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Cdc48/p97 and Shp1/p47 regulate autophagosome biogenesis in concert with ubiquitin-like Atg8.

Krick R, Bremer S, Welter E, Schlotterhose P, Muehe Y, Eskelinen EL, Thumm M - J. Cell Biol. (2010)

Bottom Line: Interaction with Shp1 requires an FK motif within the N-terminal non-ubiquitin-like Atg8 domain.Based on our data, we speculate that autophagosome formation, in contrast to Golgi reassembly, requires a complex in which Atg8 functionally substitutes ubiquitin.This, for the first time, would give a rationale for use of the ubiquitin-like Atg8 during macroautophagy and would explain why Atg8-PE delipidation is necessary for efficient macroautophagy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry II, Georg-August University, D-37073 Goettingen, Germany.

ABSTRACT
The molecular details of the biogenesis of double-membraned autophagosomes are poorly understood. We identify the Saccharomyces cerevisiae AAA-adenosine triphosphatase Cdc48 and its substrate-recruiting cofactor Shp1/Ubx1 as novel components needed for autophagosome biogenesis. In mammals, the Cdc48 homologue p97/VCP and the Shp1 homologue p47 mediate Golgi reassembly by extracting an unknown monoubiquitinated fusion regulator from a complex. We find no requirement of ubiquitination or the proteasome system for autophagosome biogenesis but detect interaction of Shp1 with the ubiquitin-fold autophagy protein Atg8. Atg8 coupled to phosphatidylethanolamine (PE) is crucial for autophagosome elongation and, in vitro, mediates tethering and hemifusion. Interaction with Shp1 requires an FK motif within the N-terminal non-ubiquitin-like Atg8 domain. Based on our data, we speculate that autophagosome formation, in contrast to Golgi reassembly, requires a complex in which Atg8 functionally substitutes ubiquitin. This, for the first time, would give a rationale for use of the ubiquitin-like Atg8 during macroautophagy and would explain why Atg8-PE delipidation is necessary for efficient macroautophagy.

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Shp1 affects autophagosome biogenesis. (a and b) Fluorescence microscopy of starved cells showed defective vacuolar uptake of GFP-Atg8. No GFP-Atg8–positive autophagosomes accumulated in the cytosol. (c) Electron microscopy of starved shp1Δ cells showed no vacuolar accumulation of autophagic bodies. (d) Lysed spheroplasts of starved cells were trypsin digested with and without detergent. Immunoblots with GFP antibodies showed proteolysis-resistant GFP-Atg8 (inside autophagosomes) in ypt7Δ but not in wild-type and shp1Δ cells. GFP-Atg8 breakdown yields GFP*. (e) Cells with a GFP-Atg8–positive PAS punctum were scored in fluorescence microscopy. The mean and SD of two experiments are shown, with >200 cells analyzed per strain. (f) To analyze Atg8-PE, extracts were separated in 6 M urea SDS-PAGE and immunoblotted with anti-Atg8.
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fig3: Shp1 affects autophagosome biogenesis. (a and b) Fluorescence microscopy of starved cells showed defective vacuolar uptake of GFP-Atg8. No GFP-Atg8–positive autophagosomes accumulated in the cytosol. (c) Electron microscopy of starved shp1Δ cells showed no vacuolar accumulation of autophagic bodies. (d) Lysed spheroplasts of starved cells were trypsin digested with and without detergent. Immunoblots with GFP antibodies showed proteolysis-resistant GFP-Atg8 (inside autophagosomes) in ypt7Δ but not in wild-type and shp1Δ cells. GFP-Atg8 breakdown yields GFP*. (e) Cells with a GFP-Atg8–positive PAS punctum were scored in fluorescence microscopy. The mean and SD of two experiments are shown, with >200 cells analyzed per strain. (f) To analyze Atg8-PE, extracts were separated in 6 M urea SDS-PAGE and immunoblotted with anti-Atg8.

Mentions: Next, we examined at which step Shp1 affects macroautophagy. The last step is intravacuolar lysis of autophagic bodies dependent on vacuolar acidification and proteinases. Light and electron microscopy showed no vacuolar accumulation of autophagic bodies in starved shp1Δ cells (Fig. 3, a–c). Fluorescence microscopy further demonstrated that, in contrast to wild-type cells, the autophagic cargo GFP-Atg8 did not reach the vacuole in shp1Δ cells (Fig. 3, a and b). GFP-Atg8–positive autophagosomes did not accumulate in the cytosol and were also not detected in shp1Δ cells by electron microscopy (unpublished data). The presence of mature carboxypeptidase Y (Fig. S1 c) in starved shp1Δ cells ruled out that disturbed vacuolar proteolysis caused the GFP-Atg8 degradation defect. Shp1 thus affects either biogenesis of autophagosomes or their vacuolar fusion. We distinguished between these possibilities in a protease protection experiment with spheroplasts hypotonically lysed under conditions that preserved the integrity of autophagosomes. Vacuolar fusion of autophagosomes requires Ypt7. Accordingly, the part of GFP-Atg8 enclosed in autophagosomes is protease protected in ypt7Δ, but not in wild-type, cells because of the rapid vacuolar fusion of autophagosomes (Fig. 3 d). The absence of protease-protected GFP-Atg8 in starved shp1Δ cells indicated defective autophagosome biogenesis or closure (Fig. 3 d). Many S. cerevisiae Atg proteins colocalize at the pre-autophagosomal structure (PAS), the site of autophagosome biogenesis. However, strong cytosolic staining masked detection of Cdc48 and Shp1 at the PAS in direct and indirect fluorescence microscopy. Because Shp1 is dispensable for proaminopeptidase I maturation, it may function in elongation of the isolation membrane, a role proposed for Atg8 (Nakatogawa et al., 2007; Xie et al., 2008). We thus examined whether Shp1 affects localization or lipidation of Atg8. Upon starvation, 42% of shp1Δ and 32% of wild-type cells showed GFP-Atg8–positive PAS punctae (Fig. 3 e), indicating normal Atg8 PAS recruitment. Also, Atg8-PE was formed in shp1Δ cells, and compared with wild type, the Atg8 level was slightly increased, most likely as a result of the autophagic defect (Fig. 3 f).


Cdc48/p97 and Shp1/p47 regulate autophagosome biogenesis in concert with ubiquitin-like Atg8.

Krick R, Bremer S, Welter E, Schlotterhose P, Muehe Y, Eskelinen EL, Thumm M - J. Cell Biol. (2010)

Shp1 affects autophagosome biogenesis. (a and b) Fluorescence microscopy of starved cells showed defective vacuolar uptake of GFP-Atg8. No GFP-Atg8–positive autophagosomes accumulated in the cytosol. (c) Electron microscopy of starved shp1Δ cells showed no vacuolar accumulation of autophagic bodies. (d) Lysed spheroplasts of starved cells were trypsin digested with and without detergent. Immunoblots with GFP antibodies showed proteolysis-resistant GFP-Atg8 (inside autophagosomes) in ypt7Δ but not in wild-type and shp1Δ cells. GFP-Atg8 breakdown yields GFP*. (e) Cells with a GFP-Atg8–positive PAS punctum were scored in fluorescence microscopy. The mean and SD of two experiments are shown, with >200 cells analyzed per strain. (f) To analyze Atg8-PE, extracts were separated in 6 M urea SDS-PAGE and immunoblotted with anti-Atg8.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3101598&req=5

fig3: Shp1 affects autophagosome biogenesis. (a and b) Fluorescence microscopy of starved cells showed defective vacuolar uptake of GFP-Atg8. No GFP-Atg8–positive autophagosomes accumulated in the cytosol. (c) Electron microscopy of starved shp1Δ cells showed no vacuolar accumulation of autophagic bodies. (d) Lysed spheroplasts of starved cells were trypsin digested with and without detergent. Immunoblots with GFP antibodies showed proteolysis-resistant GFP-Atg8 (inside autophagosomes) in ypt7Δ but not in wild-type and shp1Δ cells. GFP-Atg8 breakdown yields GFP*. (e) Cells with a GFP-Atg8–positive PAS punctum were scored in fluorescence microscopy. The mean and SD of two experiments are shown, with >200 cells analyzed per strain. (f) To analyze Atg8-PE, extracts were separated in 6 M urea SDS-PAGE and immunoblotted with anti-Atg8.
Mentions: Next, we examined at which step Shp1 affects macroautophagy. The last step is intravacuolar lysis of autophagic bodies dependent on vacuolar acidification and proteinases. Light and electron microscopy showed no vacuolar accumulation of autophagic bodies in starved shp1Δ cells (Fig. 3, a–c). Fluorescence microscopy further demonstrated that, in contrast to wild-type cells, the autophagic cargo GFP-Atg8 did not reach the vacuole in shp1Δ cells (Fig. 3, a and b). GFP-Atg8–positive autophagosomes did not accumulate in the cytosol and were also not detected in shp1Δ cells by electron microscopy (unpublished data). The presence of mature carboxypeptidase Y (Fig. S1 c) in starved shp1Δ cells ruled out that disturbed vacuolar proteolysis caused the GFP-Atg8 degradation defect. Shp1 thus affects either biogenesis of autophagosomes or their vacuolar fusion. We distinguished between these possibilities in a protease protection experiment with spheroplasts hypotonically lysed under conditions that preserved the integrity of autophagosomes. Vacuolar fusion of autophagosomes requires Ypt7. Accordingly, the part of GFP-Atg8 enclosed in autophagosomes is protease protected in ypt7Δ, but not in wild-type, cells because of the rapid vacuolar fusion of autophagosomes (Fig. 3 d). The absence of protease-protected GFP-Atg8 in starved shp1Δ cells indicated defective autophagosome biogenesis or closure (Fig. 3 d). Many S. cerevisiae Atg proteins colocalize at the pre-autophagosomal structure (PAS), the site of autophagosome biogenesis. However, strong cytosolic staining masked detection of Cdc48 and Shp1 at the PAS in direct and indirect fluorescence microscopy. Because Shp1 is dispensable for proaminopeptidase I maturation, it may function in elongation of the isolation membrane, a role proposed for Atg8 (Nakatogawa et al., 2007; Xie et al., 2008). We thus examined whether Shp1 affects localization or lipidation of Atg8. Upon starvation, 42% of shp1Δ and 32% of wild-type cells showed GFP-Atg8–positive PAS punctae (Fig. 3 e), indicating normal Atg8 PAS recruitment. Also, Atg8-PE was formed in shp1Δ cells, and compared with wild type, the Atg8 level was slightly increased, most likely as a result of the autophagic defect (Fig. 3 f).

Bottom Line: Interaction with Shp1 requires an FK motif within the N-terminal non-ubiquitin-like Atg8 domain.Based on our data, we speculate that autophagosome formation, in contrast to Golgi reassembly, requires a complex in which Atg8 functionally substitutes ubiquitin.This, for the first time, would give a rationale for use of the ubiquitin-like Atg8 during macroautophagy and would explain why Atg8-PE delipidation is necessary for efficient macroautophagy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry II, Georg-August University, D-37073 Goettingen, Germany.

ABSTRACT
The molecular details of the biogenesis of double-membraned autophagosomes are poorly understood. We identify the Saccharomyces cerevisiae AAA-adenosine triphosphatase Cdc48 and its substrate-recruiting cofactor Shp1/Ubx1 as novel components needed for autophagosome biogenesis. In mammals, the Cdc48 homologue p97/VCP and the Shp1 homologue p47 mediate Golgi reassembly by extracting an unknown monoubiquitinated fusion regulator from a complex. We find no requirement of ubiquitination or the proteasome system for autophagosome biogenesis but detect interaction of Shp1 with the ubiquitin-fold autophagy protein Atg8. Atg8 coupled to phosphatidylethanolamine (PE) is crucial for autophagosome elongation and, in vitro, mediates tethering and hemifusion. Interaction with Shp1 requires an FK motif within the N-terminal non-ubiquitin-like Atg8 domain. Based on our data, we speculate that autophagosome formation, in contrast to Golgi reassembly, requires a complex in which Atg8 functionally substitutes ubiquitin. This, for the first time, would give a rationale for use of the ubiquitin-like Atg8 during macroautophagy and would explain why Atg8-PE delipidation is necessary for efficient macroautophagy.

Show MeSH
Related in: MedlinePlus