Lost in traffic? The K(+) channel of lily pollen, LilKT1, is detected at the endomembranes inside yeast cells, tobacco leaves, and lily pollen

Abstract

Fertilization in plants relies on fast growth of pollen tubes through the style tissue toward the ovules. This polarized growth depends on influx of ions and water to increase the tube's volume. K(+) inward rectifying channels were detected in many pollen species, with one identified in Arabidopsis. Here, an Arabidopsis AKT1-like channel (LilKT1) was identified from Lilium longiflorum pollen. Complementation of K(+) uptake deficient yeast mutants was only successful when the entire LilKT1 C-terminus was replaced by the AKT1 C-terminus. No signals were observed in the plasma membrane (PM) of pollen tubes after expression of fluorescence-tagged LilKT1 nor were any LilKT1-derived peptides detectable in the pollen PM by mass spectrometry analysis. In contrast, fluorescent LilKT1 partly co-localized with the lily PM H(+) ATPase LilHA2 in the PM of tobacco leaf cells, but exhibited a punctual fluorescence pattern and also sub-plasma membrane localization. Thus, incorporation of LilKT1 into the pollen PM seems tighter controlled than in other cells with still unknown trafficking signals in LilKT1's C-terminus, resulting in channel densities below detection limits. This highly controlled incorporation might have physiological reasons: an uncontrolled number of K(+) inward channels in the pollen PM will give an increased water influx due to the raising cytosolic K(+) concentration, and finally, causing the tube to burst.

Keywords: K+ channel; Lilium longiflorum; heterologous expression; pollen; tip growth; trafficking; yeast mutant.

FIGURE 1

Sequence alignment of LilKT1 with plant inward-rectifying K⁺ channels. (A) Alignment of LilKT1 with AKT1 channel subunit from Arabidopsis thaliana showing the typical Shaker-like channel motifs and domains: transmembrane domains S1–S6 (bold, gray), binding domain for cyclic nucleotides (cNBD, blue), V and S (magenta) are important residues in the C-linker domain, ankyrin repeat domain (dark blue), putative 14-3-3 binding domain (green). Peptides underlined and bold were identified by LC-MS/MS. Di-acidic motifs at the C-terminus were indicated as red and bold. Mutated di-acidic motifs are underlined. (B) Phylogenetic tree of well-known shaker-like plant K⁺ channel subunits and the first eight protein sequences with the highest homology to LilKT1 (BLAST search, see Table S2 for accession numbers).

FIGURE 2

Functional complementation assay and expression of LilKT1 in yeast K⁺ uptake mutant. (A) PLY240 mutants expressing HA-yLilKT1 could not grow at low K⁺ concentrations. Control experiments were performed with the wild-type strain PLY232 containing the empty expression vector pGREG535. (B) HA-tagged LilKT1 was detected in the membrane fraction of PLY 240 after induction with galacatose (+). The respective organelle membrane markers, Pma1p (plasma membrane H⁺ ATPase), Dpm1p (dolichol phosphate mannose synthase of ER) and the Vma1p subunit of the vacuolar V-type ATPase (V1-subunit) were detected in non-induced yeast cells. Arrowheads mark the expected molecular weights. Additional protein signals in the Pma1p lane are caused by secondary antibody. 40 μg protein per lane.

FIGURE 3

Co-expression of LilKT1 with AKT1 and KAT1. (A) Drop assay with K⁺ uptake deficient yeast strain PLY240 complemented with AKT1, KAT1 as well as with the combinations LilKT1/AKT1 and LilKT1/KAT1. (B) Growth curves of the transformed PLY240 yeast cells show slower growth rates of double-transformed (AKT1/LilKT1) compared to single-transformed (AKT1) yeast. (C) Growth of PLY240 mutants transformed with KAT1, or KAT1/LilKT1 n = 3 ± S.D. Channels were cloned in their respective plasmids: LilKT1 in pGREG535, KAT1 in pYES and AKT1 in pFL61. Medium in (B,C) contained 100 mM KCl.

FIGURE 4

Co-sedimentation of LilKT1 with yeast organelle markers. (A) Distribution of membrane protein and sucrose concentration in a continuous sucrose gradient collected after 16 h centrifugation (n = 5 ± S.D.) (B) Typical pattern of membrane proteins along the sucrose gradient. 30 μl of sucrose-adjusted fraction loaded per lane. (C) Typical distribution of plasma membrane (Pma1p), ER (Dpm1p), and vacuole (Vma1p) marker enzymes in the sucrose gradient. (D) Plasma membrane fractions (3–8) of three gradients were pooled, proteins were precipitated and transferred to NC membranes after SDS-PAGE. The proteins AKT1, Pma1p and the HA-tag of LilKT1 were detected with respective antibodies in the pooled fractions of PM fractions (underlined) of PLY240 yeast mutants expressing AKT1 or LilKT1 alone and the combination of AKT1 and LilKT1 (n.d., not determined).

FIGURE 5

Localization of fluorescence-tagged LilKT1 in yeast protoplasts. (A) N-terminally GFP fused LilKT1 was expressed in yeast cells and localizes in structures surrounding the nucleus (ER). Upper panel: yeast cells, lower panel: yeast cell protoplasts. Fluorescence images are on the left and bright field images merged with fluorescence images on the right. (B) Localization of GFP::LilKT1 co-expressed with AKT1 in yeast cells. Co-expression with AKT1 shows the localization in similar compartments as expression of LilKT1 alone (A).

FIGURE 6

Functional complementation of yeast mutants with modified LilKT1 proteins. (A) Single (LilKT1_K840D) and double mutations (LilKT1_G797D/K840D) of the di-acidic motifs in the C-terminus of LilKT1. (B) Expression of LilKT1 with a total deletion of the C-terminus (ΔCtermLilKT1) and expression of chimeric protein with the N-terminus of LilKT1 and the C-terminus of AKT1 (chimLilKT1) did partially complement the growth deficiency of PLY240 in low K⁺ media. (C) Detection of the C-terminal deleted and chimeric LilKT1 in the cytosolic (CF) and microsomal (MF) fraction of yeast cells (PLY240) using an anti-HA-tag antibody (1:10,000) and a goat anti-mouse IgG AP-conjugated antibody (1:50,000). Predicted molecular weight s for ΔCtermLilKT1 and chimLilKT1 are 47.6 and 106.8 kDa, respectively. Channels were cloned into pGREG535. (D) Fluorescence localization of GFP::yLilKT1/AKT1 chimera in yeast protoplasts (left) and cells (right). Bar = 5 μm.

FIGURE 7

Localization of fluorescence-tagged LilKT1 in lily pollen. Localization of YFP fused to the C-terminus (A,B) or to the N-terminus (C,D) of LilKT1 by confocal laser-scanning microscopy. Lily pollen grains were transformed by particle bombardment with the respective plasmids, incubated for 24 h and finally transferred to germination medium. Arrows mark typical fluorescent structures inside the cytosol. Bright field images (A,C) of the respective confocal fluorescence images (B,D). Bar = 10 μm.

FIGURE 8

Localization of fluorescence-tagged LilKT1 in tobacco epidermis cells. Nicotiana tabacum leaves were transiently transformed by Agro-infiltration and fluorescence was monitored by confocal laser-scanning microscopy 2 days after transformation. (A) Bright field image. (B) Chloroplast autofluorescence in red. (C) Localization of the PM H⁺ ATPase LilHA2 fused to eCFP in magenta. (D) Fluorescence image of LilKT1 with N-terminal fusion of eYFP as green signals. (E) Merged fluorescence images of LilHA2 (magenta) and LilKT1 signals (green), overlapping signals in yellow. (F) Detailed view of the indicated image part of (E). Bar = 10 μm.

FIGURE 9

Details of fluorescence localization of LilKT1 in tobacco epidermis cells. Single fluorescence images of eCFP::LilHA2 (magenta) and eYFP::LilKT1 (green) were merged. Yellow signals indicate a co-localization of LilHA2 and LilKT1 in the plasma membrane. (A) Overview of a epidermal cell showing internal localization (green signals) of LilKT1. Boxes indicate the regions of the detailed images of (B,C). (B) Detail showing the sub-plasma membrane localization of LilKT1 (arrows). (C) Localization of LilKT1 in intercellular strands (green, arrow heads) connecting LilKT1 spots (yellow) in the plasma membrane. Bar = 10 μm.