Ybr063c (Cnm1) has the characteristics of a molecular tether. (A) Overexpressed mCherry-Ybr063c is localized only to areas of proximity between the nuclear envelope and mitochondria. The nuclear envelope was visualized with Nsg1-GFP and mitochondria by the blue mitochondrial dye (MitoView 405). Insets show an enlarged region of a single nucleus and mitochondria interface with mCherry-Ybr063c signal present where the two organelles connect. Scale bar, 5 µm. (B) Ybr063c is a membrane protein embedded in the lipid bilayer. Enriched mitochondrial fractions from cells overexpressing Ybr063c tagged with 3HA on its N terminus were either treated by CE or left untreated (UT). Following this, they were separated into membrane proteins in the pellet (P) or soluble proteins in the supernatant (S). (C) Overexpression (OE) of Ybr063c drives clustering of mitochondria around the nucleus. The nuclear membrane was visualized by Nsg1-GFP and mitochondria were stained using a red dye (MitoTracker Orange). Scale bar, 5 µm. (D) Quantitation of the proximity between mitochondria and nucleus from C is shown as the percentage of mitochondrial signal that overlaps with the nuclear envelope signal in both a strain that overexpresses Ybr063c (OE Ybr063c) and a control strain. Bars represent standard deviation. n = 500; ***, P = 7.24e−73. (E) EM images of the extended contact sites between nucleus and the mitochondria that are formed by overexpressing Ybr063c (OE Ybr063c, highlighted by a green outline). Scale bars, 200 nm. (F) Tomograms of nucleus–mitochondria contacts in yeast overexpressing Ybr063c (OE Ybr063c, left). Scale bars, 300 nm. Insets show high densities that may indicate molecular tethers (arrowheads). Scale bars, 50 nm. 3D segmentations of the contact site area seen in the tomograms (right). The nucleus membrane is marked in yellow, and the mitochondrial membrane is marked in green. Dashed lines on tomograms indicate the area that is seen in the 3D segmentation. M, mitochondrion; N, nucleus.
