Growth phenotype of yeast ΔtrkΔtrk2 mutants transformed with different K⁺ channels or a K⁺ transporter. Yeast cells were transformed with genes encoding either homologous TRK1, human Kir2.1 channel, or the viral channels Kcv or Kesv, as well as with mutants in which the second TMD of Kesv was extended at position 113 by 3 (Kesv¹¹³VVV) or 2 valines (Kesv¹¹³VV). In addition, yeast cells were transformed with a chimera in which the N terminus of Kcv was replaced with the N terminus of Kesv (Kcv(NT)Kesv), or vice versa (Kesv(NT)Kcv). All yeasts were grown on medium containing either 100 mM or 0.5 mM K⁺. Only yeast transformed with Kesv and Kesv(NT)Kcv failed to grow on low-K⁺ medium.

Differential colocalization of Kcv:GFP and Kesv:GFP with MitoTracker in HEK293 cells. (A) (Upper) Color-coded structural elements of Kcv:GFP chimera comprising TM1 and TM2, N-terminal domain (NT) (all in light gray), pore (orange), and GFP (green). Confocal image of exemplary HEK293 cell expressing Kcv:GFP (Aa) and staining of the same cell with MitoTracker red (Ab). Overlay of the 2 images in which colocalization of the 2 colors is shown in white (Ac). Inset magnifies a region of the cell and shows that the green and red fluorescence are well separated. The apparent colocalization only results from an insufficient resolution of the red-stained mitochondria and the green-stained perinuclear ring. (Ad) Scatter plot of green and red pixels from region of interest (borders of green fluorescing cell). The yellow bars show thresholds for both colors. The dashed line provides linear regression solution. Pearson's colocalization coefficient in the present example is 0.12. (B) (Upper) Color-coded structural elements of Kesv:GFP chimera comprising TM1 and TM2, N-terminal domain (NT) (all in dark gray), pore (blue), and GFP (green). Confocal image of exemplary HEK293 cell expressing Kesv:GFP (Ba) and staining of the same cell with MitoTracker red (Bb). Overlay of the 2 images in which colocalization of the 2 colors is shown in white (Bc). Inset magnifies a region of the cell and shows that the green and red fluorescence colocalize. (Bd) Scatter plot of green and red pixels as in Fig. 2A. Pearson's colocalization coefficient in this example is 0.36.

Import of Kesv into isolated mitochondria in vitro. (A) Kesv:GFP and GFP were synthesized in the presence of [³⁵S]methionine in reticulocyte lysate and incubated with isolated yeast mitochondria for 10 min at 25°C. The mitochondria were subsequently re-isolated by centrifugation, resuspended in the absence (lane 2) or presence of 600 mM NaCl (lane 3), and again re-isolated. The proteins were separated by SDS/PAGE and analyzed using a PhosphorImager. An aliquot of the reticulocyte lysate was included as a standard (lane 1). (B) ³⁵S-labeled Kesv was synthesized in reticulocyte lysate and incubated with isolated yeast mitochondria for 10 min. As indicated, the samples contained valinomycin (-ΔΨ), or the mitochondria were subsequently treated with proteinase K (+ PK). The mitochondria were re-isolated and analyzed by SDS/PAGE. (C) Radiolabeled Kesv was imported into mitochondria as in B, and the mitochondria were re-isolated and lysed in the presence of digitonin. The proteins were separated by blue native PAGE. (D) Kesv was imported into mitochondria that were in parallel isolated from a tom22Δ strain (14) or from the corresponding WT strain. Samples were removed after different times of incubation as indicated and treated with proteinase K. The proteins were separated by SDS/PAGE, and the relative amounts were determined using a PhosphorImager. The highest value was set to 100% (control).

The N terminus does not determine targeting of viral K⁺ channels. Upper in A and B: color-coded structural elements of chimeras (see Fig. 2). The location of mutations is indicated by red bars. (A and B) Images of GFP (green) and MitoTracker (red) channels from confocal images of exemplary HEK293 cells expressing different chimeras of Kesv:GFP or Kcv:GFP. Insets show overlay of the green and red channels from areas indicated. White pixels highlight areas of maximal colocalization. (A) Kesv:GFP channel with mutations (R³E, R⁴E) in the N terminus. (B) Chimera of N terminus of Kesv plus Kcv (Kesv(NT)Kcv:GFP). (C) Pearson's colocalization coefficient (P) for GFP and MitoTracker red in HEK293 cells expressing the constructs listed.

The second TMD determines targeting of viral K⁺ channels. (A–C) (Top) color-coded structural elements of chimeras (see Fig. 2). The location of the insertion of the 3-amino acids IVL is indicated in yellow. (A–C) Images of GFP (green) and MitoTracker (red) channels from confocal images of exemplary HEK293 cells expressing different chimeras of Kesv:GFP or Kcv:GFP. Insets show overlay of the green and red channels from areas indicated. White pixels highlight areas of maximal colocalization. (A) Chimera of Kcv:GFP in which the pore of this channel has been replaced with the pore of channel Kesv. Chimera of Kesv:GFP in which TM2 has been extended at position 113 (B) or at position 108 (C) by the 3-aa IVL. (D) Pearson's colocalization coefficient (P) for GFP and MitoTracker red in HEK293 cells expressing Kesv:GFP constructs in which TM2 was extended in the positions indicated. P was determined as shown in Fig. 2. The filled bars illustrate extensions by amino acids motive VVV. For the remaining extensions the amino acids motives VV (bar a), IVL (bar b), and IVLIVL (bar c) were used. Mean values ± standard deviation from ≥10 images.