Figure 2.
Mitochondrial-derived multilamellar structures are enriched for Tom70-GFP and exclude Tim50-mCherry. (A–E) Dual-immuno tomography obtained from five 90-nm cell sections of a yeast cell expressing Tom70-yEGFP and Tim50-mCherry treated with 200 nM Rap. Secondary antibodies conjugated to 6-nm or 10-nm gold particles targeted primary antibodies for GFP or mCherry, respectively. Scale bar = 200 nm. See related Video 1. (A) Tomograph of the immune-reactive surface of the middle section (cell section #3). White arrowheads point to 6-nm gold particles, red arrowheads point to 10-nm gold particles. (B) Tomograph of the mid-plane of the middle section (cell section #3). Yellow arrow: mitochondria, white arrow: multilamellar structure. (C) Model overlay of section 3. Purple labels mitochondria, green labels the outer layer of doublet membranes, while cyan labels the internal layer of doublet membranes in the mitochondrial-derived multilamellar structure. (D) 3D model of the dual-immuno tomography described above. The mitochondrial-derived multilamellar structure is wire-modeled with the limiting membrane of the outer double-membrane labeled in green and the inner doublet membrane labeled in cyan. 6-nm gold particles appearing on the outer surface are overlayed with green spheres, while 6-nm gold particles appearing within the multilamellar structure are overlayed with cyan spheres. 10-nm gold particles are overlayed with red spheres. Mitochondria: purple, ER: yellow. (E) Same 3D model as shown in D rotated vertically 90°. (F) Quantification of the total anti-GFP 6-nm and anti-mCherry 10-nm immunogold conjugated secondary antibodies that labeled the indicated cell structures from a thin-section TEM analysis of 178 cell sections from yeast expressing Tom70-yEGFP and Tim50-mCherry that were treated with 200 nM Rap.

Mitochondrial-derived multilamellar structures are enriched for Tom70-GFP and exclude Tim50-mCherry. (A–E) Dual-immuno tomography obtained from five 90-nm cell sections of a yeast cell expressing Tom70-yEGFP and Tim50-mCherry treated with 200 nM Rap. Secondary antibodies conjugated to 6-nm or 10-nm gold particles targeted primary antibodies for GFP or mCherry, respectively. Scale bar = 200 nm. See related Video 1. (A) Tomograph of the immune-reactive surface of the middle section (cell section #3). White arrowheads point to 6-nm gold particles, red arrowheads point to 10-nm gold particles. (B) Tomograph of the mid-plane of the middle section (cell section #3). Yellow arrow: mitochondria, white arrow: multilamellar structure. (C) Model overlay of section 3. Purple labels mitochondria, green labels the outer layer of doublet membranes, while cyan labels the internal layer of doublet membranes in the mitochondrial-derived multilamellar structure. (D) 3D model of the dual-immuno tomography described above. The mitochondrial-derived multilamellar structure is wire-modeled with the limiting membrane of the outer double-membrane labeled in green and the inner doublet membrane labeled in cyan. 6-nm gold particles appearing on the outer surface are overlayed with green spheres, while 6-nm gold particles appearing within the multilamellar structure are overlayed with cyan spheres. 10-nm gold particles are overlayed with red spheres. Mitochondria: purple, ER: yellow. (E) Same 3D model as shown in D rotated vertically 90°. (F) Quantification of the total anti-GFP 6-nm and anti-mCherry 10-nm immunogold conjugated secondary antibodies that labeled the indicated cell structures from a thin-section TEM analysis of 178 cell sections from yeast expressing Tom70-yEGFP and Tim50-mCherry that were treated with 200 nM Rap.

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