Journal: The Journal of Cell Biology

Article Title: The conserved oligomeric Golgi complex is involved in double-membrane vesicle formation during autophagy

doi: 10.1083/jcb.200904075

Figure Lengend Snippet: The Cvt, autophagy, and pexophagy pathways are defective in cog mutants. (A) The Cvt pathway is defective in COG deletion mutants. The wild-type (WT; BY4742), cog1Δ , cog5Δ , cog6Δ , cog7Δ , and cog8Δ cells were grown in rich medium, and rapamycin was added to the culture at a final concentration of 0.2 µg/ml. After 2 h, cell extracts were prepared and subjected to immunoblot analysis with anti-Ape1 or anti-YFP antisera. (B) Autophagic activity in the COG deletion mutants. The wild-type (YTS223), cog1Δ (YTS224), cog5Δ (YTS225), cog6Δ (YTS226), cog7Δ (YTS227), cog8Δ (YTS228), and atg1Δ (YTS243) cells expressing Pho8Δ60 were grown in YPD medium and incubated in SD-N for 3 h to induce autophagy. Cell extracts were prepared for measuring the Pho8Δ60-dependent alkaline phosphatase activity. Error bars indicate SD. (C) The Cvt pathway is defective in cog2-1 and cog3-2 mutants. The wild-type (BY4742), cog2-1 , and cog3-2 cells were grown in rich medium at 24°C and shifted to SMD. After incubation at either 24 or 38°C for 30 min, cells were labeled with [ 35 S]methionine/cysteine and subjected to a nonradioactive chase at the same temperature for 2 h. Ape1 was immunoprecipitated and subjected to SDS-PAGE. (D) GFP-Atg8 processing is defective in cog temperature-sensitive mutants. Wild-type (BY4742), cog2-1 , cog3-2 , cog4-1 (BCR29), and cog4-2 (BCR47) mutants were grown at permissive temperature until OD 600 = 0.8. After incubation at either 24°C or nonpermissive temperatures (37 or 39°C) for 30 min, rapamycin was added for 2 h. Aliquots were collected, and protein extracts were subjected to immunoblotting analysis. (E) Autophagic activity in temperature-sensitive cog mutants. Wild-type (YTS223), cog2-1 (YTS230), cog3-2 (YTS231), and cog2-1 and cog3-2 strains harboring the pCuCOG2HA(416) and pCuCOG3HA(416) plasmids, respectively, expressing Pho8Δ60 were grown in YPD medium and incubated in SD-N at either 24 or 37°C for 3 h. Cell lysates were used to measure Pho8Δ60 activity. (F) COG complex subunits are required for pexophagy. The wild-type (IRA001), atg1Δ (IRA002), cog2-1 (WLY90), and cog3-2 (WLY91) cells expressing Pex14-GFP were grown under conditions to induce peroxisome proliferation as described in Materials and methods. After incubation at either 24°C or nonpermissive temperatures for 30 min, the cultures were shifted to SD-N. For cog1Δ (WLY175) and cog6Δ (WLY174) strains, the cells were grown at 30°C and after being induced for peroxisome proliferation were shifted to SD-N at the same temperature. Protein extracts were prepared at the indicated time points and resolved by SDS-PAGE.

Article Snippet: The anti-YFP was labeled with 0.8-nm ultrasmall gold particles (Aurion) that were detected by silver enhancement, whereas the anti-Ape1 was labeled with 10 nm colloidal gold–conjugated goat anti–rabbit IgG (British Biocell).

Techniques: Concentration Assay, Western Blot, Activity Assay, Expressing, Incubation, Labeling, Immunoprecipitation, SDS Page

Journal: The Journal of Cell Biology

Article Title: The conserved oligomeric Golgi complex is involved in double-membrane vesicle formation during autophagy

doi: 10.1083/jcb.200904075

Figure Lengend Snippet: Anterograde movement of Atg9 is defective in the cog2-1 strain. cog2-1 Atg9-3GFP (WLY227) cells expressing RFP-Ape1 were grown to OD 600 = 0.8 at 24°C or shifted to 37°C for 1 h. For autophagy-inducing conditions, rapamycin was added for 1 h at either 24 or 37°C before imaging. DIC, differential interference contrast. Bar, 2.5 µm. (B) Quantification of Atg9 PAS localization. The colocalization percentages were quantified in cells containing both Atg9-3GFP and RFP-Ape1 signals from three independent repeat experiments.

Article Snippet: The anti-YFP was labeled with 0.8-nm ultrasmall gold particles (Aurion) that were detected by silver enhancement, whereas the anti-Ape1 was labeled with 10 nm colloidal gold–conjugated goat anti–rabbit IgG (British Biocell).

Techniques: Expressing, Imaging

Journal: The Journal of Cell Biology

Article Title: The conserved oligomeric Golgi complex is involved in double-membrane vesicle formation during autophagy

doi: 10.1083/jcb.200904075

Figure Lengend Snippet: The cog mutants affect sequestering vesicle formation. (A) cog2-1 cells were grown in SMD and shifted to 38°C in SMD or SD-N for 1.5 h. The cells were analyzed by immunoelectron microscopy and labeled with anti-YFP antibody followed by immunogold as described in Materials and methods. The white arrowheads mark clusters of GFP-Atg8, and the black arrow marks a phagophore. Bars, 200 nm. The inset shows an image immunostained with affinity-purified antiserum to Ape1, demonstrating that the electron-dense structure is a Cvt complex. Bar, 100 nm. (B) Cog2 is involved in Cvt vesicle formation. Spheroplasts from the wild-type ( vam3Δ ; WLY118), cog2-1 (WLY117), and atg1Δ (WLY119) strains were incubated at 37°C for 20 min, pulse labeled with [ 35 S]methionine/cysteine for 10 min, and subjected to a nonradioactive chase for 30 min. The prApe1-containing pellet fractions were obtained after osmotically lysing the spheroplasts and centrifugation at 5,000 g . The pellets were treated with proteinase K (PK) in the presence or absence of 0.2% Triton X-100 for 20 min on ice. The resulting samples were TCA precipitated and immunoprecipitated with Ape1 or Pgk1 antiserum and resolved by SDS-PAGE. T, total; P5 and S5, pellet and supernatant fractions, respectively, after 5,000 g centrifugation.

Article Snippet: The anti-YFP was labeled with 0.8-nm ultrasmall gold particles (Aurion) that were detected by silver enhancement, whereas the anti-Ape1 was labeled with 10 nm colloidal gold–conjugated goat anti–rabbit IgG (British Biocell).

Techniques: Immuno-Electron Microscopy, Labeling, Affinity Purification, Incubation, Centrifugation, Immunoprecipitation, SDS Page

Journal: The Journal of Cell Biology

Article Title: The conserved oligomeric Golgi complex is involved in double-membrane vesicle formation during autophagy

doi: 10.1083/jcb.200904075

Figure Lengend Snippet: Cog2 localizes to the PAS. (A) Cog2-GFP (WLY190) and cog2-1–GFP (WLY188) strains expressing RFP-Ape1 were grown in SMD at 24°C or shifted to 37°C for 30 min before imaging. For autophagy-inducing conditions, cells were either grown at 24°C or shifted to 37°C for 30 min, and rapamycin was added for an additional 30 min. DIC, differential interference contrast. Bars, 2.5 µm. (B) Quantification of Cog2-GFP and cog2-1–GFP PAS localization. The colocalization percentages were quantified in cells containing both GFP and RFP-Ape1 signals from three independent repeat experiments.

Article Snippet: The anti-YFP was labeled with 0.8-nm ultrasmall gold particles (Aurion) that were detected by silver enhancement, whereas the anti-Ape1 was labeled with 10 nm colloidal gold–conjugated goat anti–rabbit IgG (British Biocell).

Techniques: Expressing, Imaging

Journal: The Journal of Cell Biology

Article Title: The conserved oligomeric Golgi complex is involved in double-membrane vesicle formation during autophagy

doi: 10.1083/jcb.200904075

Figure Lengend Snippet: COG subunits associate with Atg proteins. (A and B) HA-Cog4 (A; WLY208) and HA-Cog2 (B; WLY209) cells transformed with the indicated plasmids expressing tagged Atg proteins were grown in selective SMG medium to OD 600 = 1.0. Cell lysates were prepared and subjected to affinity isolation or immunoprecipitation (IP) with either anti-HA or anti-Myc antibody as described in Materials and methods. Plasmids expressing PA (pRS424-CuProtA), PA-tagged Atg17 (pProtA-Apg17(424)), Atg20 (pProtA-Cvt20(424)), Atg24 (pProtA-Cvt13(424)), and Myc-tagged Atg12 (pMyc-Apg12(426)) were used as indicated. The eluted proteins were separated by SDS-PAGE and detected with monoclonal anti-HA antibody (A) or immunoblotting with anti-HA and anti-Myc antibodies (B). For each experiment, ∼1% of the total cell lysate or 10% of the total eluate was loaded. WB, Western blot. (C) Cog2-GFP colocalizes with RFP-Ape1 in the MKO ( ATG11 ATG19 ) strain. The MKO ( ATG11 ATG19 ; WLY205) cells expressing chromosomally tagged Cog2-GFP and a plasmid-based RFP-Ape1 were grown in selective SMD to OD 600 = 0.8 and observed by fluorescence microscopy. DIC, differential interference contrast. Bar, 2.5 µm.

Article Snippet: The anti-YFP was labeled with 0.8-nm ultrasmall gold particles (Aurion) that were detected by silver enhancement, whereas the anti-Ape1 was labeled with 10 nm colloidal gold–conjugated goat anti–rabbit IgG (British Biocell).

Techniques: Transformation Assay, Expressing, Isolation, Immunoprecipitation, SDS Page, Western Blot, Plasmid Preparation, Fluorescence, Microscopy

Journal: EMBO Reports

Article Title: Two distinct regulatory pathways govern Cct2-Atg8 binding in the process of solid aggrephagy

doi: 10.1038/s44319-024-00275-7

Figure Lengend Snippet: ( A ) ape1 ∆ atg5 ∆ or ape1 ∆ yeast cells co-expressing Ape1 WT or Ape1 P22L with empty vector (EV), 3×HA-Cct1, 3×HA-Cct2, or 3×HA-Cct4 were grown to log phase under nutrient-rich conditions. Samples were analyzed by immunoblot for detecting the maturation of PrApe1 into mApe1. Pgk1 served as a loading control. ( B ) Quantification of mApe1 to PrApe1+mApe1 ratio from ( A ). Data are presented as means ± SD (Data represent the results of three independent experiments). *** P < 0.001; NS not significant; two-tailed Student’s t tests were used. P < 0.0001 (WT atg5 Δ vs. WT Cct2); p = 0.7014 (WT EV vs. WT Cct2); P > 0.9999 (WT Cct1 vs. WT Cct2); P = 0.6838 (WT Cct4 vs. WT Cct2); P < 0.0001 (P22L atg5 Δ vs. P22L Cct2); p = 0.0001 (P22L EV vs. P22L Cct2); P < 0.0001 (P22L Cct1 vs. P22L Cct2); P < 0.0001 (P22L Cct4 vs. P22L Cct2). ( C ) The indicated yeast strains were plated in 3-fold serial dilution onto YPD at 30 °C or 37 °C for 2 days. ( D ) ape1 ∆ atg5 ∆, ape1 ∆, ape1 ∆ cct1-ts , or ape1 ∆ cct4-ts yeast cells co-expressing Ape1 P22L with empty vector (EV) or 3×HA-Cct2 were grown to the early log phase at 30 °C and then subjected to 37 °C for 2 h. Samples were analyzed by immunoblot for detecting the maturation of PrApe1 into mApe1. Pgk1 served as a loading control. ( E ) Quantification of mApe1 to PrApe1+mApe1 ratio from ( D ). Data are presented as means ± SD (Data represent the results of three independent experiments). *** P < 0.001; NS not significant; two-tailed Student’s t tests were used. P < 0.0001 ( atg5 Δ vs. Cct2); P = 0.0001 (EV vs. Cct2); P = 0.0001 ( cct1-ts EV vs. Cct2); P = 0.3923 ( cct1-ts WT vs. Cct2); P = 0.0001 ( cct4-ts EV vs. Cct2); P = 0.2921 ( cct4-ts WT vs. Cct2). ( F ) ape1 ∆ atg5 ∆, ape1 ∆, ape1 ∆ cct1-ts , or ape1 ∆ cct4-ts yeast cells co-expressing RFP-Ape1 P22L and Vph1-GFP with either an empty vector (EV) or 3×HA-Cct2 were grown to log phase at 30 °C. These yeast strains were then cultured at 37 °C for 2 h under full medium conditions. Images of cells were obtained using an inverted fluorescence microscope. Scale bar, 2 µm. ( G ) Cells from ( F ) were quantified for the vacuolar localization of RFP-Ape1 P22L. n = 300 cells were pooled from three independent experiments. Data are presented as means ± SD. *** P < 0.001; NS not significant; two-tailed Student’s t tests were used. P < 0.0001 ( atg5 Δ vs. Cct2); P = 0.0001 (EV vs. Cct2); P < 0.0001 ( cct1-ts EV vs. Cct2); P = 0.9552 ( cct1-ts WT vs. Cct2); P = 0.0001 ( cct4-ts EV vs. Cct2); P = 0.7581 ( cct4-ts WT vs. Cct2). ( H ) ape1 ∆ atg5 ∆, ape1 ∆, or ape1 ∆ 3×HA-AID-Cct2 yeast cells were treated with 0.5 mM IAA for 0 h or 2 h. Samples were analyzed by immunoblot for detecting the maturation of PrApe1 into mApe1 and the levels of 3×HA-AID-Cct2 protein were detected by using Anti-HA antibody. Pgk1 served as a loading control. ( I ) Quantification of mApe1 to PrApe1+mApe1 ratio from ( H ). Data are presented as means ± SD (Data represent the results of three independent experiments). *** P < 0.001; NS not significant; two-tailed Student’s t tests were used. P < 0.0001 ( atg5 Δ vs. ape1 Δ); P < 0.0001 ( atg5 Δ + IAA vs. ape1 Δ); P = 0.9397 ( ape1 Δ + IAA vs. ape1 Δ); P = 0.9020 ( AID-Cct2 vs. ape1 Δ); P < 0.0001 ( AID-Cct2 + IAA vs. ape1 Δ). .

Article Snippet: Rabbit anti-Ape1 , GL Biochem Ltd., Shanghai , .

Techniques: Expressing, Plasmid Preparation, Western Blot, Control, Two Tailed Test, Serial Dilution, Cell Culture, Fluorescence, Microscopy

Journal: EMBO Reports

Article Title: Two distinct regulatory pathways govern Cct2-Atg8 binding in the process of solid aggrephagy

doi: 10.1038/s44319-024-00275-7

Figure Lengend Snippet: ( A ) BY4741, cct1-ts , or cct4-ts yeast cells expressing Atg11-3×FLAG were grown to log phase at 30 °C and then subjected to 37 °C for 2 h. Samples were analyzed by immunoblot for detecting the expression of Atg11-3×FLAG and α-Tubulin. Pgk1 served as a loading control. The data are representative of three independent experiments. ( B ) GST pulldowns were performed by using purified GST, GST-Cct1, GST-Cct2, or GST-Cct4 with His-Atg8 protein from E. Coli . The data are representative of three independent experiments. ( C ) ape1 ∆ cells co-expressing HA-Cct1, HA-Cct2, HA-Cct4, HA-Cct5, or HA-Cct8 with FLAG-Atg8 and Ape1 P22L were grown to the log phase under nutrient-rich conditions. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and then analyzed by western blot using anti-HA antibody. The data are representative of three independent experiments. ( D ) ape1 ∆, ape1 ∆ atg8 ∆, ape1 ∆ atg11 ∆, ape1 ∆ atg15 ∆, ape1 ∆ atg2 ∆ atg15 ∆, or ape1 ∆ pep4 ∆ cells co-expressing Cct2-GFP and Vph1-mCherry in the presence of Ape1 P22L were grown to OD 600 = 0.8. These yeast strains were then cultured in nutrient-rich medium for 6 h. Images of cells were obtained using an inverted fluorescence microscope. Scale bar, 2 µm. ( E ) Cells from ( D ) were quantified for the vacuolar localization of Cct2-GFP. n = 300 cells were pooled from three independent experiments. Data are presented as means ± SD. *** P < 0.001; two-tailed Student’s t tests were used. P < 0.0001( atg8 Δ vs. WT); P < 0.0001( atg11 Δ vs. WT); P < 0.0001( atg15 Δ vs. WT); P < 0.0001( atg2 Δ& atg15 Δ vs. WT); P < 0.0001( pep4 Δ vs. WT) in ( E ).

Article Snippet: Rabbit anti-Ape1 , GL Biochem Ltd., Shanghai , .

Techniques: Expressing, Western Blot, Control, Purification, Immunoprecipitation, Cell Culture, Fluorescence, Microscopy, Two Tailed Test

Journal: EMBO Reports

Article Title: Two distinct regulatory pathways govern Cct2-Atg8 binding in the process of solid aggrephagy

doi: 10.1038/s44319-024-00275-7

Figure Lengend Snippet: ( A ) ape1 ∆ cells co-expressing 3×HA-Cct2 and Atg11-3×FLAG with Ape1 P22L were grown to the early log phase under nutrient-rich conditions. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and then analyzed by western blot using anti-HA antibody. The data are representative of three independent experiments. ( B ) Secondary structure diagram of Atg11. ( C ) atg11 ∆ cells co-expressing an empty vector (EV), WT HA-Atg11, HA-Atg11-CC1∆, HA-Atg11-CC2∆, HA-Atg11-CC3∆, or HA-Atg11-CC4∆ with 3×FLAG-Cct2 were grown to the early log phase. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and then analyzed by western blot using anti-HA antibody. The data are representative of two independent experiments. ( D ) In vitro GST pulldowns were performed using Atg11 R1, R2, R3, or R4 with GST-Cct2 purified from E. coli . Protein samples were separated by SDS-PAGE and detected using Coomassie blue staining. The data are representative of two independent experiments. ( E ) Cells co-expressing an empty vector, WT 3×HA-Cct2, 3×HA-Cct2(2-210aa∆, D1∆), 3×HA-CCT2(211-361aa∆, D2∆), or 3×HA-CCT2(362-527 aa∆, D3∆) with Atg11-3×FLAG were grown to the log phase. Cell lysates were immunoprecipitated with anti-HA agarose beads and then analyzed by western blot using anti-FLAG antibody. The red arrows indicate the bands corresponding to the wild-type (WT) and specific mutants of the 3×HA-Cct2 protein. The data are representative of two independent experiments. ( F ) ape1 ∆ cells co-expressing an empty vector (EV), WT 3×HA-Cct2, 3×HA-Cct2 D473A , or 3×HA- Cct2 I487E with Atg11-3×FLAG were grown to the early log phase in the presence of Ape1 P22L under nutrient-rich conditions. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and then analyzed by western blot using anti-HA antibody. The data are representative of three independent experiments. ( G ) In vitro GST pulldowns were performed using Atg11 CC4 with GST, GST-Cct2, GST-Cct2 D473A , or GST-Cct2 I487E purified from E. coli . Protein samples were separated by SDS-PAGE and detected using Coomassie blue staining. The data are representative of two independent experiments. .

Article Snippet: Rabbit anti-Ape1 , GL Biochem Ltd., Shanghai , .

Techniques: Expressing, Immunoprecipitation, Western Blot, Plasmid Preparation, In Vitro, Purification, SDS Page, Staining

Journal: EMBO Reports

Article Title: Two distinct regulatory pathways govern Cct2-Atg8 binding in the process of solid aggrephagy

doi: 10.1038/s44319-024-00275-7

Figure Lengend Snippet: ( A ) ape1 ∆ yeast cells expressing RFP-Ape1 P22L were grown to log phase. FRAP analysis was performed at the indicated time points. Scale bar, 2 µm. ( B ) Quantification of the normalized RFP-Ape1 P22L fluorescence signal (mean ± SD) in ( A ) (>30 cells from three independent experiments. ( C ) BY4741 yeast cells expressing Cu 2+ -inducible GFP-47Q plasmids were cultured in nutrient-rich medium for 12 h to induce the formation of solid aggregates. FRAP analysis was performed at the indicated time points. Scale bar: 2 µm. ( D ) Quantification of the normalized GFP-47Q fluorescence signal (mean ± SD) in ( C ) (>30 cells from three independent experiments). ( E ) Yeast cells co-expressing Cu 2+ -inducible GFP-47Q plasmids with an empty vector (EV), 3×HA-Cct2, or 3×HA-Cct2 variants were grown to an OD 600 = 0.6. Subsequently, 0.1 mM CuSO 4 was added to the cells to induce GFP-47Q to form solid aggregates for 12 h. Samples were analyzed by immunoblot for the cleavage of GFP-47Q. Pgk1 served as a loading control. ( F ) Quantification of GFP to GFP-47Q + GFP ratio from ( E ). Data are presented as means ± SD (Data represent the results of three independent experiments). *** P < 0.001; two-tailed Student’s t tests were used. P = 0.0005 (EV vs. Cct2); P = 0.0002 (D473A vs. Cct2); P = 0.0002 (I487E vs. Cct2). ( G ) In vitro GST pulldowns were performed using His-Atg8 with GST, GST-Cct2, GST-Cct2 D473A, or I487E purified from E. coli . Protein samples were separated by SDS-PAGE and detected using Coomassie blue staining. The data are representative of two independent experiments. ( H ) In vitro GST pulldowns were performed using Atg11 CC4 with GST, GST-Cct2, GST-Cct2 mVLL (AIM motif mutant) purified from E. coli . Protein samples were separated by SDS-PAGE and detected using Coomassie blue staining. The data are representative of two independent experiments. ( I , J ) ape1 ∆, atg2 ∆ ape1 ∆, or atg14 ∆ ape1 ∆ cells co-expressing 3×HA-Cct2 with FLAG-Atg8 in the presence of Ape1 P22L were grown to the log phase under nutrient-rich conditions. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and then analyzed by western blot using anti-HA antibody. The data are representative of three independent experiments.

Article Snippet: Rabbit anti-Ape1 , GL Biochem Ltd., Shanghai , .

Techniques: Expressing, Fluorescence, Cell Culture, Plasmid Preparation, Western Blot, Control, Two Tailed Test, In Vitro, Purification, SDS Page, Staining, Mutagenesis, Immunoprecipitation

Journal: EMBO Reports

Article Title: Two distinct regulatory pathways govern Cct2-Atg8 binding in the process of solid aggrephagy

doi: 10.1038/s44319-024-00275-7

Figure Lengend Snippet: ( A ) ape1 ∆ or ape1 ∆ atg5 ∆ yeast cells co-expressing RFP-Ape1 P22L, Vph1-GFP with empty vector (EV), WT 3×HA-Cct2, or 3×HA-Cct2 variants were grown to log phase under nutrient-rich conditions. Images of cells were obtained using an inverted fluorescence microscope. Scale bar, 2 µm. ( B ) Cells from ( A ) were quantified for the vacuolar localization of RFP-Ape1 P22L. n = 300 cells were pooled from three independent experiments. Data are presented as means ± SD. *** P < 0.001; two-tailed Student’s t tests were used. P < 0.0001 ( atg5 Δ vs. Cct2); p = 0.0001 (EV vs. Cct2); P = 0.0002 (Cct2 D473A vs. Cct2); P = 0.0002 (Cct2 I487E vs. Cct2). ( C ) ape1 ∆ atg5 ∆ or ape1 ∆ yeast cells co-expressing WT Ape1 or Ape1 P22L with empty vector (EV), 3×HA-Cct2 WT, or the indicated 3×HA-Cct2 variants were grown to log phase under nutrient-rich conditions. Samples were analyzed by immunoblot for detecting the maturation of PrApe1 into mApe1. Pgk1 served as a loading control. ( D ) Quantification of mApe1 to PrApe1 ratio from ( C ). Data are presented as means ± SD (Data represent the results of three independent experiments). *** P < 0.001; NS not significant; two-tailed Student’s t tests were used. P < 0.0001 (WT atg5 Δ vs. WT Cct2); P = 0.6407 (WT EV vs. WT Cct2); P = 0.4033 (WT Cct2 D473A vs. WT Cct2); P = 0.7400 (WT Cct2 I487E vs. WT Cct2); P < 0.0001(P22L atg5 Δ vs. P22L Cct2); P < 0.0001(P22L EV vs. P22L Cct2); P < 0.0001(P22L Cct2 D473A vs. P22L Cct2); P < 0.0001(P22L Cct2 I487E vs. P22L Cct2). ( E ) ape1 ∆ Ape1 P22L yeast cells co-expressing Vph1-mCherry with Cct2-GFP, Cct2 D473A -GFP, or Cct2 I487E -GFP were grown to OD 600 = 0.8, and then further cultured in nutrient-rich medium for 6 h. Images of cells were obtained using an inverted fluorescence microscope. Scale bar, 2 µm. ( F ) Cells from ( E ) were quantified for the vacuolar localization of Cct2-GFP. n = 300 cells were pooled from three independent experiments. Data are presented as means ± SD. *** P < 0.001; two-tailed Student’s t tests were used. P < 0.0001(Cct2 D473A vs. WT); P < 0.0001(Cct2 I487E vs. WT). ( G ) ape1 ∆ atg8 ∆ cells co-expressing an empty vector (EV), 3×HA-Cct2, 3×HA-Cct2 D473A , or 3×HA-Cct2 I487E with 3×FLAG-Atg8 in the presence of Ape1 P22L were grown to the log phase under nutrient-rich medium. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and then analyzed by western blot using anti-HA antibody. The data are representative of three independent experiments. ( H ) ape1 ∆ or ape1 ∆ atg11 ∆ cells co-expressing 3×HA-Cct2 and FLAG-Atg8 were grown to log phase in the presence of Ape1 P22L under nutrient-rich conditions. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and then analyzed by western blot using anti-HA antibody. The data are representative of three independent experiments. ( I ) ape1 ∆ or ape1 ∆ atg8 ∆ cells co-expressing 3×HA-Cct2 and Atg11-3×FLAG were grown to log phase in the presence of Ape1 P22L under nutrient-rich conditions. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and then analyzed by western blot using anti-HA antibody. The data are representative of three independent experiments. .

Article Snippet: Rabbit anti-Ape1 , GL Biochem Ltd., Shanghai , .

Techniques: Expressing, Plasmid Preparation, Fluorescence, Microscopy, Two Tailed Test, Western Blot, Control, Cell Culture, Immunoprecipitation

Journal: EMBO Reports

Article Title: Two distinct regulatory pathways govern Cct2-Atg8 binding in the process of solid aggrephagy

doi: 10.1038/s44319-024-00275-7

Figure Lengend Snippet: ( A ) The indicated yeast cells were grown to the log phase in nutrient-rich medium. Samples were analyzed by immunoblot for detecting the maturation of PrApe1 into mApe1. Pgk1 served as a loading control. ( B ) Quantification of mApe1 to PrApe1+mApe1 ratio from ( A ). Data are presented as means ± SD (Data represent the results of three independent experiments). *** P < 0.001; NS, not significant; two-tailed Student’s t tests were used. P < 0.0001 (WT atg5 Δ vs. WT Cct2); P = 0.5412 (WT EV vs. WT Cct2); P < 0.0001 (WT atg11 Δ vs. WT Cct2); P < 0.0001 (P22L atg5 Δ vs. P22L Cct2); P < 0.0001 (P22L EV vs. P22L Cct2); P < 0.0001 (P22L atg11 Δ vs. P22L Cct2). ( C ) The indicated yeast cells were grown to an OD 600 = 0.6. Subsequently, 0.1 mM CuSO 4 was added to the cells to induce GFP-47Q to form solid aggregates for 12 h. Samples were analyzed by immunoblot for the cleavage of GFP-47Q. Pgk1 served as a loading control. ( D ) Quantification of GFP to GFP-47Q + GFP ratio from ( C ). Data are presented as means ± SD (Data represent the results of three independent experiments). *** P < 0.001; two-tailed Student’s t tests were used. P < 0.0001 (EV vs. Cct2); P < 0.0001 ( atg11 Δ vs. Cct2). ( E ) ape1 ∆ yeast cells co-expressing RFP-Ape1 P22L with Atg1-2✕GFP, GFP-Atg8, Atg11-2✕GFP, or Cct2-GFP were grown to the log phase in nutrient-rich medium. Images of cells were obtained using an inverted fluorescence microscope. Scale bar, 2 µm. ( F ) Cells from ( E ) were quantified for the number of cells in which RFP-Ape1 P22L colocalized with the indicated GFP-fused proteins. n = 300 cells were pooled from three independent experiments. Data are shown as mean ± SD. ( G ) ape1 ∆ yeast cells co-expressing RFP-Ape1 P22L and Atg11-2✕GFP with 3✕HA-Cct2, 3✕HA-Cct2 D473A, or 3✕HA-Cct2 I487E were grown to the log phase in nutrient-rich medium. Images of cells were obtained using an inverted fluorescence microscope. Scale bar, 2 µm. ( H ) Cells from ( G ) were quantified for the number of cells in which RFP-Ape1 P22L colocalized with Atg11-2✕GFP. n = 300 cells were pooled from three independent experiments. Data are shown as mean ± SD. *** P < 0.001; two-tailed Student’s t tests were used. P < 0.0001 (D473A vs. WT); P < 0.0001 (I487E vs. WT).

Article Snippet: Rabbit anti-Ape1 , GL Biochem Ltd., Shanghai , .

Techniques: Western Blot, Control, Two Tailed Test, Expressing, Fluorescence, Microscopy

Journal: EMBO Reports

Article Title: Two distinct regulatory pathways govern Cct2-Atg8 binding in the process of solid aggrephagy

doi: 10.1038/s44319-024-00275-7

Figure Lengend Snippet: ( A ) ape1 ∆ cells co-expressing 3×HA-Cct2 and Atg1-3×FLAG were grown to the log phase in the presence of Ape1 P22L under nutrient-rich conditions. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and then analyzed by western blot using anti-HA antibody. The data are representative of three independent experiments. ( B ) In vitro kinase assays were performed using the GST-Cct2 purified from E. coli as substrates and WT Atg1-3×FLAG purified from yeast cells expressing Ape1 P22L under nutrient-rich conditions as the protein kinase. Phosphorylation of GST-Cct2 was detected using anti-thioP antibody. The data are representative of two independent experiments. ( C ) In vitro kinase assays were performed using the GST-Cct2 purified from E. coli as substrates with WT (wild-type) or KD (kinase-dead) Atg1-3×FLAG purified from yeast cells expressing Ape1 P22L under nutrient-rich conditions as a protein kinase. Phosphorylation of GST-Cct2 was detected using anti-thioP antibody. The data are representative of two independent experiments. ( D ) In vitro kinase assays were performed using the GST-Cct2 or its variants purified from E. coli as substrates with wild-type (WT) Atg1-3×FLAG or Atg1 kinase dead (KD) purified from yeast cells expressing Ape1 P22L under nutrient-rich conditions as the protein kinase. Phosphorylation of GST-Cct2 was detected using anti-thioP antibody. The data are representative of two independent experiments. ( E ) Eight typical Atg1 phosphorylation sites on GST-Cct2 D3 domain. ( F ) An in vitro kinase assays were performed using GST, GST-Cct2, or eight indicated GST-Cct2 variants were purified from E. coli as substrates and purified Atg1-3×FLAG from yeast cells expressing Ape1 P22L under nutrient-rich conditions as a protein kinase. The phosphorylation of Cct2 and these variants were detected by anti-thioP antibody. The data are representative of two independent experiments. ( G ) In vitro kinase assays were performed using the GST-Cct2 or GST-Cct2 S412A-S470A purified from E. coli as substrates with Atg1-3×FLAG purified from yeast cells expressing Ape1 P22L under nutrient-rich conditions as a protein kinase. Phosphorylation of Cct2 and Cct2 S412A-S470A were detected using anti-thioP antibody. The data are representative of two independent experiments. .

Article Snippet: Rabbit anti-Ape1 , GL Biochem Ltd., Shanghai , .

Techniques: Expressing, Immunoprecipitation, Western Blot, In Vitro, Purification

Journal: EMBO Reports

Article Title: Two distinct regulatory pathways govern Cct2-Atg8 binding in the process of solid aggrephagy

doi: 10.1038/s44319-024-00275-7

Figure Lengend Snippet: ( A ) ape1 ∆ or ape1 ∆ atg5 ∆ yeast cells co-expressing RFP-Ape1 P22L, Vph1-GFP with empty vector (EV), 3×HA-Cct2 WT or 3×HA-CCT2 S412A-S470A were grown to the log phase under nutrient-rich conditions. Images of cells were obtained using an inverted fluorescence microscope. Scale bar, 2 µm. ( B ) Cells from ( A ) were quantified for the vacuolar localization of RFP-Ape1 P22L. n = 300 cells were pooled from three independent experiments. Data are presented as means ± SD. *** P < 0.001; two-tailed Student’s t tests were used. P < 0.0001 ( atg5 Δ vs. Cct2); P = 0.0003(EV vs. Cct2); P = 0.0006(Cct2 2A vs. Cct2). ( C ) ape1 ∆ atg5 ∆ or ape1 ∆ yeast cells co-expressing Ape1 WT or Ape1 P22L with empty vector (EV), wild-type 3×HA-Cct2 (WT), or 3×HA-Cct2 S412A-S470A (2A) were grown to the log phase under nutrient-rich conditions. Samples were analyzed by immunoblot for detecting the maturation of PrApe1 into mApe1. Pgk1 served as a loading control. ( D ) Quantification of mApe1 to PrApe1 ratio from ( C ). Data are presented as means ± SD (Data represent the results of three independent experiments). *** P < 0.001; NS, not significant; two-tailed Student’s t tests were used. P < 0.0001 (WT atg5 Δ vs. WT Cct2); P = 0.7945 (WT EV vs. WT Cct2); P = 0.8705 (P22L Cct2 S412A-S470A vs. WT Cct2); P < 0.0001 (P22L atg5 Δ vs. P22L Cct2); P = 0.0012 (P22L EV vs. P22L Cct2); P = 0.0007 (P22L Cct2 S412A-S470A vs. P22L Cct2). ( E ) ape1 ∆ atg8 ∆ cells co-expressing an empty vector (EV), 3×HA-Cct2, 3×HA-Cct2 S412A-S470A (2A) with FLAG-Atg8 were grown into the log phase under nutrient-rich conditions. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and then analyzed by western blot using anti-HA antibody. The data are representative of three independent experiments. ( F ) ape1 ∆ atg1 ∆ atg8 ∆ cells co-expressing an empty vector (EV), 3×HA-Cct2, 3×HA-Cct2 S412A-S470A (2A) with FLAG-Atg8 were grown to the log phase under nutrient-rich conditions. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and then analyzed by western blot using anti-HA antibody. The data are representative of three independent experiments. ( G ) In vitro GST pulldowns were performed using His-Atg8 with GST, GST-Cct2, GST-Cct2 S412A-S470A (2A) purified from E. coli . Protein samples were separated by SDS-PAGE and detected using Coomassie blue staining. The data are representative of two independent experiments. ( H ) In vitro kinase assays were performed using GST-Cct2 WT or 2A (S412A-S470A) purified from E. coli as substrates, with WT or KD Atg1-3×FLAG purified from yeast cells expressing Ape1 P22L under nutrient-rich conditions as the protein kinase. After that, in vitro GST pulldowns were performed using phosphorylated products enriched by GST beads in vitro with His-Atg8 protein purified from E. coli . Protein samples were separated by SDS-PAGE, and then detected using Coomassie blue staining. The data are representative of three independent experiments. .

Article Snippet: Rabbit anti-Ape1 , GL Biochem Ltd., Shanghai , .

Techniques: Expressing, Plasmid Preparation, Fluorescence, Microscopy, Two Tailed Test, Western Blot, Control, Immunoprecipitation, In Vitro, Purification, SDS Page, Staining

Journal: EMBO Reports

Article Title: Two distinct regulatory pathways govern Cct2-Atg8 binding in the process of solid aggrephagy

doi: 10.1038/s44319-024-00275-7

Figure Lengend Snippet: ( A ) Yeast cells co-expressing Cu 2+ -inducible GFP-47Q plasmids with an empty vector (EV), 3×HA-Cct2, or Cct2 S412A-S470A (2A) were grown to an OD 600 = 0.6. Subsequently, 0.1 mM CuSO 4 was added to the cells to induce GFP-47Q to form solid aggregates over 12 h. Samples were analyzed by immunoblot for the cleavage of GFP-47Q. Pgk1 served as a loading control. ( B ) Quantification of GFP to GFP-47Q + GFP ratio from ( A ). Data are presented as means ± SD (Data represent the results of three independent experiments). *** P < 0.001; two-tailed Student’s t tests were used. P < 0.0001 (EV vs. Cct2); P < 0.0001 (S412A-S470A vs. Cct2). ( C ) ape1 ∆ cells co-expressing an empty vector, 3×HA-Cct2, or 3×HA-Cct2 S412A-S470A with Atg11-3×FLAG in the presence of Ape1 P22L were grown to the log phase under nutrient-rich medium. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and then analyzed by western blot using anti-HA antibody. The data are representative of three independent experiments. ( D ) ape1 ∆ or ape1 ∆ atg1 ∆ cells co-expressing 3×HA-Cct2 with Atg11-3×FLAG in the presence of Ape1 P22L were grown to the log phase under nutrient-rich conditions. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and then analyzed by western blot using anti-HA antibody. The data are representative of three independent experiments. ( E ) In vitro GST pulldowns were performed using Atg11 CC4 domain with GST, GST-Cct2, or GST-Cct2 S412A-S470A purified from E. coli . Protein samples were separated by SDS-PAGE and detected using Coomassie blue staining. The data are representative of two independent experiments. ( F ) In vitro phosphorylation assays were performed using GST-Cct2 WT or 2A(S412A-S470A) purified from E. coli as substrates, with WT or KD Atg1-3×FLAG, purified from yeast cells, in the presence of Ape1 P22L under nutrient-rich conditions as the protein kinase. After that, in vitro GST pulldowns were performed using phosphorylated products enriched by GST beads in vitro with Atg11 CC4 protein purified from E. coli . Protein samples were separated by SDS-PAGE, and then detected using Coomassie blue staining. The data are representative of three independent experiments. ( G ) In vitro phosphorylation assays were performed using GST-Cct2 WT or 2A(S412A-S470A) purified from E. coli as substrates, with WT or KD Atg1-3×FLAG, purified from yeast cells, in the presence of Ape1 P22L under nutrient-rich conditions as the protein kinase. After that, in vitro GST pulldowns were performed using phosphorylated products enriched by GST beads in vitro with the same amount of His-Atg8 protein and Atg11 CC4 protein purified from E. coli . Protein samples were separated by SDS-PAGE, and then detected using Coomassie blue staining. The data are representative of three independent experiments.

Article Snippet: Rabbit anti-Ape1 , GL Biochem Ltd., Shanghai , .

Techniques: Expressing, Plasmid Preparation, Western Blot, Control, Two Tailed Test, Immunoprecipitation, In Vitro, Purification, SDS Page, Staining

Journal: EMBO Reports

Article Title: Two distinct regulatory pathways govern Cct2-Atg8 binding in the process of solid aggrephagy

doi: 10.1038/s44319-024-00275-7

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rabbit anti-Ape1 , GL Biochem Ltd., Shanghai , .

Techniques: Recombinant, Software, Fluorescence, Microscopy, Imaging