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An Atg9-containing compartment that functions in the early steps of autophagosome biogenesis.

Mari M, Griffith J, Rieter E, Krishnappa L, Klionsky DJ, Reggiori F - J. Cell Biol. (2010)

Bottom Line: Eukaryotes use the process of autophagy, in which structures targeted for lysosomal/vacuolar degradation are sequestered into double-membrane autophagosomes, in numerous physiological and pathological situations.The key questions in the field relate to the origin of the membranes as well as the precise nature of the rearrangements that lead to the formation of autophagosomes.We show that these clusters translocate en bloc next to the vacuole to form the phagophore assembly site (PAS), where they become the autophagosome precursor, the phagophore.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology and Institute of Biomembranes, University Medical Centre Utrecht, Utrecht University, 3584 CX Utrecht, The Netherlands.

ABSTRACT
Eukaryotes use the process of autophagy, in which structures targeted for lysosomal/vacuolar degradation are sequestered into double-membrane autophagosomes, in numerous physiological and pathological situations. The key questions in the field relate to the origin of the membranes as well as the precise nature of the rearrangements that lead to the formation of autophagosomes. We found that yeast Atg9 concentrates in a novel compartment comprising clusters of vesicles and tubules, which are derived from the secretory pathway and are often adjacent to mitochondria. We show that these clusters translocate en bloc next to the vacuole to form the phagophore assembly site (PAS), where they become the autophagosome precursor, the phagophore. In addition, genetic analyses indicate that Atg1, Atg13, and phosphatidylinositol-3-phosphate are involved in the further rearrangement of these initial membranes. Thus, our data reveal that the Atg9-positive compartments are important for the de novo formation of the PAS and the sequestering vesicle that are the hallmarks of autophagy.

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Model for the role of the Atg9 reservoirs in double-membrane vesicle formation. The Atg9 reservoirs, which often are adjacent to mitochondria, act as a pre-PAS. Association with the prApe1 oligomer in nutrient-rich conditions (Cvt pathway) and probably cellular signals during starvation (autophagy) induces the translocation of one or more Atg9 reservoirs into close proximity with the vacuole. This relocalization event triggers the recruitment of the rest of the Atg proteins to a reservoir, leading to the formation of the PAS. Successive fusion of the tubulovesicular membranes composing the PAS and possibly acquisition of additional membrane from other Atg9 reservoirs and/or other sources creates a double-membrane vesicle.
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fig10: Model for the role of the Atg9 reservoirs in double-membrane vesicle formation. The Atg9 reservoirs, which often are adjacent to mitochondria, act as a pre-PAS. Association with the prApe1 oligomer in nutrient-rich conditions (Cvt pathway) and probably cellular signals during starvation (autophagy) induces the translocation of one or more Atg9 reservoirs into close proximity with the vacuole. This relocalization event triggers the recruitment of the rest of the Atg proteins to a reservoir, leading to the formation of the PAS. Successive fusion of the tubulovesicular membranes composing the PAS and possibly acquisition of additional membrane from other Atg9 reservoirs and/or other sources creates a double-membrane vesicle.

Mentions: In addition to the similar morphology between the Atg9 reservoirs and the PAS (Figs. 1, 6, S1, and S4), one of our unpredicted discoveries has been the en bloc translocation of the reservoirs to form the PAS next to the vacuole. Our time-lapse fluorescence microscopy showed that the Atg machinery can be recruited to a single Atg9 reservoir (Fig. 9 and Videos 1 and 2). When the Atg9 movement was triggered from the reservoirs to the PAS, we did not observe an increase in the number of isolated Atg9-containing vesicles and tubules in the cytoplasm (Fig. 8). These data support the notion that the Atg9 reservoirs move as clusters. This observation fits with our previous studies showing Atg9 present in cytoplasmic clusters (Reggiori et al., 2004a, 2005) and the demonstration that Atg9 self-interacts (Reggiori et al., 2005; He et al., 2008). An alternative model is that the PAS is generated by a small cluster of vesicles and/or tubules that results from the fragmentation of an Atg9 reservoir, but we do not consider this likely because we have never seen such a scission event during the live-cell imaging experiments, and small clusters comprising less than six vesicular and/or tubular profiles have only rarely been observed in our IEM preparations (see Materials and methods). All together, our data allow us to postulate a model where at least one Atg9 reservoir acts as a pre-PAS and where a change in localization of this compartment determines the biogenesis of the PAS (Fig. 10). The movement of an Atg9 reservoir in close proximity to the vacuole triggers the hierarchical recruitment of the remaining Atg proteins that mediate the rearrangement of these vesicles and tubules into what becomes the phagophore. It remains to be determined which factors on the vacuole limiting membrane or adjacent to it induce the Atg machinery assembly. Nonetheless, at this time we cannot conclusively rule out alternative models, and this hypothesis has to be experimentally demonstrated in future.


An Atg9-containing compartment that functions in the early steps of autophagosome biogenesis.

Mari M, Griffith J, Rieter E, Krishnappa L, Klionsky DJ, Reggiori F - J. Cell Biol. (2010)

Model for the role of the Atg9 reservoirs in double-membrane vesicle formation. The Atg9 reservoirs, which often are adjacent to mitochondria, act as a pre-PAS. Association with the prApe1 oligomer in nutrient-rich conditions (Cvt pathway) and probably cellular signals during starvation (autophagy) induces the translocation of one or more Atg9 reservoirs into close proximity with the vacuole. This relocalization event triggers the recruitment of the rest of the Atg proteins to a reservoir, leading to the formation of the PAS. Successive fusion of the tubulovesicular membranes composing the PAS and possibly acquisition of additional membrane from other Atg9 reservoirs and/or other sources creates a double-membrane vesicle.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3101592&req=5

fig10: Model for the role of the Atg9 reservoirs in double-membrane vesicle formation. The Atg9 reservoirs, which often are adjacent to mitochondria, act as a pre-PAS. Association with the prApe1 oligomer in nutrient-rich conditions (Cvt pathway) and probably cellular signals during starvation (autophagy) induces the translocation of one or more Atg9 reservoirs into close proximity with the vacuole. This relocalization event triggers the recruitment of the rest of the Atg proteins to a reservoir, leading to the formation of the PAS. Successive fusion of the tubulovesicular membranes composing the PAS and possibly acquisition of additional membrane from other Atg9 reservoirs and/or other sources creates a double-membrane vesicle.
Mentions: In addition to the similar morphology between the Atg9 reservoirs and the PAS (Figs. 1, 6, S1, and S4), one of our unpredicted discoveries has been the en bloc translocation of the reservoirs to form the PAS next to the vacuole. Our time-lapse fluorescence microscopy showed that the Atg machinery can be recruited to a single Atg9 reservoir (Fig. 9 and Videos 1 and 2). When the Atg9 movement was triggered from the reservoirs to the PAS, we did not observe an increase in the number of isolated Atg9-containing vesicles and tubules in the cytoplasm (Fig. 8). These data support the notion that the Atg9 reservoirs move as clusters. This observation fits with our previous studies showing Atg9 present in cytoplasmic clusters (Reggiori et al., 2004a, 2005) and the demonstration that Atg9 self-interacts (Reggiori et al., 2005; He et al., 2008). An alternative model is that the PAS is generated by a small cluster of vesicles and/or tubules that results from the fragmentation of an Atg9 reservoir, but we do not consider this likely because we have never seen such a scission event during the live-cell imaging experiments, and small clusters comprising less than six vesicular and/or tubular profiles have only rarely been observed in our IEM preparations (see Materials and methods). All together, our data allow us to postulate a model where at least one Atg9 reservoir acts as a pre-PAS and where a change in localization of this compartment determines the biogenesis of the PAS (Fig. 10). The movement of an Atg9 reservoir in close proximity to the vacuole triggers the hierarchical recruitment of the remaining Atg proteins that mediate the rearrangement of these vesicles and tubules into what becomes the phagophore. It remains to be determined which factors on the vacuole limiting membrane or adjacent to it induce the Atg machinery assembly. Nonetheless, at this time we cannot conclusively rule out alternative models, and this hypothesis has to be experimentally demonstrated in future.

Bottom Line: Eukaryotes use the process of autophagy, in which structures targeted for lysosomal/vacuolar degradation are sequestered into double-membrane autophagosomes, in numerous physiological and pathological situations.The key questions in the field relate to the origin of the membranes as well as the precise nature of the rearrangements that lead to the formation of autophagosomes.We show that these clusters translocate en bloc next to the vacuole to form the phagophore assembly site (PAS), where they become the autophagosome precursor, the phagophore.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology and Institute of Biomembranes, University Medical Centre Utrecht, Utrecht University, 3584 CX Utrecht, The Netherlands.

ABSTRACT
Eukaryotes use the process of autophagy, in which structures targeted for lysosomal/vacuolar degradation are sequestered into double-membrane autophagosomes, in numerous physiological and pathological situations. The key questions in the field relate to the origin of the membranes as well as the precise nature of the rearrangements that lead to the formation of autophagosomes. We found that yeast Atg9 concentrates in a novel compartment comprising clusters of vesicles and tubules, which are derived from the secretory pathway and are often adjacent to mitochondria. We show that these clusters translocate en bloc next to the vacuole to form the phagophore assembly site (PAS), where they become the autophagosome precursor, the phagophore. In addition, genetic analyses indicate that Atg1, Atg13, and phosphatidylinositol-3-phosphate are involved in the further rearrangement of these initial membranes. Thus, our data reveal that the Atg9-positive compartments are important for the de novo formation of the PAS and the sequestering vesicle that are the hallmarks of autophagy.

Show MeSH
Related in: MedlinePlus