Figure 3. In vivo characterization of Ssb1Δ601-13 shows complementation of pleiotropic ssb1,2Δ phenotypes.

(a) Schematic overview of Ssb domains and constructs used. Ssb1Δ601–13 lacks 13 C-terminal residues. (b) Growth analysis of Ssb-GFP constructs. Wild type (wt) or ssb1,2Δ cells transformed with empty vector or Ssb-GFP versions were adjusted to OD₆₀₀=0.4 and spotted in fivefold serial dilutions onto a YPD-plate, which was incubated at 30 °C for two days. (c) Cellular distribution of yEGFP-Ssb1 and yEGFP-Ssb1Δ601–13 analysed by confocal fluorescence microscopy. Transformed cells were grown to early exponential phase and applied onto agarose slices. Differential interference contrast (DIC) with scale bar=5 μm; DAPI was used for nuclei staining, overlay merges DAPI and GFP. (d) For polysome profiling wt cells transformed with empty vector (grey) or ssb1,2Δ cells transformed with either empty vector (blue) or Ssb1 constructs (green, orange) were grown to early exponential phase. Lysates were adjusted and 18 A₂₆₀ units on top of a 15–45% (w/v) sucrose gradient were ultracentrifuged followed by gradient fractionation from top to bottom and OD₂₅₄ monitoring (top). Fractions were analysed via immunoblotting (bottom). Wt profiles at the background serve as control. (e) Protein expression of wt or ssb1,2Δ cells transformed with either empty vector or Ssb1 constructs. Cells were grown to early exponential phase, lysed and protein levels were adjusted followed by immunoblotting. (f) Quantification of Rpl17A protein levels as shown exemplarily in e. Rpl17A signals were normalized to Pgk1 loading control and wt cells transformed with empty vector were set as 100%. Error bars represent s.e.m. of at least three independent experiments. (g) Quantitative analysis of aggregates isolated from wt and ssb1,2Δ cells transformed with empty vector or Ssb1 constructs. Samples were blotted followed by Ponceau S staining and immunological detection.