Figure 5: Rsp5 conjugates K48-linked ubiquitin chains to its heat-induced substrates in vivo in an Ubp2 and 3-dependent manner. | Nature Communications

Figure 5: Rsp5 conjugates K48-linked ubiquitin chains to its heat-induced substrates in vivo in an Ubp2 and 3-dependent manner.

From: Deubiquitinase activity is required for the proteasomal degradation of misfolded cytosolic proteins upon heat-stress

Figure 5

(a) Schematic representation of two possible models to explain the role of Rsp5 in the assembly of K48 chains after HS. The I537D mutant of Rsp5 that impairs mostly poly- but not monoubiquitination should only strongly affect the editing pathway. (b) Cells that expressed 3HARsp5 or 3HARsp5-I537D together with empty or MYCubiquitin-K48 only constructs were heat shocked for 20 min (40 °C) and crosslinked with 1% formaldehyde for the remaining 10 min before lysis with SDS and the anti-HA IP. Western blot analysis of both IP and input samples are shown. (c,d) WT and ubp2Δ (c) or ubp3Δ (d) cells that expressed 3HARsp5 together with empty, MYCubiquitin-K48 only or MYCubiquitin-K63 only constructs were heat shocked (40 °C, 20 min) or not, and crosslinked with 1% formaldehyde before lysis with SDS and IP with anti-HA antibody-conjugated magnetic beads. Western blot analysis of both IP and Input samples are shown. (e,f) As in b,c, the ubp2Δ (e) or ubp3Δ (f) cells carried out either an empty control plasmid or a plasmid that expressed the WT or catalytically inactive deubiquitinases.

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