Figure 1.

pI215l acts as an E2-ubiquitin conjugating enzyme. (A) Results from an in vitro ubiquitination assay showed that recombinant pI215L binds one or two ubiquitin molecules, in an ATP- and Mg²⁺-dependent manner, and in the presence of an E1 ubiquitin-activating enzyme (UBA1). Reaction mixtures were incubated 2 hours at 37 °C, quenched with a non-reducing protein loading buffer, and then subjected to polyacrylamide gel electrophoresis. (B) When the ubiquitination assay was performed using an ubiquitin that is mutated in all lysine residues (Ub^NoK), the di-ubiquitinated forms of pI215L were not detected. (C) The residue cysteine-85 is essential for the E2-like activity of pI215L. Site-directed mutagenesis revealed that replacement of Cys-85 by an alanine residue led to an absence of ubiquitinated species, whereas the substitution of the Cys-162 or Cys-189 residue does not hamper the formation of ubiquitinated forms of pI215L. (D) pI215L forms thioester bonds with ubiquitin in a wide range of temperatures, although mono-ubiquitinated forms of pI215L were less detectable at 4 °C and 24 °C. (E) pI215L binds ubiquitin in a broad range of pH values, with mono-ubiquitinated forms only found at a pH value of 7.5. (F) Poly-ubiquitinated forms of I215L were detected after a short incubation period of 1 min, whereas the mono-ubiquitinated forms were detected later (5 min), showing increased concentrations in longer incubation times (e.g. 30, 60 minutes). (G) Mono- and poly-ubiquitinated forms of pI215L were mainly found in the Triton X-100-soluble fractions harvested at 6 and 16 hpi. In detergent-insoluble fractions, only the di-ubiquitinated form of pI215L was faintly detected (asterisks). Blots of Fig. 1(D to F) were cropped to improve clarity, full-length blots are presented in supplementary Figure S1. Fig. 1(G) is composed by two individual blots obtained from soluble and insoluble fractions.