Figure 2. Dma2 ubiquitin ligase activity is required for viability of cla4 mutants and for normal septin organization. (A) A dma1∆ dma2∆ cla4∆ mutant strain (cla4-75 allele) was transformed with an empty vector or with a plasmid encoding pDma2^WT, pDma2^C451A or pDma2^R299A. Dma2 proteins were Myc-tagged while Dma2^R299A was expressed from a low- (CEN) or high- (2 μ) copy plasmid. Cells were incubated at 25°C or 37°C for 3 d (left). Extracts from (A) were analyzed by sequential blotting with anti-Myc (right) then anti-Pgk1 as loading control. (B) dma1∆ dma2∆ mutants transformed as in (A) were transformed with a plasmid expressing GFP-Cdc3. Cells were grown at 25°C, shifted to 37°C for 3 h, and percentages of budded cells showing abnormal septin ring morphology or position were scored by microscopy (left; n = 200). Examples of cells scored as having abnormal septin rings are shown on the right. Scale bar is 5 μm.