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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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Atg9 is transported through part of the secretory pathway. (A) Atg9 is translocated into the ER. The wild-type (MMY126) and sec12 (MMY129) cells expressing Sec63-mChe-V5 and carrying the pGalATG9GFP416 plasmid were grown in SMD at 24°C before being transferred into a galactose-containing medium. Cultures were subsequently split and separately incubated at either 24°C or 37°C for 2 h before imaging. No fluorescence signal was detected when cells were grown in the presence of glucose (not depicted). (B) Atg9 passes through the Golgi before reaching its final destination. Wild-type (MMY125) and sec7 (MMY127) cells expressing genomically mChe-V5–tagged Sec7 and Sec7ts, respectively, and transformed with the pGalATG9GFP416 plasmid were analyzed as in A. Arrows highlight the colocalization between Atg9-GFP and Sec7ts-mChe in sec7ts cells at 37°C. DIC, differential interference contrast. Bars, 2 µm.
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fig4: Atg9 is transported through part of the secretory pathway. (A) Atg9 is translocated into the ER. The wild-type (MMY126) and sec12 (MMY129) cells expressing Sec63-mChe-V5 and carrying the pGalATG9GFP416 plasmid were grown in SMD at 24°C before being transferred into a galactose-containing medium. Cultures were subsequently split and separately incubated at either 24°C or 37°C for 2 h before imaging. No fluorescence signal was detected when cells were grown in the presence of glucose (not depicted). (B) Atg9 passes through the Golgi before reaching its final destination. Wild-type (MMY125) and sec7 (MMY127) cells expressing genomically mChe-V5–tagged Sec7 and Sec7ts, respectively, and transformed with the pGalATG9GFP416 plasmid were analyzed as in A. Arrows highlight the colocalization between Atg9-GFP and Sec7ts-mChe in sec7ts cells at 37°C. DIC, differential interference contrast. Bars, 2 µm.

Mentions: To follow the Atg9 biosynthetic route, Atg9-GFP expression was put under the control of the galactose-inducible GAL1 promoter. Transfer into a galactose-containing medium for 2 h allowed us to detect Atg9 clusters (Fig. 4). No intermediate structures, e.g., mitochondria or ER, were visualized even at earlier time points, probably due to the rapid transport of Atg9 after synthesis. To further explore whether Atg9 is transported through the secretory pathway, we used a strain carrying a thermosensitive allele of SEC12, which blocks ER exit (Barlowe and Schekman, 1993). At a permissive temperature, the localization pattern of Atg9-GFP was indistinguishable from that observed in wild-type cells (Fig. 4 A). At a restrictive temperature, in contrast, the transport block between the ER and Golgi apparatus resulted in the accumulation of newly synthesized Atg9 in the ER, as revealed by colocalization with the specific protein marker Sec63 (Fig. 4 A; Deshaies et al., 1991). This transport block was reversible, which indicates that Atg9-GFP was not amassed in a terminal structure but rather accumulated in a transport intermediate (unpublished data). To examine the role of the Golgi complex in Atg9 transport, we used a thermosensitive sec7 allele, which blocks protein traffic from this organelle but does not affect localization of the protein (Franzusoff and Schekman, 1989; Jackson and Casanova, 2000). At 24°C, Atg9-GFP was again normally distributed to several cytoplasmic puncta, and those were only rarely positive for the late Golgi compartment protein marker Sec7 (Fig. 4 B; Losev et al., 2006). In contrast, at 37°C, newly synthesized Atg9-GFP was present in circular structures, often positive for Sec7ts tagged with Discosoma red fluorescent protein (dsRed), which occasionally had an elongated conformation (Fig. 4 B, arrows). These structures are likely Berkeley bodies, aberrant Golgi generated as a result of the sec7 sorting defect (Novick et al., 1980). Again, the transport block was reversible, which indicates that Atg9-GFP was not accumulated in a terminal structure (unpublished data). We concluded that Atg9 is translocated into the ER and reaches its final destination, the Atg9 clusters, probably after passing through the Golgi.


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)

Atg9 is transported through part of the secretory pathway. (A) Atg9 is translocated into the ER. The wild-type (MMY126) and sec12 (MMY129) cells expressing Sec63-mChe-V5 and carrying the pGalATG9GFP416 plasmid were grown in SMD at 24°C before being transferred into a galactose-containing medium. Cultures were subsequently split and separately incubated at either 24°C or 37°C for 2 h before imaging. No fluorescence signal was detected when cells were grown in the presence of glucose (not depicted). (B) Atg9 passes through the Golgi before reaching its final destination. Wild-type (MMY125) and sec7 (MMY127) cells expressing genomically mChe-V5–tagged Sec7 and Sec7ts, respectively, and transformed with the pGalATG9GFP416 plasmid were analyzed as in A. Arrows highlight the colocalization between Atg9-GFP and Sec7ts-mChe in sec7ts cells at 37°C. DIC, differential interference contrast. Bars, 2 µm.
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Related In: Results  -  Collection

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fig4: Atg9 is transported through part of the secretory pathway. (A) Atg9 is translocated into the ER. The wild-type (MMY126) and sec12 (MMY129) cells expressing Sec63-mChe-V5 and carrying the pGalATG9GFP416 plasmid were grown in SMD at 24°C before being transferred into a galactose-containing medium. Cultures were subsequently split and separately incubated at either 24°C or 37°C for 2 h before imaging. No fluorescence signal was detected when cells were grown in the presence of glucose (not depicted). (B) Atg9 passes through the Golgi before reaching its final destination. Wild-type (MMY125) and sec7 (MMY127) cells expressing genomically mChe-V5–tagged Sec7 and Sec7ts, respectively, and transformed with the pGalATG9GFP416 plasmid were analyzed as in A. Arrows highlight the colocalization between Atg9-GFP and Sec7ts-mChe in sec7ts cells at 37°C. DIC, differential interference contrast. Bars, 2 µm.
Mentions: To follow the Atg9 biosynthetic route, Atg9-GFP expression was put under the control of the galactose-inducible GAL1 promoter. Transfer into a galactose-containing medium for 2 h allowed us to detect Atg9 clusters (Fig. 4). No intermediate structures, e.g., mitochondria or ER, were visualized even at earlier time points, probably due to the rapid transport of Atg9 after synthesis. To further explore whether Atg9 is transported through the secretory pathway, we used a strain carrying a thermosensitive allele of SEC12, which blocks ER exit (Barlowe and Schekman, 1993). At a permissive temperature, the localization pattern of Atg9-GFP was indistinguishable from that observed in wild-type cells (Fig. 4 A). At a restrictive temperature, in contrast, the transport block between the ER and Golgi apparatus resulted in the accumulation of newly synthesized Atg9 in the ER, as revealed by colocalization with the specific protein marker Sec63 (Fig. 4 A; Deshaies et al., 1991). This transport block was reversible, which indicates that Atg9-GFP was not amassed in a terminal structure but rather accumulated in a transport intermediate (unpublished data). To examine the role of the Golgi complex in Atg9 transport, we used a thermosensitive sec7 allele, which blocks protein traffic from this organelle but does not affect localization of the protein (Franzusoff and Schekman, 1989; Jackson and Casanova, 2000). At 24°C, Atg9-GFP was again normally distributed to several cytoplasmic puncta, and those were only rarely positive for the late Golgi compartment protein marker Sec7 (Fig. 4 B; Losev et al., 2006). In contrast, at 37°C, newly synthesized Atg9-GFP was present in circular structures, often positive for Sec7ts tagged with Discosoma red fluorescent protein (dsRed), which occasionally had an elongated conformation (Fig. 4 B, arrows). These structures are likely Berkeley bodies, aberrant Golgi generated as a result of the sec7 sorting defect (Novick et al., 1980). Again, the transport block was reversible, which indicates that Atg9-GFP was not accumulated in a terminal structure (unpublished data). We concluded that Atg9 is translocated into the ER and reaches its final destination, the Atg9 clusters, probably after passing through the Golgi.

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