Intracellular localization and physiological function of a rice Ca²⁺-permeable channel OsTPC1

Abstract

Two-pore channels (TPCs) are cation channels with a voltage-sensor domain conserved in plants and animals. Rice OsTPC1 is predominantly localized to the plasma membrane (PM), and assumed to play an important role as a Ca²⁺-permeable cation channel in the regulation of cytosolic Ca²⁺ rise and innate immune responses including hypersensitive cell death and phytoalexin biosynthesis in cultured rice cells triggered by a fungal elicitor, xylanase from Trichoderma viride. In contrast, Arabidopsis AtTPC1 is localized to the vacuolar membrane (VM). To gain further insights into the intracellular localization of OsTPC1, we stably expressed OsTPC1-GFP in tobacco BY-2 cells. Confocal imaging and membrane fractionation revealed that, unlike in rice cells, the majority of OsTPC1-GFP fusion protein was targeted to the VM in tobacco BY-2 cells. Intracellular localization and functions of the plant TPC family is discussed.

The TPC Family Cation Channels

Despite the molecular diversity of signaling components among plants and animals, Ca²⁺ has been established as a common second messenger in most eukaryotic cells.^1,2 Though electrophysiological studies have revealed several types of Ca²⁺-permeable channels localized at the plasma membrane (PM) and vacuolar membrane (VM) in many plant species,³ their molecular identity are still largely unknown. Homologs of the major voltage-dependent Ca²⁺ channels (VDCCs) are not found in plants.³ In turn, the two-pore channel (TPC) family, originally isolated from rat,⁴ is homologous to a half structure of the α1-subunit of vertebrate VDCCs.⁵

Human TPCs mediate nicotinic acid adenine dinucleotide phosphate-induced Ca²⁺ release from acidic organelles in HEK293 cells.⁶ Arabidopsis AtTPC1 shows a slow-activating vacuolar cation channel activity^7,8 and be involved in the sucrose-induced Ca²⁺ rise,⁹ abscisic acid-induced repression of germination¹⁰ and the stomatal response to extracellular Ca²⁺ changes.^7,10 Tobacco NtTPC1s have roles in increasing Ca²⁺ concentrations, defense-related gene expression, and regulation of hypersensitive cell death triggered by cryptogein, an elicitor from an oomycete, in tobacco BY-2 cells.¹¹ Rice and wheat TPC1s appear to function in responses to abiotic stresses.^12,13

Role of OsTPC1 in Xylanase Elicitor-Triggered Defense Responses in Rice

Characterization of the retrotransposon-insertional knockout mutant of rice Ostpc1 revealed that OsTPC1 affected the sensitivity to a proteinaceous elicitor, xylanase from Trichoderma viride (TvX), and plays a role in the regulation of various TvX-induced defense responses including Ca²⁺ mobilization, production of reactive oxygen species (ROS), hypersensitive cell death, and biosynthesis of phytoalexins as well as activation of a MAP kinase in cultured rice cells.^14,15

Membrane fractionation by two-phase partitioning and immunoblot analyses revealed that OsTPC1 is localized predominantly at the PM. Whole cell patch clamp analyses of OsTPC1 heterologously expressed in HEK293T cells showed its voltage-dependent Ca²⁺-permeability, indicating that OsTPC1 plays a crucial role in TvX-induced Ca²⁺ influx as a PM Ca²⁺-permeable channel consequently required for the regulation of defense responses in cultured rice cells.¹⁵

On the other hand, [Ca²⁺]_cyt changes induced by chitin fragments (N-acetyl-chitooligosaccharides) in Ostpc1 cells was almost comparable to the wild type cells.¹⁵ The temporal patterns of increased [Ca²⁺]_cyt are significantly different between the molecular species of the elicitors as triggers: [Ca²⁺]_cyt increase triggered by chitin fragments is large and transient,¹⁶ while that induced by TvX is much smaller but sustained.¹⁵ These consequences of the knockout of OsTPC1 were similar at least in part with those of Arabidopsis AtTPC1,⁸ suggesting that OsTPC1 may not be important in the signal transduction of general pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) such as chitin fragments and flg22, but may be one of the multiple Ca²⁺-permeable channels activated by some specific elicitors.

Intracellular Localization of OsTPC1

Molecular and electrophysiological studies have shown that Arabidopsis TPC1 is mainly localized at the VM and function as a slow-activating vacuolar cation channel.^17-19 In contrast, TPCs in monocots including OsTPC1 have been suggested to be localized at the PM and confirmed by several groups independently.^13-15,20 To resolve this discrepancy, and confirm the intracellular localization of TPC family proteins, we introduced a GFP construct fused to the coding sequence of the C-terminus of OsTPC1 (OsTPC1-GFP) into tobacco BY-2 cells, which is a suitable tool for studying intracellular localization and dynamics of proteins,²¹ and examined its intracellular localization using confocal laser scanning microscopy as described elsewhere.^22-24 FM4–64 was used as a VM marker.^23,25 When GFP alone was expressed, it is localized to the nucleus and the cytoplasm (Fig. 1A, left column). In contrast, OsTPC1-GFP appeared to co-localize with FM4–64, suggesting the localization of OsTPC1-GFP at the VM (Fig. 1A, right column).

Figure 1. Subcellular localization of OsTPC1-GFP in tobacco BY-2 cells. (A) Confocal images of tobacco BY-2 cells transformed with 35S::OsTPC1-GFP (right column) and 35S::GFP (left column) plasmids are shown. Confocal fluorescence images (b–d, f–h) and differential interference contrast (DIC) images (a, e) of tobacco BY-2 cells expressing GFP (a–d) or OsTPC1-GFP (e–h) stained with the fluorescent styryl membrane probe FM4–64 (Molecular Probes, Carlsbad, CA, USA). Scale bar: 20 μm. FM4–64 was kept as a 17 mM stock solution in sterile water, and used at a final concentration of 4.25 μM. Three-day-old BY-2 cells were treated with FM4–64 for 3 h and washed twice with the culture medium at room temperature to label the VM.^23,25 (B) The PM and the VM fractions of tobacco BY-2 cells were prepared using an aqueous two-phase partitioning¹⁵ and a sucrose/sorbitol method,²⁶ respectively. To detect the GFP fluorescence in the SDS-PAGE gels, unheated protein samples (20 μg per lane) of each fraction were subjected to SDS-PAGE and the fluorescence of OsTPC1-GFP were detected using a 473 nm excitation laser through a 520 nm LP emission filter^24,27 using the fluoro image analyzer FLA3000G (GE Healthcare Bio-sciences, Piscataway, CA, USA). Thereafter, the OsTPC1 protein was detected using an affinity-purified anti-OsTPC1 antibody. The purified fractions were assessed using a polyclonal antibody of plasma membrane H⁺-ATPase and vacuolar H⁺-ATPase subunit A (COSMO BIO).

Furthermore, we isolated the PM fractions from tobacco BY-2 cells as described for rice cells.¹⁵ For the purification of the VM fraction, we used the experimental protocol optimized for BY-2 cells.²⁶ Proteins of each fraction was analyzed by SDS-PAGE without thermal denaturation, and OsTPC1-GFP was detected both by immunoblot analysis using specific antibody against OsTPC1¹⁵ and fluorescence image analysis of GFP.^24,27 Unlike the localization in the rice cells, the majority of OsTPC1-GFP expressed in tobacco BY-2 cells was detected in the VM fraction (Fig. 1B). These results suggest that OsTPC1 is predominantly localized to the PM in rice cells,¹⁵ while mainly targeted to the VM when heterologously expressed in tobacco BY-2 cells.

The human TPC2 is localized to the lysosomal membrane. A mutant of TPC2 lacking a di-leucine motif in its N-terminal has recently been shown to be localized to the PM instead of the lysosomal membrane, suggesting that this motif is required for its localization to acidic organelles.²⁸ Plant TPC family members including OsTPC1 possess a similar motif in their N-terminal tail, which is consistent with the localization to the VM. The PM localization of OsTPC1 in cultured rice cells^15,20 suggests some differences in sorting machinery of the TPC family membrane proteins among plant species. Unknown components that interact with OsTPC1 may regulate the intracellular localization of OsTPC1.

Some membrane proteins show dual targeting. A tobacco water channel, aquaporin1 (NtAQP1) shows a dual localization in the PM and the chloroplast envelope, which creates a dual function as a water channel (at the PM) or a CO₂ channel (at the chloroplast envelope).^29-31 Interestingly, though the VM marker protein was not detected in the PM fraction, a small portion of OsTPC1-GFP was also detected in the PM fraction in addition to the VM faction (Fig. 1B). This may suggest possible dual localization of OsTPC1-GFP in the VM and the PM of tobacco BY-2 cells. Some members of the TPC family may possibly be targeted both to the PM and VM. Localization and functions of the TPC family cation channels is an emerging subject that warrants further analysis. Searches for the in vivo interactors of these channel proteins may provide new insights to further unveil the roles of the TPC family in planta.