FIGURE 8.

Immunoblot analysis and CTP-dependent DAG kinase activities of wild-type and mutant forms of Dgk1p. dgk1Δ mutant cells bearing the indicated wild-type (WT) and mutant galactose-inducible DGK1 alleles on the YCplac111 plasmid (see Table 1) were grown to exponential phase in synthetic medium with 2% raffinose as the carbon source. The expression of the DGK1 alleles was induced by the addition of 2% galactose to the growth medium. The cells were then incubated for 24 h, followed by the preparation of cell extracts and the membrane fraction. A, 10 μg of each membrane fraction was subjected to immunoblot analysis using anti-Dgk1p antibodies (1 μg/ml). The positions of the full-length (F) and truncated (T) forms of Dgk1p are indicated. The blot shown is representative of two independent experiments. B, each cell extract was used for the measurement of DAG kinase activity. The data shown are means ± S.D. from triplicate enzyme determinations from two independent experiments.