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The conserved oligomeric Golgi complex is involved in double-membrane vesicle formation during autophagy.

Yen WL, Shintani T, Nair U, Cao Y, Richardson BC, Li Z, Hughson FM, Baba M, Klionsky DJ - J. Cell Biol. (2010)

Bottom Line: The morphological hallmark of this process is the formation of double-membrane autophagosomes that sequester cytoplasm.COG subunits localized to the phagophore assembly site and interacted with Atg (autophagy related) proteins.In addition, mutations in the COG genes resulted in the mislocalization of Atg8 and Atg9, which are critical components involved in autophagosome formation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA.

ABSTRACT
Macroautophagy is a catabolic pathway used for the turnover of long-lived proteins and organelles in eukaryotic cells. The morphological hallmark of this process is the formation of double-membrane autophagosomes that sequester cytoplasm. Autophagosome formation is the most complex part of macroautophagy, and it is a dynamic event that likely involves vesicle fusion to expand the initial sequestering membrane, the phagophore; however, essentially nothing is known about this process including the molecular components involved in vesicle tethering and fusion. In this study, we provide evidence that the subunits of the conserved oligomeric Golgi (COG) complex are required for double-membrane cytoplasm to vacuole targeting vesicle and autophagosome formation. COG subunits localized to the phagophore assembly site and interacted with Atg (autophagy related) proteins. In addition, mutations in the COG genes resulted in the mislocalization of Atg8 and Atg9, which are critical components involved in autophagosome formation.

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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 OD600 = 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 OD600 = 0.8 and observed by fluorescence microscopy. DIC, differential interference contrast. Bar, 2.5 µm.
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fig7: 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 OD600 = 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 OD600 = 0.8 and observed by fluorescence microscopy. DIC, differential interference contrast. Bar, 2.5 µm.

Mentions: To overexpress COG subunits, we chromosomally replaced their endogenous promoters with the GAL1 promoter and an N-terminal HA tag and performed a series of protein A (PA) affinity purification experiments. Either a PA-tagged Atg protein or PA alone was coexpressed in combination with HA-COG subunits. Cells were grown in synthetic minimal medium with galactose (SMG) to induce COG subunit overexpression. Cell extracts were prepared, and PA-tagged Atg proteins and associated proteins were affinity isolated. The recovered immunocomplex was resolved by SDS-PAGE, and the presence of HA-COG subunits was analyzed by Western blotting using anti-HA antibody. HA-Cog1 bound to PA-Atg17 and PA-Atg20, whereas HA-Cog3 was coprecipitated with PA-Atg17 and PA-Atg24 (unpublished data). HA-Cog4 was able to bind PA-Atg17, PA-Atg20, and PA-Atg24 (Fig. 7 A). HA-COG subunits did not bind to PA alone, indicating that the interactions with HA-COG subunits were dependent on Atg17, Atg20, or Atg24 fused to PA. In addition, these interactions were absent when the affinity isolation was performed with combined cell lysates from two different strains each expressing an individual tagged protein; thus, the interactions we detected did not occur after lysis (unpublished data).


The conserved oligomeric Golgi complex is involved in double-membrane vesicle formation during autophagy.

Yen WL, Shintani T, Nair U, Cao Y, Richardson BC, Li Z, Hughson FM, Baba M, Klionsky DJ - J. Cell Biol. (2010)

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 OD600 = 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 OD600 = 0.8 and observed by fluorescence microscopy. DIC, differential interference contrast. Bar, 2.5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2812853&req=5

fig7: 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 OD600 = 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 OD600 = 0.8 and observed by fluorescence microscopy. DIC, differential interference contrast. Bar, 2.5 µm.
Mentions: To overexpress COG subunits, we chromosomally replaced their endogenous promoters with the GAL1 promoter and an N-terminal HA tag and performed a series of protein A (PA) affinity purification experiments. Either a PA-tagged Atg protein or PA alone was coexpressed in combination with HA-COG subunits. Cells were grown in synthetic minimal medium with galactose (SMG) to induce COG subunit overexpression. Cell extracts were prepared, and PA-tagged Atg proteins and associated proteins were affinity isolated. The recovered immunocomplex was resolved by SDS-PAGE, and the presence of HA-COG subunits was analyzed by Western blotting using anti-HA antibody. HA-Cog1 bound to PA-Atg17 and PA-Atg20, whereas HA-Cog3 was coprecipitated with PA-Atg17 and PA-Atg24 (unpublished data). HA-Cog4 was able to bind PA-Atg17, PA-Atg20, and PA-Atg24 (Fig. 7 A). HA-COG subunits did not bind to PA alone, indicating that the interactions with HA-COG subunits were dependent on Atg17, Atg20, or Atg24 fused to PA. In addition, these interactions were absent when the affinity isolation was performed with combined cell lysates from two different strains each expressing an individual tagged protein; thus, the interactions we detected did not occur after lysis (unpublished data).

Bottom Line: The morphological hallmark of this process is the formation of double-membrane autophagosomes that sequester cytoplasm.COG subunits localized to the phagophore assembly site and interacted with Atg (autophagy related) proteins.In addition, mutations in the COG genes resulted in the mislocalization of Atg8 and Atg9, which are critical components involved in autophagosome formation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA.

ABSTRACT
Macroautophagy is a catabolic pathway used for the turnover of long-lived proteins and organelles in eukaryotic cells. The morphological hallmark of this process is the formation of double-membrane autophagosomes that sequester cytoplasm. Autophagosome formation is the most complex part of macroautophagy, and it is a dynamic event that likely involves vesicle fusion to expand the initial sequestering membrane, the phagophore; however, essentially nothing is known about this process including the molecular components involved in vesicle tethering and fusion. In this study, we provide evidence that the subunits of the conserved oligomeric Golgi (COG) complex are required for double-membrane cytoplasm to vacuole targeting vesicle and autophagosome formation. COG subunits localized to the phagophore assembly site and interacted with Atg (autophagy related) proteins. In addition, mutations in the COG genes resulted in the mislocalization of Atg8 and Atg9, which are critical components involved in autophagosome formation.

Show MeSH
Related in: MedlinePlus