Figure 7. Overexpression of Fzo1.

(A) Total protein extracts of fzo1Δ cells transformed with an empty vector (fzo1Δ), pRS314-FZO1 (WT) or pRS414-TEF-FZO1 (FZO1 o.e.) were analyzed by anti-Fzo1 and anti-Pgk1 immunoblotting. Fzo1 is overexpressed about 50 fold in FZO1 o.e. as compared to WT conditions. (B) Serial dilutions of cells from A grown in the presence of glucose or glycerol as the sole carbon source at 30°C. Lack or overexpression of Fzo1 both abolishes respiration and, therefore, growth on glycerol, consistent with inhibition of mitochondrial fusion. (C) Mitochondrial morphology in WT and FZO1 o.e. cells. Left: Representative morphologies. Right: Percentage of WT and FZO1 o.e. cells with indicated mitochondrial morphologies. Error bars represent the s.d. from three independent experiments. (D) Left: TEM analysis of in vitro outer membrane fusion reactions performed with mitochondria isolated from wild-type cells or cells overexpressing Fzo1. Note that mitochondria from Fzo1 o.e. cells are smaller than from wild-type cells. Right: Effect of Fzo1 overexpression on outer membrane fusion and attachment in vitro. (E) Slices through tomographic volume of mitochondrial attached intermediates upon Fzo1 overexpression (abortive, 74%, see Table 1); outer membrane distance (blue bracket) and densities between outer membranes (red arrows) are indicated. (F) 3D rendering of two closely apposed mitochondria shown in E.

DOI: http://dx.doi.org/10.7554/eLife.14618.020

Figure 7—figure supplement 1. Absence or accumulation of Fzo1.

(A) Tomographic slice of in vitro attachment reactions with mitochondria isolated from fzo1△ cells; scale bar 100 nm. (B) Levels of Ugo1 and Mgm1 in whole-cell extracts prepared from wildtype or Fzo1-overexpressing cells. Ugo1 levels did not vary, which is consistent with an imbalance with mitofusins upon Fzo1 overexpression. Note that the ratio between long and short forms of Mgm1 was slightly shifted toward the short form in cells overexpressing Fzo1. This may contribute to the changes in cristae morphology upon Fzo1 overexpression as seen in the electron micrographs shown in (D). (C) Mitochondrial morphology in representative WT and FZO1 o.e. cells. (D) Electron micrographs from Figure 7D at higher resolution. Fusion intermediates (fused outer membranes, separated inner membranes) and attached intermediates (attached outer membranes) are indicated by red and green arrowheads, respectively. (E-F) in vitro mitochondrial attachment upon Fzo1 overexpression. (E) Method summary: Mitochondria isolated from cells expressing either mito-GFP or mito-mCherry were mixed in equal amounts and processed for in vitro attachment reactions before analysis by fluorescence microscopy. Attached mitochondria (red-red; green-green; green-red) were counted in reactions stopped before centrifugation (t −10), after centrifugation (t 0) or after 10 min incubation on ice (t +10). (F) Ratios of attached mitochondria from wild-type (WT, blue) or Fzo1 overexpressing (FZO1 o.e., red) cells at t −10, t 0 and t +10. Ratios were normalized to the WT attachment at t 0. Mitochondria from Fzo1-overexpressing mitochondria were attached twice as frequently than mitochondria from wild-type cells at all time points, both after and before centrifugation (t −10). With Fzo1 overexpression, centrifugation stimulated in vitro attachment by a factor of two, compared to wild-type (t 0). In contrast, incubation on ice (t +10) had a weak effect on attachment in both wild-type and Fzo1 overexpressing conditions (compare t +10 with t 0), consistent with the requirement of this step for the transition from tethering to docking rather than for de novo attachment in vitro.

DOI: http://dx.doi.org/10.7554/eLife.14618.021

Figure 7—figure supplement 2. Cryo-ET of mitochondria from Fzo1-overexpressing cells.

(A–C) Minor population of attached intermediates (Other, 26%, see Table 1). (A) Slice through tomographic volume. (B) Stack representation of slices through tomographic volume with details of mitochondrial contacts. One out of 20 slices is shown. Red arrows, interstitial densities. All scale bars 100 nm. (C) 3D rendering of two closely apposed mitochondria. Rings of densities around the edge of contact areas are less extensive than in wild-type. (D–E) Cryo-ET of major population of attached intermediates (Abortive, 74%, see Table 1) (D) Histogram of closest outer membrane distances in attached mitochondria. (E) Slices through tomographic volume of the mitochondria shown in Figure 7F. One out of 20 slices is shown. Red arrows, interstitial density between outer membranes. All scale bars 100 nm.

DOI: http://dx.doi.org/10.7554/eLife.14618.022