SUMMARY Macroautophagy mediates the degradation of long-lived proteins and organelles via the de novo formation of double-membrane autophagosomes that sequester cytoplasm and deliver it to the vacuole/lysosome; however, relatively little is known about autophagosome biogenesis. Atg8, a phosphatidylethanolamine-conjugated protein, was previously proposed to function in autophagosome membrane expansion, based on the observation that it mediates liposome tethering and hemifusion in vitro. We show here that with physiological concentrations of phosphatidylethanolamine, Atg8 does not act as a fusogen. Rather, we provide evidence for the involvement of exocytic Q/t-SNAREs in autophagosome formation, acting in the recruitment of key autophagy components to the site of autophagosome formation, and in regulating the organization of Atg9 into tubulovesicular clusters. Additionally, we found that the endosomal Q/t-SNARE Tlg2 and the R/v-SNAREs Sec22 and Ykt6 interact with Sso1-Sec9, and are required for normal Atg9 transport. Thus, multiple SNARE-mediated fusion events are likely to be involved in autophagosome biogenesis.

f/f/p mice was decreased by 50% compared with wild-type mice, whereas baseline ventilation and the hypoxic ventilatory response were normal. In addition, Lmx1b f/f/p mice rapidly became hypothermic when exposed to an ambient temperature of 4°C, decreasing core temperature to 30°C within 120 min. This failure of thermoregulation was caused by impaired shivering and nonshivering thermogenesis, whereas thermosensory perception and heat conservation were normal. Finally, intracerebroventricular infusion of 5-HT stimulated baseline ventilation, and rescued the blunted hypercapnic ventilatory response. These data identify a previously unrecognized role of 5-HT neurons in the CO 2 chemoreflex, whereby they enhance the response of the rest of the respiratory network to CO 2 . We conclude that the proper function of the 5-HT system is particularly important under conditions of environmental stress and contributes significantly to the hypercapnic ventilatory response and thermoregulatory cold defense.

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The Atg8/LC3/GABARAP protein family has been implicated in membrane remodeling events on the growing autophagosome. In particular, each of these proteins can form a protein-lipid conjugate that has been shown in vitro to drive liposome aggregation and in some cases membrane fusion. Furthermore, yeast Atg8 has been described as a curvature sensing protein, through its natural capacity to concentrate on highly curved membranes. A key advance with yeast Atg8, was the introduction of Giant Unilamellar Vesicles (GUVs) as an in vitro support that could allow membrane deformation and tethering to be observed by simple microscopy. Further, micromanipulation of an individual GUV could be used to create local areas of curvature to follow Atg8 partitioning. Here, we use a recently developed method to decorate GUVs with the mammalian Atg8 protein GABARAPL1 and establish the generality of the observations made on yeast Atg8. Then we apply double micromanipulation, the capture and positioning of two independently prepared GUVs, to test elements of the mechanism, speed and reversibility of mammalian Atg8 protein-mediated tethering. We find that the membranes adhere through GABARAPL1/GABARAPL1 homotypic trans-interactions. On a single membrane with two regions with significantly different curvatures we observed that the regions of higher curvature can be enriched up to 10 times in GABARAPL1 compared to the planar regions. This approach has the potential to allow the formation and study of specific topographically-controlled interfaces involving Atg8-proteins and their targets on apposing membranes.