Figure 3

Functional complementation with GlRpn10. (a) S. cerevisiae rpn10∆ strain was transformed individually with each of the constructs expressing the proteins shown in Panel b. The growth of these transformed yeast cells was monitored by spot test using serial dilutions on YCM plates lacking uracil and containing galactose and canavanine. To ensure that equal number of cells have been used, spotting was also done on YCM plates lacking uracil and containing glucose. All the plates were incubated at 30 °C. (b) Schematic diagrams of GlRpn10, ScRpn10, and different deletion variants of these two proteins. The regions corresponding to the two domains, VWA and the UIM, are denoted in blue and green respectively. The K residues within the VWA domain of ScRpn10 are marked and their respective positions are indicated above. (c) Western blot using anti-ubiquitin antibody of the total cell extract of wild-type, rpn10∆ and rpn10∆ transformed with the above mentioned constructs. The composition of the growth medium is same as given in (a) above, except that these transformants were grown in liquid medium. Extracts were loaded in the following order: lane 1, Wild-type transformed with vector; lane 2, rpn10∆ transformed with vector; lane 3, rpn10∆ cells expressing GlRpn10; lane 4, rpn10∆ cells expressing ScRpn10; lane 5, rpn10∆ cells expressing ScRpn10*; lane 6, rpn10∆ cells expressing GlRpn10* and lane 7, rpn10∆ cells expressing GlRpn10•. 3-PGK was used as loading control.