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. 2000 Nov 27;151(5):1025-34.
doi: 10.1083/jcb.151.5.1025.

Dissection of autophagosome biogenesis into distinct nucleation and expansion steps

H Abeliovich  1 , W A Dunn Jr, J Kim, D J Klionsky
Affiliations

Dissection of autophagosome biogenesis into distinct nucleation and expansion steps

H Abeliovich et al. J Cell Biol. .

Abstract

Rapamycin, an antifungal macrolide antibiotic, mimics starvation conditions in Saccharomyces cerevisiae through activation of a general G(0) program that includes widespread effects on translation and transcription. Macroautophagy, a catabolic membrane trafficking phenomenon, is a prominent part of this response. Two views of the induction of autophagy may be considered. In one, up-regulation of proteins involved in autophagy causes its induction, implying that autophagy is the result of a signal transduction mechanism leading from Tor to the transcriptional and translational machinery. An alternative hypothesis postulates the existence of a dedicated signal transduction mechanism that induces autophagy directly. We tested these possibilities by assaying the effects of cycloheximide and specific mutations on the induction of autophagy. We find that induction of autophagy takes place in the absence of de novo protein synthesis, including that of specific autophagy-related proteins that are up-regulated in response to rapamycin. We also find that dephosphorylation of Apg13p, a signal transduction event that correlates with the onset of autophagy, is also independent of new protein synthesis. Finally, our data indicate that autophagosomes that form in the absence of protein synthesis are significantly smaller than normal, indicating a role for de novo protein synthesis in the regulation of autophagosome expansion. Our results define the existence of a signal transduction-dependent nucleation step and a separate autophagosome expansion step that together coordinate autophagosome biogenesis.

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Figures

Figure 1
Figure 1
A, Rapamycin-induced bypass of the vac8Δ prAPI trafficking block occurs in the presence of cycloheximide. Deletion mutants (vac8Δ, ΗΑY394; apg1Δ, ΗΑY395) were grown overnight at 30°C and pulse-labeled for 10 min with 35S-labeled cysteine and methionine. Chase solution was added, followed by addition of cycloheximide to 40 μg/ml in SD medium, or mock SD medium to the same volume. After a 5-min incubation, the cultures were diluted fourfold with SD medium containing rapamycin (0.2 μg/ml final concentration, diluted from a 1 mg/ml stock in 90% ethanol/10% Tween-20) or SD medium alone. Samples (2 A600 equivalents) were removed at 0, 1, and 2 h after addition of rapamycin. HAY382 cells (vac8Δ vam3ts) were grown overnight at 26°C and shifted to 38°C for 15 min before labeling and treatment as above. Control lane: cells were treated as above except that cycloheximide was added 5 min before the addition of label for 10 min, followed by chase, processing, and immunoprecipitation as above. B, Cycloheximide blocks specific induction of API and Aut7p by rapamycin. Wild-type (SEY6210) cells were grown to an A600 of 0.6. They were treated with or without cycloheximide (10 μg/ml from a 50 mg/ml stock in ethanol) for 5 min before administration of rapamycin (2 μg/ml final concentration) or mock drug vehicle to the same volume. The cultures were further incubated for 2 h, precipitated with 10% TCA, and processed for Western blotting as described in Materials and Methods. 20 μg protein were loaded per lane and blots were probed for API.
Figure 6
Figure 6
Model for induction of autophagy by rapamycin. Tor activity prevents dephosphorylation of Apg13p, by an unknown mechanism. Inhibition of Tor by the rapamycin–RBP complex then results in rapid dephosphorylation of Apg13p. Dephosphorylated Apg13p acts on Apg1p to mediate autophagosome nucleation. In the absence of new protein synthesis, this results in abnormally small autophagosomes. Under normal conditions, however, inhibition of Tor also upregulates the transcription of starvation- and autophagy-specific genes, and the ensuing increase in levels of specific proteins, such as Aut7p, allows the expansion of autophagosomes.
Figure 2
Figure 2
Cycloheximide does not block a rapamycin-induced, Apg1p-independent dephosphorylation of Apg13p. A, D3Y101 cells (apg13Δ) cells harboring plasmid YEp351[APG13] were grown in SD medium to A600 of 0.6 and treated with or without cycloheximide (10 μg/ml final concentration) for 5 min before administration of rapamycin (0.2 μg/ml final concentration). Cells were incubated for 2 h, treated with 10% TCA, and cell extracts were prepared for Western blotting as above. Blots were probed with specific antisera for Apg13p and API by Western blotting. B, Wild-type and apg1Δ cells harboring plasmid YEp351[APG13] were grown in SD and treated with or without rapamycin for 2 h before processing as above.
Figure 3
Figure 3
Induction of autophagosomes in the presence of cycloheximide results in the accumulation of abnormally small autophagosomes. TDY27 (A–F; vam3ts) or TVY1 (G and H; pep4Δ) yeast were grown to A600 of 0.5 at 26°C (TDY27) or 30°C (TVY1) and shifted to 38°C for 15 min (TDY27) or directly processed at 30°C (TVY1). The cells were then treated with or without cycloheximide (10 μg/ml final concentration) for 5 min before addition of rapamycin (0.2 μg/ml final concentration) or mock drug vehicle. Cells were incubated a further 1.5 h at 38°C before permanganate fixation for EM as described in Materials and Methods. Arrows denote autophagosomes and autophagic bodies, arrowheads identify the small autophagosomes and autophagic bodies that are seen in cycloheximide + rapamycin-treated cells. A and B, no treatment; C, E, and G, rapamycin alone; D, F, and H, rapamycin + cycloheximide. Bar, 1 μM. N, Nucleus; V, vacuole; m, mitochondria.
Figure 4
Figure 4
Induction of aberrantly shaped autophagosomes occurs in aut7Δ vam3ts cells upon treatment with rapamycin. EM analysis of HAY398 cells (aut7Δ vam3ts) after rapamycin treatment at nonpermissive temperature. Yeast were grown overnight at 26°C to A600 of 0.5 and shifted to 38°C for 15 min. The cells were then treated with or without cycloheximide (10 μg/ml final concentration) for 5 min before addition of rapamycin (0.2 μg/ml final concentration) or mock drug vehicle. Cells were incubated a further 1.5 h at 38°C before permanganate fixation for EM as described in Materials and Methods. A, no treatment; B and D, rapamycin; C, rapamycin + cycloheximide. Arrowheads denote the small autophagosomes visualized in these cells. Bar, 1 μM. N, Nucleus; V, vacuole; m, mitochondria.
Figure 5
Figure 5
The Cvt bypass phenotype is correlated with partial survival under nitrogen starvation conditions. A, Immunoblots of prAPI maturation in nitrogen starvation medium. SEY6210 (wild-type), THY119 (cvt3), WPHYD7 (aut7Δ), WPHYD2 (aut2Δ), and HAY395 (apg1Δ) strains were grown to midlog phase and shifted to SD-N medium for 0, 1, 2, 4, and 6 h. At each time point, 3 A600 units of cells were precipitated in 10% TCA, washed twice with acetone, and processed for Western blotting as described in Materials and Methods. B, Viability of yeast strains in nitrogen starvation medium. SEY6210 (wild-type), THY119 (cvt3), WPHYD7 (aut7Δ), WPHYD2 (aut2Δ), and HAY395 (apg1Δ) strains were grown to midlog phase in SMD and transferred to SD-N medium. Aliquots were removed at the indicated times and spread onto YPD plates in triplicate. The number of colonies was counted after two to three days.
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References

    1. Abeliovich H., Grote E., Novick P., Ferro-Novick S. Tlg2p, a yeast syntaxin homolog that resides on the Golgi and endocytic structures. J. Biol. Chem. 1998;273:11719–11727. - PubMed
    1. Abeliovich H., Darsow T., Emr S.D. A t-SNARE/Sec1p complex composed of Tlg2p and Vps45p is required for cytoplasm to vacuole targeting of aminopeptidase I. EMBO (Eur. Mol. Biol. Organ.) J. 1999;18:6005–6016. - PMC - PubMed
    1. Baba M., Takeshige K., Baba N., Ohsumi Y. Ultrastructural analysis of the autophagic process in yeastdetection of autophagosomes and their characterization. J. Cell Biol. 1994;124:903–913. - PMC - PubMed
    1. Baba M., Osumi M., Scott S.V., Klionsky D.J., Ohsumi Y. Two distinct pathways for targeting proteins from the cytoplasm to the vacuole/lysosome. J. Cell Biol. 1997;139:1687–1695. - PMC - PubMed
    1. Barbet N.C., Schneider U., Helliwell S.B., Stansfield I., Tuite M.F., Hall M.N. Tor controls translation initiation and early G1 progression in yeast. Mol. Biol. Cell. 1996;7:25–42. - PMC - PubMed

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