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. 2004 Aug;87(2):858–872. doi: 10.1529/biophysj.103.037671

FIGURE 5.

FIGURE 5

Refined SKOR/KAT1 chimeras analyzed in yeast growth tests and by coexpression with SKOR in oocytes. In the KAT1 channel several parts of the C-terminus were replaced by equivalent parts from SKOR. (A) The division of each C-terminus into six regions is represented schematically. The position of the first amino acid of each region is indicated. The separation (A–G) illustrates the division made in Fig. 4. (B) In the chimeras containing the KAT1 core and the chimeric C-termini, C-terminal parts originating from KAT1 are illustrated by white rectangles, and parts from SKOR by black rectangles. Additionally, the origin of the different C-terminal parts is indicated by a six-letters patch, “xxxxxx”, where “x” can be “k” (of KAT1 origin) or “s” (of SKOR origin). The six white rectangles and kkkkkk (B, a2) represent the KAT1 channel, the six black rectangles and ssssss (B, d4) the chimera KAT1-CtSKOR (cf. Fig. 2 C). Only chimeras containing the cNBD from SKOR (xxsxxx) were tested. Note that the C-terminus of KAT1 is shorter than that of SKOR because it does not contain an ankyrin domain. The chimeras were expressed in the potassium uptake deficient yeast strain Wagf2 and analyzed in drop tests (10 μl, OD600 adjusted to 0.1) on minimal medium supplemented with 2 mM K+. The presented results are representative for at least three repeats. (C) Coexpression (1:1 cRNA mixtures) of SKOR with chimeric mutants in Xenopus oocytes. The chimeric mutants are built of the core (Nterm-S6) of dnSKOR (= SKOR-G292R-D293N-A296V) and the chimeric C-termini as displayed in panel B. Current amplitudes (ISS) were measured at the end of 2-s voltage pulses to +50 mV. To compare results obtained from different oocyte batches, the data were normalized to the mean value of the control SKOR/H2O (solid column). Data are shown as means ± SD (n = 5–18, six different oocyte batches). The asterisks indicate combinations where the mutant showed a strong dominant-negative effect, i.e., ≈80% reduction in the K+ current amplitude (Student's t-test, P < 10−7). The expression of five constructs (marked with x) was verified in 35S-protein-labeling experiments.