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1.
Figure 5.

Figure 5. From: Atg21 Is a Phosphoinositide Binding Protein Required for Efficient Lipidation and Localization of Atg8 during Uptake of Aminopeptidase I by Selective Autophagy.

Arginine343 and arginine344 are required for function of Atg21. (A) atg21Δ cells (PSY7) expressing GFP-Atg21 (pPS123) but not GFP-Atg21R343,344K (pPS125) mature prApe1. (B) GFP-Atg21R343,344K does not localize to the vacuole and punctate structures. DIC, differential interference contrast.

Per E. Strømhaug, et al. Mol Biol Cell. 2004 August;15(8):3553-3566.
2.
Figure 2.

Figure 2. From: Atg21 Is a Phosphoinositide Binding Protein Required for Efficient Lipidation and Localization of Atg8 during Uptake of Aminopeptidase I by Selective Autophagy.

Atg21 is associated with the vacuole and prevacuolar structures. Wild-type (SEY6210) cells were chromosomally tagged with GFP at the ATG18 (PSY62), ATG21 (PSY63), and YGR223c (PSY100) loci, respectively, and stained with FM 4-64 to label the vacuoles. Cells were grown to early mid-log phase in YPD before viewing. DIC, differential interference contrast.

Per E. Strømhaug, et al. Mol Biol Cell. 2004 August;15(8):3553-3566.
3.
Figure 4.

Figure 4. From: Atg21 Is a Phosphoinositide Binding Protein Required for Efficient Lipidation and Localization of Atg8 during Uptake of Aminopeptidase I by Selective Autophagy.

Atg21 binds to phosphoinositides in vitro. (A) MBP, MBP-Atg18, MBP-Atg21, and MBP-Ygr223c were purified from E. coli as described in MATERIALS AND METHODS. Protein (1 μg) was loaded on a gel, and the gel was stained with Coomassie Brilliant Blue G. (B) PIP Strips (Echelon) containing lipids immobilized on nitrocellulose were incubated with 100 ng/ml MBP, MBP-Atg18, MBP-Atg21, or MBP-Ygr223c as described in MATERIALS AND METHODS.

Per E. Strømhaug, et al. Mol Biol Cell. 2004 August;15(8):3553-3566.
4.
Figure 3.

Figure 3. From: Atg21 Is a Phosphoinositide Binding Protein Required for Efficient Lipidation and Localization of Atg8 during Uptake of Aminopeptidase I by Selective Autophagy.

Atg18, Atg21, and Ygr223c localization is dependent on PtdIns 3-kinase activity. Strains PSY291, PSY292, PSY293, PSY62, PSY63, PSY100, PSY146, and PSY290 expressing Atg18-GFP, Atg21-GFP, or Ygr223c-GFP from the respective chromosomal loci in the vps34ts and wild-type SEY6210 (A) or fab1-2ts background (B) were grown at 26°C to early mid-log phase in SMD and shifted to 38°C for 10 (A) or 40 min (B). Cells were viewed by fluorescence microscopy as described in the legend to Figure 2. Loss of PtdIns 3-kinase but not PtdIns(3)P 5-kinase activity at the nonpermissive temperature resulted in loss of localization of Atg18 and Atg21. DIC, differential interference contrast.

Per E. Strømhaug, et al. Mol Biol Cell. 2004 August;15(8):3553-3566.
5.
Figure 9.

Figure 9. From: Atg21 Is a Phosphoinositide Binding Protein Required for Efficient Lipidation and Localization of Atg8 during Uptake of Aminopeptidase I by Selective Autophagy.

Atg21 is required for Atg5-GFP accumulation at the PAS. (A) Cells lacking both Atg8 and Atg21 do not mature prApe1 upon starvation. WT (SEY6210), single mutants (WPHYD7, WPHYD2, AHY001, and PSY7) and double mutants (PSY139, PSY128, and PSY190) were grown in YPD to mid-log phase and starved for 4 and 6 h before being analyzed by Western blot by using an antibody against Ape1. (B) Atg21 is not required for conjugation of Atg12 to Atg5. WT (PSY66) and atg21Δ (PSY155) cells expressing Atg5-HA from the chromosomal locus were grown to early mid-log phase in YPD and subjected to Western blot by using an antibody against HA. (C) Atg21 is required for Atg5-GFP accumulation at the PAS. atg8Δ (PSY285) and atg8Δ atg21Δ (PSY286) cells expressing Atg5-GFP from the chromosomal locus and RFP-prApe1 from an integrating plasmid were grown to early mid-log phase in YPD before being viewed by fluorescence microscopy as described in the legend to Figure 6. DIC, differential interference contrast.

Per E. Strømhaug, et al. Mol Biol Cell. 2004 August;15(8):3553-3566.
6.
Figure 8.

Figure 8. From: Atg21 Is a Phosphoinositide Binding Protein Required for Efficient Lipidation and Localization of Atg8 during Uptake of Aminopeptidase I by Selective Autophagy.

The conjugation system responsible for posttranslational modification of Atg8 is functional in the atg21Δ mutant. (A) Atg4 is able to recognize Atg8 and carry out proteolytic cleavage at the C terminus. A plasmid encoding Atg8-GFP was transformed into the wild-type (SEY6210), atg4Δ (WPHYD2) or atg21Δ (PSY5) strain. Cells were grown in SMD to early log phase (A600 = 0.6) and then either retained in SMD or nitrogen starved in SD-N medium for 3 h. Protein extracts were analyzed by Western blot by using antiserum to Ape1 or anti-GFP monoclonal antibodies. The asterisk denotes a contaminating band that cross-reacts with the anti-GFP antibody. (B) Atg7 E1-like and Atg3 E2 enzymes are able to activate and conjugate Atg8. A yeast two-hybrid analysis was carried out as described in MATERIALS AND METHODS.

Per E. Strømhaug, et al. Mol Biol Cell. 2004 August;15(8):3553-3566.
7.
Figure 6.

Figure 6. From: Atg21 Is a Phosphoinositide Binding Protein Required for Efficient Lipidation and Localization of Atg8 during Uptake of Aminopeptidase I by Selective Autophagy.

Localization of Atg proteins to the PAS in atg21Δ cells. (A) Atg19-CFP and Atg11-YFP colocalize in atg21Δ cells. PSY6 (atg21Δ) cells were transformed with plasmids expressing Atg19-CFP (pCVT19CFP(414)) and YFP-Atg11 (pPS97), grown in SMD to mid-log phase and analyzed by fluorescent microscopy. (B) GFP-Atg8 is not localized to the PAS in atg21Δ cells. atg21Δ cells expressing Atg1-GFP (PSY227) or Atg14-GFP (PSY228) from the chromosomal loci, and also expressing RFP-prApe1 from the endogenous promoter on an integrated plasmid, or atg21Δ atg8Δ cells expressing GFP-Atg8 (PSY226) behind the endogenous promoter from an integrated plasmid, and also expressing RFP-prApe1 from the endogenous promoter on a centromeric plasmid, were grown in YPD to mid-log phase before viewing. Localization of Atg2-GFP was essentially the same as that shown for Atg1-GFP. (C) Transport of GFP-Atg8 to the vacuole is reduced in atg21Δ cells. Wild-type (WT, SEY6210) and atg21Δ cells (PSY7) were transformed with a plasmid expressing GFP-Atg8 from the CUP1-promoter (pCuGFP-AUT7(416)), grown to mid-log phase in SMD before the addition of 1 mM phenylmethylsulfonyl fluoride and starved for nitrogen for up to 8 h as indicated. DIC, differential interference contrast.

Per E. Strømhaug, et al. Mol Biol Cell. 2004 August;15(8):3553-3566.
8.
Figure 7.

Figure 7. From: Atg21 Is a Phosphoinositide Binding Protein Required for Efficient Lipidation and Localization of Atg8 during Uptake of Aminopeptidase I by Selective Autophagy.

Lipidation of Atg8 is reduced in atg21Δ cells. (A) Atg8 lipidation in rich medium versus nitrogen starvation medium. Cells were grown in YPD to early mid-log phase and then starved for 3 h in SD-N. Atg8-PE was separated from Atg8 by 12% SDS page gels containing 6% urea (Kirisako et al., 2000). All strains are of BY4742 background. (B) Deletion of ATG21 antagonizes the accumulation of Atg8-PE in autophagy mutants. WT (SEY6210), atg21Δ (PSY7), atg18Δ (JGY3), atg18Δ atg21Δ (PSY167), atg9Δ (JKY007), and atg9Δ atg21Δ (PSY191) cells were starved for 4 h in SD-N, and Atg8-PE was analyzed by Western blot. (C) Deletion of ATG21 antagonizes the accumulation of GFP-Atg8 at the PAS in autophagy mutants. Strains used in B containing a plasmid expressing GFP-Atg8 from the CUP1 promoter were grown to mid-log phase in SMD and then starved for 4 h in SD-N. (D) atg21Δ cells have a reduced rate of Atg8 lipidation. Cells of BY4742 background were labeled and Atg8 immunoprecipitated as described in MATERIALS AND METHODS and analyzed by 12% SDS-urea gels.

Per E. Strømhaug, et al. Mol Biol Cell. 2004 August;15(8):3553-3566.
9.
Figure 1.

Figure 1. From: Atg21 Is a Phosphoinositide Binding Protein Required for Efficient Lipidation and Localization of Atg8 during Uptake of Aminopeptidase I by Selective Autophagy.

Atg21 is required for the Cvt pathway. (A) atg21Δ cells were grown in YPD and shifted to SD-N to induce autophagy. The atg21Δ cells mature prApe1 under starvation conditions more rapidly than atg11Δ, whereas no maturation is seen in atg18Δ cells. (B) Pho8Δ60, a marker for nonspecific autophagy, is delivered to the vacuole of atg21Δ cells in nitrogen starvation conditions. WT (TN124), atg18Δ (JGY20), and atg21Δ (PSY107) cells were grown as described in A. Cells were shifted to SD-N, samples collected, and protein extracts assayed for alkaline phosphatase activity. The activity in the wild-type sample was set to 100% and the other activities normalized relative to wild-type. The atg21Δ results represent the mean and S.E. of three experiments, whereas the atg18Δ results are based on duplicate samples from the same experiment. (C) atg21Δ and ygr223cΔ cells are not deficient in peroxisome degradation. Cells were shifted from oleic acid-containing medium to SD-N, and pexophagy was monitored by Western blot by using an antibody against peroxisomal thiolase (Fox3). The result for the ygr223cΔ strain was essentially the same as that shown for atg21Δ. (D) Atg21 is required for Cvt vesicle formation. atg21Δ pep4Δ (PSY2) cells were converted to spheroplasts and subjected to a protease protection assay performed as described in MATERIALS AND METHODS.

Per E. Strømhaug, et al. Mol Biol Cell. 2004 August;15(8):3553-3566.

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