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. 2012 Oct 4;7(10):e45194. doi: 10.1371/journal.pone.0045194

Figure 3. KlDoa10 Nt- and Ct- fragments interact and form a functional ubiquitin ligase.

Figure 3

A. Co-immunoprecipitation (co-IP) analysis of digitonin-solubilized microsomes. HA-tagged Nt-KlDoa10 and 13MYC-tagged Ct-KlDoa10 proteins were either individually expressed or co-expressed in K. lactis doa10Δ cells, and HA-Nt-KlDoa10 was precipitated with anti-HA agarose beads. Precipitates were analyzed by immunoblotting with anti-MYC or anti-HA. B. Control demonstrating specificity of the co-IP protocol. HA-epitope-tagged Ubc7 was precipitated with an anti-HA antibody from digitonin-solubilized microsomes derived from S. cerevisiae cells expressing MYC-tagged full-length Doa10 or a C-terminally truncated derivative, Doa101–950. Precipitates were analyzed by immunoblotting with anti-MYC or anti-HA antibodies. Co-precipitation is only observed for full-length Doa10. The strain (MHY3000) was made by T. Ravid and M.H. (unpublished). C. Co-expression of KlDoa10 Nt- and Ct-fragment reconstitutes Doa10 E3 ligase activity toward the model membrane substrate Deg1-Vma12-KanMX in an S.cerevisiae doa10Δ strain. Serial dilutions of S. cerevisiae transformants expressing the indicated epitope-tagged KlDoa10 fragment(s), FLAG-tagged Nt and 13MYC-tagged Ct, were spotted onto minimal plates lacking histidine and tryptophan (SD-his-trp) with or without G418 added. ScDoa10, S. cerevisiae (doa10Δ; Deg1-Vma12-KanMX) strain expressing functional ScDoa10 from a plasmid served as a control. Upper and lower panels are from the same plate. D. Co-expression of KlDoa10 Nt- and Ct- fragment reconstitutes E3 ligase activity toward the soluble model substrate Deg1-Ura3 in S.cerevisiae doa10Δ cells. Serial dilutions of the indicated transformants were spotted onto SD-his-trp and SD-his-trp-ura plates. Upper and lower panels are from the same plate. E. Endogenous split-KlDoa10 is active toward the model membrane substrate Deg1-Vma12-KanMX. Serial dilutions of WT or doa10Δ K. lactis cells transformed with a Deg1-Vma12-KanMX reporter plasmid (+) or an empty control plasmid (−) were spotted onto SD-leu and SD-leu+G418 plates.