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. 2023 Jan 27;42(7):e112309. doi: 10.15252/embj.2022112309

Figure 6. MitoStores form proximity to mitochondria.

Figure 6

  1. After 4.5 h of the galactose‐induced expression of clogger and Pdb1‐RFP, cells were incubated for 4 h in the absence of galactose. Whereas Hsp104‐GFP was distributed throughout the cytosol after this chase period, Pdb1‐RFP showed a distribution pattern characteristic of mitochondrial proteins. Scale bars, 2 μm.
  2. To verify that stored proteins are chased into the mitochondrial matrix, Aim17‐GFP11 was co‐expressed with a clogger for 4.5 h in cells containing mitochondria with Oxa1‐GFP1‐10. Cells were washed in galactose‐free buffer and further incubated for 4 h before fluorescence was measured to quantify the matrix‐localized Aim17 proteins. Data are displayed as mean ± standard deviations from n = 3 independent biological replicates.
  3. Clogger was expressed for 4.5 h before colocalization of MitoStores (visualized by Pdb1‐RFP), and mitochondria (stained with matrix‐residing mito‐GFP) were assessed. Scale bars, 2 μm.
  4. Clogger was expressed for 4.5 h in Δhsp42 cells before Hsp104‐GFP was visualized. Please note that in the absence of Hsp42, no aggregates were detected. Scale bars, 5 μm.
  5. Clogger and Mam33‐HA were co‐expressed for 4.5 h in wild‐type and Δhsp42 cells. Mitochondria were isolated and subjected to Western blotting to detect mature (m) and precursor (pre) forms of Mam33‐HA. The matrix protein Tim16 served as a loading control.
  6. The mitochondria used for (E) were treated with proteinase K (PK) to remove surface‐exposed proteins. Mitochondrial membranes were lysed with NP‐40 and soluble (S) and aggregated (P) proteins were separated by centrifugation. T, total.
  7. To measure the toxicity of clogger expression, clogger proteins were induced for 24 h with lactate medium containing 0.5% galactose in the mutants indicated. Aliquots were removed, and the number of living cells was assessed by a plating assay on glucose‐containing plates. The resistance of Δrpn4 to clogger expression depends on Hsp104 and Hsp42. Data are displayed as mean ± standard deviations from n = 3 independent biological replicates.
  8. Schematic representation of cytosolic MitoStores.

Source data are available online for this figure.