

Autophagy is a lysosome-mediated degradative system that is a highly conserved pathway present in all eukaryotes. In all cells, double-membrane autophagosomes form and engulf cytoplasmic components, delivering them to the lysosome for degradation. Autophagy is essential for cell health and can be activated to function as a recycling pathway in the absence of nutrients or as a quality-control pathway to eliminate damaged organelles or even to eliminate invading pathogens. Autophagy was first identified as a pathway in mammalian cells using morphological techniques, but the Atg (autophagy-related) genes required for autophagy were identified in yeast genetic screens. Despite tremendous advances in elucidating the function of individual Atg proteins, our knowledge of how autophagosomes form and subsequently interact with the endosomal pathway has lagged behind. Recent progress toward understanding where and how both the endocytotic and autophagic pathways overlap is reviewed here.A utophagy is a lysosome-mediated pathway for the degradation of cytosolic proteins and organelles, which is essential for cell homeostasis, development, and for the prevention of several human diseases and infection (Choi et al. 2013). Importantly, autophagy cannot occur without an active lysosome. However, it is becoming increasingly recognized that the endosomal pathway plays a greater role than just providing the degradative enzymes found in the lysosome. Recent data suggest that in mammalian cells multiple contributions from several stages of the endocytic pathway are essential for efficient autophagy. Here we outline the autophagic pathway and then address the recent data on how different endosomal compartments contribute to autophagy, and the molecular machinery required for the interaction of the endosome and lysosome during the formation, and consumption of the autophagosome. Given the model emerging that the amino-acidsensitive autophagic pathway originates from the endoplasmic reticulum (ER), several questions arise, including how do recognition and productive interaction occur between an ERderived membrane and endosomes? How are these interactions mediated, and which are essential for efficient autophagy?

Autophagy is the main cellular catabolic process responsible for degrading organelles and large protein aggregates. It is initiated by the formation of a unique membrane structure, the phagophore, which engulfs part of the cytoplasm and forms a double-membrane vesicle termed the autophagosome. Fusion of the outer autophagosomal membrane with the lysosome and degradation of the inner membrane contents complete the process. The extent of autophagy must be tightly regulated to avoid destruction of proteins and organelles essential for cell survival. Autophagic activity is thus regulated by external and internal cues, which initiate the formation of well-defined autophagy-related protein complexes that mediate autophagosome formation and selective cargo recruitment into these organelles. Autophagosome formation and the signaling pathways that regulate it have recently attracted substantial attention. In this review, we analyze the different signaling pathways that regulate autophagy and discuss recent progress in our understanding of autophagosome biogenesis.

Autophagy is a major intracellular trafficking pathway that delivers proteins and organelles from the cytoplasm into lysosomes for consequential degradation and recycling. Mammalian Atg8s are key autophagic factors that undergo a unique ubiquitin-like conjugation to the lipid phase of the autophagosomal membrane. In addition to their activity in autophagosome formation, several Atg8s directly bind p62/SQSTM1. Here we show that LC3 and GATE-16 differ in their mode of p62 binding. While the soluble form of both LC3 and GATE-16 bind p62, only the lipidated form of LC3 is directly involved in p62 recruitment into autophagosomes. Moreover, by utilizing chimeras of LC3 and GATE-16 where their N-terminus was swapped, we determined the regions responsible for this differential binding. Accordingly, we found that the chimera of GATE-16 containing the LC3 N-terminal region acts similarly to wild-type LC3 in recruiting p62 into autophagosomes. We therefore propose that LC3 is responsible for the final stages of p62 incorporation into autophagosomes, a process selectively mediated by its N-terminus.

SignificanceThe contribution of SNARE-mediated membrane fusion to the different stages along the autophagic process is not well understood. In this work we demonstrate that inhibition of SNARE priming leads to impairment of the autophagic flux manifested by the accumulation of mature autophagosomes that do not fuse with the lysosomes. Our results suggest that SNARE priming is essential for the fusion of the autophagosome with the lysosome but not for autophagosome formation.