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

Figure 2. Competitive inhibition of mitochondrial protein import in Δrpn4 cells remodels the cytosolic chaperone system.

Figure 2

  1. The clogger was expressed in wild‐type (WT) and Δrpn4 (Δ) cells for 4.5 h with medium containing 0.5% galactose. Medium was exchanged for a noninducing lactate medium. Precursor (pre) and mature (m) forms of the b 2Δ‐DHFR clogger were visualized by Western blotting using a DHFR‐specific antibody.
  2. The clogger was induced for 6 h with 0.5% galactose before cellular proteomes were measured by mass spectrometry. See Materials and Methods for further details.
  3. 3,550 proteins were measured in all samples of the three replicates.
  4. Principal component analysis. Presence of Rpn4 and clogger expression caused specific changes in the proteome. The different shapes of the data points indicate the three different biological replicates.
  5. GO Term Analysis was done using the GOrilla tool (http://cbl‐gorilla.cs.technion.ac.il/; Eden et al, 2009). The absence of Rpn4 caused a broad reduction in the components of the proteasome‐ubiquitin system. The number of proteins in the indicated GO components is stated as “Counts.”
  6. Comparison of the proteome of wild‐type and Δrpn4 cells after clogger expression for 6 h. Positions of proteins of the proteasome and chaperone systems are indicated in blue and green, respectively. Please note that Hsp104 and Hsp42 (indicated in magenta) show a higher abundance in Δrpn4 cells. See Dataset EV1 for details.
  7. Signals of Hsp104 and Hsp42 before and after clogger induction in wild‐type and Δrpn4 cells. The cytosolic soluble DHFR protein was expressed for control. The strains were grown in lactate medium and clogger expression was induced with 0.5% galactose for 6 h.
  8. Wild‐type carrying plasmids for the GAL‐induced expression of the clogger and for the constitutive expression (from TPI promoter) of Hsp42 and Hsp104 were grown on lactate medium to mid‐log phase and used to inoculate cultures with synthetic lactate (no clogger expression) or lactate plus 0.5% galactose (with clogger). Cell growth was measured over time.
  9. The respective strains were grown in lactate medium. Growth was monitored upon constant agitation at 30°C.

Source data are available online for this figure.