Figure 7.

Hul5 targets proteins that are specifically ubiquitylated in the low solubility cellular fraction. (a–d) Validation of the Hul5 substrate candidates Lsm7 and Pin3 using TAP-tagged strains expressing H₈-Ubi. IMAC was performed in denaturating conditions to pull down ubiquitylated proteins, and anti-TAP or anti-ubiquitin antibody was used for Western blot analysis. The asterisk denotes unspecific signal. Corresponding signal intensities for poly- and mono-ubiquitin were measured by subtracting the background signal in control cells (a, c). Solubility of ubiquitylated Lsm7^TAP (b) and Pin3^TAP (d) were assessed by comparing both soluble and pellet fractions subjected to IMAC and analyzed by Western blots with anti-TAP (b, d) and anti-ubiquitin (b). A 20 min 45°C heat-shock (HS) was also applied to Pin3^TAP expressing cells (c, d). (e–f) Turnover of Slh1 is dependent on cytosolic Hul5. Protein levels of Slh1^TAP were monitored by Western blot after the addition of 100 μg/ml cycloheximide to both HUL5 and hul5Δ cells grown at 25°C (e), and to hul5Δ cells expressing either ^GFPHul5 or ^GFP-NLSHul5 and shifted to 38°C (f). Relative averaged signal intensities (with standard deviations) were quantified and normalized to Pgk1 levels in three independent experiments.