Figure 3

Complementation of ScRub1 by PfNEDD8. (A) Schematic for generation of rub1Δ or ubc12Δ. The wild type locus (WT locus) was replaced with a linear kanamycin cassette flanked by the homology regions of target locus (T cassette) via a double crossover homologous recombination, resulting in the integration locus (Int locus). The kanamycin coding sequence (kanR) is under the control of translation elongation factor promoter (TEF pro) and terminator (TEF ter). The horizontal arrows indicate positions of primers used in the analysis of knockout strains. (B) Confirmation of knockout. The knockout was confirmed by PCR of the gDNAs of wild type (WT), rub1Δ and ubc12Δ strains using locus-specific primers (5′ Int for 5′ integration locus, 3′ Int for 3′ integration locus, WTsp for wild type locus and Con for ScAtg18). The ethidium bromide stained agarose gel shows PCR products, with DNA markers in kbp (M). (C) Western blot analysis of complemented strains. HA-PfNEDD8 (HA-PfN8) or HA-ScRub1 (HA-rub1) were episomally expressed in rub1Δ and ubc12Δ strains. The lysates of wild type (WT), rub1Δ, ubc12Δ, HA-PfNEDD8-complemented rub1Δ or ubc12Δ (HA-PfN8), and HA-ScNEDD8-complemented rub1Δ or ubc12Δ (HA-rub1) strains were processed for western blotting using anti-HA antibodies (ab-HA). The lanes containing lysates of complemented strains show free NEDD8/Rub1 protein (marked with an asterisk), whereas the lanes with rub1Δ[HA-PfN8] and rub1Δ[HA-rub1] strains also contain a high molecular weight band (marked with an arrowhead) and some lower molecular weight conjugates, which are absent in other lanes. Phosphoglycerate kinase (anti-PGK) was used as a loading control. The protein size markers are in kDa (M).