Rice Two-Pore K+ Channels Are Expressed in Different Typesof Vacuoles W

Stanislav Isayenkov, Jean-Charles Isner, and Frans J.M. Maathuis1

University of York, Biology Department/Area 9, York YO10 5DD, United Kingdom

Potassium (K+) is a major nutrient for plant growth and development. Vacuolar K+ ion channels of the two-pore K+ (TPK)

family play an important role in maintaining K+ homeostasis. Several TPK channels were previously shown to be expressed

in the lytic vacuole (LV) tonoplast. Plants also contain smaller protein storage vacuoles (PSVs) that contain membrane

transporters. However, the mechanisms that define how membrane proteins reach different vacuolar destinations are

largely unknown. The Oryza sativa genome encodes two TPK isoforms (TPKa and TPKb) that have very similar sequences

and are ubiquitously expressed. The electrophysiological properties of both TPKs were comparable, showing inward

rectification and voltage independence. In spite of high levels of similarity in sequence and transport properties, the cellular

localization of TPKa and TPKb channels was different, with TPKa localization predominantly at the large LV and TPKb

primarily in smaller PSV-type compartments. Trafficking of TPKa was sensitive to brefeldin A, while that of TPKb was not.

The use of TPKa:TPKb chimeras showed that C-terminal domains are crucial for the differential targeting of TPKa and TPKb.

Site-directed mutagenesis of C-terminal residues that were different between TPKa and TPKb identified three amino acids

that are important in determining ultimate vacuolar destination.

INTRODUCTION

Vacuoles play essential roles in many physiologically relevant

processes in plants. Some of themore prominent roles are turgor

provision, the storage of minerals and nutrients, and cellular

signaling (Marty, 1999). All these processes have been associ-

ated with the large central lytic vacuole (LV), which can make up

to 90% of the cellular volume. In addition, storage of nutrients

can occur in vacuoles that are dedicated to this specific function.

These so-called protein storage vacuoles (PSVs) are particularly

prevalent in reproductive tissue, such as seeds, where they store

proteins and minerals for the developing embryo. However,

PSVs are also present in vegetative cells, where their numbers

vary greatly from species to species. Their role in nonreproduc-

tive tissue is not clear but may include the accumulation of

vegetative storage protein in response to developmental or

environmental cues (Jauh et al., 1998; Vitale and Hinz, 2005).

Toadequately fulfill vacuolar functions,membrane transporters,

including K+-selective cation channels, are present at the vacuolar

membrane, the tonoplast (Isayenkov et al., 2010). In Arabidopsis

thaliana, the latter are encoded by TPK1, a member of the two-

pore K+ (TPK) channel family. TPKs are characterized by a sec-

ondary structure with four transmembrane and two pore regions,

each with a GYGD domain (Czempinski et al., 2002; Schonknecht

et al., 2002; Voelker et al., 2006; Gobert et al., 2007; Latz et al.,

2007; Dunkel et al., 2008). Five TPK isoforms are present in

Arabidopsis, four of which (TPK1, TPK2, TPK3, and TPK5) localize

to the tonoplast of the LV, although expression in smaller vesicle-

like structures has also been observed (Voelker et al., 2006). Only

TPK1 has been shown to form functional ion channels; TPK1

activity is voltage independent, it has one or two EF hands

localized in the C terminus, and it is regulated by intracellular

Ca2+ (Gobert et al., 2007). TPK1 activity is further modulated by

cytoplasmic pH, phosphorylation, and 14-3-3 proteins (Gobert

et al., 2007; Latz et al., 2007), whereas the activity of Nicotiana

tabacum TPK1 was also sensitive to spermidine and spermine

(Hamamoto et al., 2008). At TPK1 has been functionally charac-

terized using overexpression, loss-of-function, and heterologous

expression analyses; it is ubiquitously expressed and has a role in

cellular K+ homeostasis and K+ release during stomatal function-

ing (Gobert et al., 2007). At TPK1 expression also affected seed

germination, and its expression is high in embryonic tissues

(Czempinski et al., 2002; Gobert et al., 2007).

A survey of the rice (Oryza sativa) genome shows the presence

of two TPK isoforms, TPKa and TPKb, that encode proteins that

are highly similar to each other (63% identity) and to At TPK1

(57% identity). To gain insight into the biophysical properties and

physiological functions of these rice TPKs, they were subjected

to patch clamp analyses and studied with respect to their

membrane localization using fluorescent protein fusions. Elec-

trophysiological assays showed a large degree of similarity

between the rice TPK isoforms. However, a distinct difference

in membrane targeting was observed, with TPKa being ex-

pressed predominantly at the LV and TPKb mostly localized in

PSVs. Little is known about the mechanistic details of how inte-

gral membrane proteins arrive at specific destinations, and this is

particularly so where targeting of different types of vacuoles is

concerned. We therefore used the rice TPK isoforms, proteins

that are highly similar in structure and transport properties, to

1 Address correspondence to fjm3@york.ac.uk.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Frans J.M.Maathuis (fjm3@york.ac.uk).WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.110.081463

The Plant Cell, Vol. 23: 756–768, February 2011, www.plantcell.org ã 2011 American Society of Plant Biologists

study the underlying mechanisms responsible for their traffic to

different membrane localisations. Our results suggest that spe-

cific C-terminal residues direct trafficking of integral tonoplast

proteins to LV- and PSV-type vacuoles.

RESULTS

TPKaandTPKbAreLocalized toDifferentTypesofVacuoles

It was previously shown that the majority of Arabidopsis TPKs

(TPK1, TPK2, TPK3, and TPK5) localized at the membrane of

the large LV (Voelker et al., 2006; Latz et al., 2007). To understand

the potential roles of TPKs in rice, we obtained the two isoforms

that show the highest expression levels (Os03g54100 and

Os07g01810), which we named TPKa and TPKb, respectively.

To assess the membrane localization of each isoform, C-terminal

enhanced yellow fluorescent protein (EYFP) translational fusions

were transiently expressed in protoplasts from rice roots and

shoots. Figure 1A (top, right panels) shows the typical expression

pattern of TPKa. Although there is some endoplasmic reticulum

(ER)-based fluorescence, the main signal follows the vacuolar

perimeter in intact protoplasts and is associated with the large LV

in osmotically disrupted cells. By contrast, TPKb is mostly con-

fined to small vesicular structures that are often hardly discernible

in intact protoplasts but become obvious using fluorescence light

(Figure 1A, bottom, right panels). Unlike the LV, which has an

acidic pH and therefore accumulates neutral red indicator, TPKb-

expressing compartments do not have an acidic pH (see Supple-

mental Figure 1 online). To confirm the validity of these strikingly

different fluorescence patterns, N-terminal EYFP fusions were

also made for each isoform. The N-terminal position of the

fluorescent protein did not change the pattern of subcellular

localization for either TPK (see Supplemental Figure 2 online).

The TPKb fluorescence patterns occurred in small vacuolar

structures that resemble PSVs (Park et al., 2004). Small PSV-type

vacuoles are common in rice mesophyll cells (Park et al., 2004)

but are better characterized and more prevalent in reproductive

tissues, such as the aleurone layer of developing rice seeds (Kim

et al., 2004). Whereas mature aleurone tissue mainly consists of

cells packed with PSVs, immature aleurone also contains cells

with large LVs. Transformation of aleurone protoplastswith TPKb

resulted in strong EYFP fluorescence inmultiple PSVs (Figure 2A)

but was not observed in protoplasts with a large central vacuole.

By contrast, TPKa-dependent fluorescence only occurred in the

subpopulation of aleurone protoplasts, where a large central

vacuole was present (Figure 2B), but not in PSV-filled cells.

Tonoplast intrinsic proteins (TIPs) were previously shown to

target different types of vacuole and as such can be used as

markers for LVs and PSVs, with g-TIP being expressed at the LV

membrane and a- and d-TIP being indicative of PSVmembranes

(Jauh et al., 1998, 1999; Hunter et al., 2007). We therefore used

Arabidopsis TIP marker lines to carry out colocalization studies

with Os TPKs. Confocal images of d-TIP-green fluorescent

protein (GFP) marker line protoplasts that were transformed

with OsTPKb-RFP showed almost 100% overlapping of red

fluorescent protein (RFP) and GFP fluorescence (Figure 3A),

providing evidence that d-TIP and TPKb colocalize to the same

compartment. By contrast, TPKa was expressed in a compart-

ment that was different from that occupied by the d-TIP marker

(Figure 3B). Transient expression of TPKb-RFP in g-TIP-YFP

protoplasts leads to distinct fluorescence patterns of RFP and

YFP (Figure 3C), indicating that TPKb and g-TIP expression

patterns are dissimilar. In addition to these marker line coex-

pression studies, we coexpressed both Os TPK isoforms in the

same cell. Figure 3D represents a typical example that shows

that the fluorescence signals obtained from TPKa-RFP and

TPKb-YFP occur in distinct and separate patterns.

These data show that, in spite of a large degree of identity at

both the nucleotide and amino acid level, TPKa and TPKb have

distinct subcellular locations, with TPKa predominantly being

expressed in the tonoplast of the large LV and TPKb predomi-

nantly being localized to PSV membranes.

TPKaandbAreVoltage-Independent, VacuolarK+Channels

with Similar Properties

The difference in membrane localization of TPKa and TPKb may

point to discrete functions for these proteins in their respective

compartments, and this in itself may rely on different channel

properties. We therefore expressed YFP fusions of both chan-

nels in leaf protoplasts derived from the Arabidopsis tpk1 tpc1

double loss-of-function mutant (Gobert et al., 2007). The tpk1

tpc1 mutant lacks both slow vacuolar and vacuolar K+ currents

and thus provides an ideal, electrically silent background to

characterize vacuolar cation channels. Typical recordings de-

picted in Figures 4A and 4B show that both rice TPK isoforms

form functional ion channels in spite of the attached C-terminal

YFP. Both channels show larger inward than outward current at

equivalent membrane voltages, even though the ionic solutions

facing each side of the tonoplast are identical. However, this

intrinsic inward rectification is more pronounced for TPKa. Out-

ward conductance values of 22 (63.6) and 21 (63.1) pS were

calculated at 100 mV (Figure 4C) for TPKa and TPKb, respec-

tively, when cytoplasmic and luminal sides of the membrane

were exposed to 100 mM KCl. Inward conductance values

calculated at 2100 mV were 54 (65.6) and 38 (64.3) for TPKa

and TPKb, respectively. Open probability of either channel was

not or only weakly voltage dependent, and channels showed a

high selectivity for K+ (data not shown). For both isoforms, the

presence of ATP plus 14-3-3 protein promoted channel open

probability (see Supplemental Figure 3 online), as was reported

previously for At TPK1 (Latz et al., 2007).

Although sequence similarity is high betweenArabidopsis TPK1

and rice TPKa and b, it diverges considerably in the C terminus.

The TPK1 C terminus contains two conserved Ca2+ binding EF

hand domains. These consist of the 29- to 30-residue helix-loop-

helix sequencesand the 12-residue loopcoremotif responsible for

Ca2+ coordination (Bihler et al., 2005). The presence of two EF

domains may explain why TPK1 activity is steeply dependent on

cytoplasmic Ca2+ (Gobert et al., 2007). TPKa and b have lesswell-

definedEFdomains, which prompted us to determinewhether the

Ca2+ dependence of these proteins is the same as that for TPK1.

Figure 4D shows a steep dependence on cytoplasmic Ca2+ for

TPK activity, as was previously reported (Gobert et al., 2007). By

contrast, TPKa activity is only moderately decreased, even when

Rice Two-Pore K+ Channels 757

cytoplasmic Ca2+ is 200 nM. Activity of TPKb is even less mod-

ulated by changes in cytoplasmic Ca2+.

Conductances with properties similar to those described

above for TPKa were also recorded from LVs (n = 8) that were

isolated from wild-type rice cells, whereas channels with TPKb

characteristics were only recorded from small vacuolar com-

partments (n = 11) that released after lysis of rice protoplasts (see

Supplemental Figure 4 online). This shows that in native tissue,

TPKa and TPKb localize to separate compartments.

TPKb Trafficking to PSVs Is Golgi Independent

Current models suggest that LV-designated proteins are se-

quentially trafficked through the ER and Golgi apparatus. The

trans-Golgi phase of this pathway relies on clathrin-coated

vesicles that link the Golgi with endosome-like prevacuolar com-

partments (Jurgens, 2004). By contrast, two different pathways

appear to function in protein trafficking to the PSV; namely, a

Golgi-dependent and a Golgi-independent pathway (Levanony

et al., 1992; Hara-Nishimura et al., 1998; Neuhaus and Rogers,

1998; Jiang and Rogers, 1998; Jiang et al., 2000; Toyooka et al.,

2000). Some storage proteins, such as lectins, are transported

through the Golgi machinery where they aggregate in dense,

non-chlathrin-coated vesicles before being released to merge

with PSVs (Hara-Nishimura et al., 1998; Toyooka et al., 2000).

Another class of storage proteins (e.g., globulins and Cys pro-

teinase) is transported into PSVs in aGolgi-independent manner.

These proteins form large aggregates in specific regions of the

smooth ER, which bud off as large vesicles that reach the PSV by

an unknown mechanism (Levanony et al., 1992).

Trafficking of integral membrane proteins to the LV tonoplast

occurs via the Golgi system. This was previously shown also to

be the case for Arabidopsis TPKs, such as TPK1 (Dunkel et al.,

2008). However, trafficking of membrane proteins to prevacuolar

and PSV-like compartments can also occur directly from the ER

(Oufattole et al., 2005). For example, vacuolar aquaporins, such

as a-TIP, bypass the Golgi machinery via formation of vesicles

that originate directly from the ER (Jiang and Rogers, 1998; Park

Figure 1. Vacuolar Localization of TPKa and TPKb Channels in Rice Protoplasts.

(A) The secondary structure of TPKa (blue) and TPKb (green) shows the position of transmembrane domains, the N-terminal 14-3-3 motif, and the

C-terminal putative EF hand motifs. Bright-field (left) and fluorescence (right) images of intact rice protoplasts and released vacuoles show TPKa:EYFP

expression in the central LV and TPKb:EYFP expression in multiple smaller PSVs. Bars = 5 mm.

(B) Distribution of TPKa- and TPKb-dependent fluorescence in LVs, PSVs, and ER in protoplasts transformed with TPKa (left) and TPKb (right). Error

bars depict SE of at least three counts using independent batches of protoplasts.

758 The Plant Cell

et al., 2004). To test whether TPKa and b reach their respective

destinations via the Golgi, we used the fungal antibiotic brefeldin

A, which interferes with protein transport from the ER to the Golgi

by disrupting Golgi membrane integrity (Nebenfuhr et al., 2002).

Transient expression of TPKa in tobacco leaf cells leads to a

typical tonoplast fluorescence pattern (Figure 5A). Treatment

with brefeldin A caused fluorescence to accumulate in membra-

nous and vesicular structures that resemble ER and are distinct

from the LV tonoplast. These data indicate that TPKa trafficking

to the LV tonoplast is brefeldin sensitive and, therefore, Golgi

dependent. To further investigate the role of the Golgi complex in

TPKa trafficking, we treated rice and barley (Hordeum vulgare)

protoplasts with brefeldin A shortly after they were transformed

with either TPKa or TPKb. Figures 5B and 5D show that brefeldin

A has a profound effect on TPKa expression patterns in rice

protoplasts, causing a shift from LV to strand-like and vesicular

PSV-type compartments. By contrast, the localization pattern of

TPKb in protoplasts remains virtually the same in the presence of

brefeldin A (Figures 5C and 5D). The differential effect of brefeldin

A on TPKa and TPKb was reproducible in barley protoplasts

(Figure 5E). These data therefore indicate that TPKa trafficking

relies on an intact Golgi system, whereas TPKb targeting to PSVs

appears to be Golgi independent. However, it is possible that

TPKb trafficking could occur via cis-Golgi only, which is less

sensitive to brefeldin A (Nebenfuhr et al., 2002).

The C Termini of Rice TPKs Are Important for Differential

Vacuolar Targeting

Protein function is often inextricably linked with cellular location;

thus, how proteins reach their destination is an important question.

To gain insight into the mechanism of different localization for TPKa

and b, a series of chimeric OsTPKa:OsTPKb proteins was con-

structed to identify potential regions that affectmembrane targeting.

Exchanging the entire cytoplasmic N-terminal domain of

TPKa, which contains the 14-3-3 binding region (Latz et al.,

2007) with the corresponding domain from TPKb led to fluores-

cence patterns that were restricted to the ER compartment (see

Supplemental Figure 5A online). A reduction of this domain,

which still included the TPKb 14-3-3 binding region, did not

significantly change this result (see Supplemental Figure 5B

online). Subsequently, we only substituted the TPKa N-terminal

section upstream of the 14-3-3 binding domain with its TPKb

counterpart. In this case too, fluorescence was predominantly

ERbased, although someweak fluorescencewas found in the LV

tonoplast (see Supplemental Figure 5C online).

To test whether membrane-spanning regions have an impact

on membrane targeting, the C-terminal half of TPKa was

substituted with the corresponding part of the TPKb protein.

No significant expression in either PSV or LV was observed with

this chimera, with virtually all fluorescence being retained in the

ER compartment (Figure 6A). We then exchanged only the forth

transmembrane domain (TMD) and C-terminal cytosolic part of

TPKa with the equivalent region of TPKb. Imaging of 40 to 50

protoplasts revealed that around 85% of protoplasts showed

PSV-located fluorescence (Figure 6B). Subsequently, chimeras

were constructed with increasingly shorter C-terminal regions

derived from TPKb. Proteins containing all four TMDs from TPKa

and the TPKb cytosolic C terminus showed fluorescence similar

to those containing TMD4 from TPKb (Figures 6B and 6C). These

observations indicate that the presence of TPKb TMDs is not

required to achieve PSV localization but that the TPKb C termi-

nus is essential. A shorter TPKb-derived C-terminal sequence,

starting before the first EF core motif, did not alter the predom-

inantly PSV-based fluorescence (Figure 6D). However, when the

first EF core motif in the TPKa sequence was left in place, some

change in fluorescence patterns was observed, although ulti-

mately a similar proportion of fluorescence was found in PSVs

(;85%; Figure 6E) 24 h after transformation, whereas, after 12 h,

around 55% of protoplasts only showed fluorescence in the ER

compartment. Thus, the first EF core motif in TPKa may be

involved in ER release, and mutations in this region delay traffic

from the ER but do not affect the ultimatemembrane destination.

A further reduction of the TPKb C-terminal region, starting just

before the second EF core motif, also greatly influenced the

observed fluorescence patterns. In this construct, an intermediate

pattern was found, with some increased ER retention and equal

numbers of protoplasts showing LV fluorescence and PSV fluo-

rescence (Figure 6F). This suggests that the sequencebetween the

two EF core motifs is critical in determining membrane targeting.

When both EF handsoriginated fromTPKa and the short remaining

TPKa sequence was substituted with its TPKb counterpart, fluo-

rescence patterns reverted to that of the native TPKa protein, with

expression almost exclusively found in the LV (Figure 6G).

Together, the results using TPKa:TPKb chimeras suggest that

the N terminus is likely involved in ER release, but not membrane

specificity. Furthermore, the TMD regions are probably also not

important for membrane targeting. By contrast, the region in the

C terminus between the EF hand core motifs and the second EF

core domain greatly affect differential targeting to LVs and PSVs.

Figure 2. Expression of TPKa- and OsTPKb-EYFP Fusions in Proto-

plasts Isolated from the Aleurone Layer of Developing Rice Seeds.

Bright-field (top panels) and epifluorescence images (bottom panels) of

protoplasts show localization of TPKa to the LV and that of TPKb to

PSVs. Bars = 5 mm.

Rice Two-Pore K+ Channels 759

Specific C-Terminal Amino Acids Are Important for LV and

PSV Targeting

Results obtained with rice TPKa:TPKb chimeras show that the

C-terminal EFmotifs are highly relevant in the ultimate destination

of these proteins in LVs and PSVs, respectively. We therefore

asked ourselves whether specific residues in these areas have an

impact on the different expression patterns of TPKa and TPKb.

Several amino acids in this region were mutated (Figure 7), and

mutant proteins were subsequently expressed in rice protoplasts

to assess their impact on fluorescence patterns that were previ-

ously obtained with native proteins. First, we determined whether

EF function itself (i.e., the binding of Ca2+ to induce a conforma-

tional change) had an impact on the observed fluorescence

patterns. We therefore reduced Ca2+ binding capacity by sub-

stituting residues in the core EF loop sequence that are known to

be essential for Ca2+ coordination (Gifford et al., 2007). In the first

EF motif, the polar Asp at position 292 in TPKa was exchanged

for a nonpolar Gly. Although the D292Gmutation is likely to affect

Ca2+ binding, the localization of TPKa was not altered (see

Supplemental Figure 6A online), with fluorescence remaining in

the tonoplast of the central LV. In the second EF, the acidic Asp

residue in position 330 and the small aliphatic Gly (G) in position

333 were substituted with a basic His (H) and a larger aliphatic Ala

(A), respectively. The singlemutations did not affect fluorescence.

Figure 3. Coexpression of TPKb and TPKa with LV and PSV Markers.

(A) OsTPKb-EYFP colocalizes with d-TIP-GFP. Protoplasts isolated from an Arabidopsis marker line with d-TIP-GFP expression in PSVs were

transiently transformed with OsTPKb-RFP. The panel shows bright-field illumination of a protoplast and confocal images of the GFP signal (green), the

RFP signal (red), and the merged GFP-RFP signal. Yellow denotes colocalization.

(B) As in (A), but coexpression of the d-TIP marker line with OsTPKa-RFP, showing fluorescence from separate compartments.

(C) OsTPKb-RFP does not colocalize with g-TIP-EYFP, a marker of LVs. Rice protoplasts were cotransformed with OsTPKb-RFP and g-TIP-EYFP. The

panel shows bright-field illumination of a protoplast and confocal images of the RFP signal (red), the EYFP signal (green), and the merged RFP-YFP

signal.

(D) OsTPKb-EYFP fluorescence is distinct from OsTPKa-RFP fluorescence. Rice protoplasts were cotransformed with OsTPKb-EYFP and OsTPKa-

RFP. The panel shows bright-field illumination of a protoplast and confocal images of the EYFP signal (green), the RFP signal (red), and themerged RFP-

YFP signal. Bars = 5 mm.

760 The Plant Cell

Proteins that contained both mutations showed increased reten-

tion in the ER compartment (see Supplemental Figure 6B online)

but no PSV fluorescence.

Next, a series of mutations was performed to study the

relevance of residues that show pronounced differences be-

tween TPKa and TPKb at conserved positions within the C

terminus. The first EFmotif in TPKa contains aGlu at position 300

that is highly conserved among TPK proteins (Figure 7; Dunkel

et al., 2008). Interestingly, TPKb is the only isoform that deviates

from this configuration, with an Asp residue at the corresponding

position (284). However, replacing the TPKb acidic Asp residue

with an acidic Glu had no effect with regards to the localization of

this protein (see Supplemental Figure 6D online). Substitution of

the aromatic Tyr in TPKa at position 338 in the second EF hand

with the much smaller aliphatic Ala from TPKb did also not affect

fluorescence patterns, with virtually all protoplasts only showing

expression in LVs (see Supplemental Figure 6C online).

We then focused on residues outside the core EF motifs that

showed differences between TPKa and TPKb. The TPKa ali-

phatic Val at position 303 (Figure 7) was mutated to the much

larger aliphatic Leu residue, as found in TPKb. This mutant TPKa

protein showed a partial shift in localization, with around 65% of

protoplasts exhibiting fluorescence in LVs, but also a substantial

number of protoplasts (around 30%) exhibiting PSV fluores-

cence (Figure 8A), suggesting a crucial role of this residue in

determining compartment destination. Changing the basic Lys of

TPKa in position 326 to an acidic Asn also generated a shift from

LV to PSV localization, with approximately equal numbers of

protoplasts showing expression in each compartment (Fig-

ure 8B). Replacement of the TPKa Asn at position 313 with a

noncharged Ser led to a similar shift away from LV expression,

but to a larger extent, with around 50% of protoplasts showing

PSV fluorescence (Figure 8C). Since these substitutions had a

significant effect on TPKa membrane localization, we also in-

vestigated expression of proteins containing multiple substitu-

tions. Double mutants that contained the K326N mutation

(K326N V303L and K326N N313S) did not generate any fluores-

cence, suggesting these are nonviable proteins. However, when

V303L and N313S occurred simultaneously, TPKa expression

was almost exclusively found in PSV compartments (Figure 8D).

Inspection of the C termini of other integral PSV membrane

proteins, such as d-TIP and a-TIP, shows a lack of similar residues

in the corresponding positions. However, mutation of the equiv-

alent Lys (326) residue in the Hv TPK1 C terminus (Figure 7) to an

Asn also induced a shift of fluorescence. Hv TPK1 transiently

expressed in either Arabidopsis, barley, or rice protoplasts shows

mainly LV localization (see Supplemental Figure 7A online). How-

ever, the K328N substitution doubled the frequency of Hv TPK1

fluorescence in PSVs (see Supplemental Figure 7B online).

DISCUSSION

TPKa and TPKb Function in Different

Cellular Compartments

TPKa and TPKb show high levels of identity in their protein

sequences. In addition, they exhibit similar electrophysiological

Figure 4. Electrophysiological Properties of TPKa and TPKb.

TPKa (A) and TPKb (B) single-channel activity recorded from excised

luminal-side-out tonoplast patches derived from central LV (A) and small

PSV-like compartments (B). Membrane voltage is depicted on the right-

hand side of each trace, and arrows on the left hand side indicate closed

levels. Solutions contained 100 mM KCl on each side of the membrane,

and vacuoles were derived frommesophyll cells of Arabidopsis tpk1 tpc1

mutants.

(C) Representative unitary I/V relationship for TPKa (open circles) and

TPKb (closed squares), showing an outward conductance of around 22

pS and an inward conductance of 54 and 38 pS for TPKa and TPKb,

respectively.

(D) Relative activity as a function of cytoplasmic [Ca2+] for At TPK1

(closed circles), Os TPKa (open circles) and Os TPKb (closed squares),

showing that the open probability of TPK1 depends on the cytoplasmic

concentration of Ca2+, whereas that of rice TPKs is hardly affected by the

cytoplasmic concentration of Ca2+. Error bars are standard deviations

(n = 3).

Rice Two-Pore K+ Channels 761

properties, with K+ selectivity, inward rectification, and voltage-

independent gating being reminiscent ofArabidopsis and tobacco

TPKs (Gobert et al., 2007; Hamamoto et al., 2008). Nevertheless,

these proteins appear to localize to different cellular organelles.

Although some ER expression was often observed for both TPKa

and TPKb, C-terminal and N-terminal fluorescent protein fusions

showed that, as a rule, TPKa vacuolar expression is localized to

the tonoplast of the central LV. By contrast, TPKb-linked fluores-

cence predominantly emanates from small spherical compart-

ments that have the hallmarks of vegetative PSVs, such as pH

neutrality (see Supplemental Figure 1 online) and d-TIP expression

(Figure 3). This distinctive patternwas observed in different organs

and cell types, such as those derived from the mesophyll and

aleurone tissues, andwas observed in both homologous (rice) and

heterologous (Arabidopsis, barley, and tobacco) expression sys-

tems. Furthermore, colocalization studies using the PSV marker

d-TIP and the LV marker g-TIP showed excellent convergence

between d-TIP and TPKb but not g-TIP and TPKb. In addition,

trafficking to the PSV of several proteins, such as a-TIP (Jiang and

Rogers, 1998, 1999; Park et al., 2004), has been shown to beGolgi

independent, and our experiments with brefeldin A confirm that

this is also the case for TPKb, but not TPKa.

The TPK N Terminus Influences TPK Trafficking

Plants contain at least two different types of vacuole. Although

progress has been made in understanding the generic sorting

and trafficking pathways that are followed for soluble proteins to

LVs andPSVs (Jurgens, 2004), little is known about themolecular

and/or structural components that determine which route is

followed.

LV-designated proteins are normally transported from the ER

through the Golgi and prevacuolar compartments (Figure 9). This

pathway hasmany similaritieswith lysosome/vacuolar trafficking

in animal and yeast cells and relies on Golgi located sorting by

receptors such as binding protein BP80 and epidermal growth

factor-like protein ELP in Arabidopsis (Matsuoka and Bednarek,

1998). Subsequent antegrade traffic involves clathrin-coated

vesicles for transfer from Golgi to prevacuolar compartments

and the LV (Figure 9).

PSVs are unique to plant cells, and PSV-destined trafficking

can occur via multiple routes (Figure 9). For soluble proteins,

trafficking from ER to the PSVs mostly occurs through the Golgi

complex: protein aggregates into dense vesicles in the cis-Golgi,

which exit the trans-Golgi to merge with PSVs (Jurgens, 2004).

Figure 5. Effect of Brefeldin A Treatment on Rice TPK Localization in Tobacco, Rice, and Barley.

(A) OsTPKa-EYFP fluorescence in tobacco epidermal cells occurs in the cell periphery, which is indicative of LV tonoplast expression (top panel).

Fluorescence of OsTPKa-EYFP in brefeldin A (bref)-treated cells accumulates in membranous structures that resemble the ER compartment (bottom

panel). Leaves were infiltrated with 0.1% DMSO (control) or brefeldin A solution 2 d after leaf inoculation.

(B) TPKa is expressed in the LVs of control rice protoplasts. Brefeldin A treatment shifts expression from LVs to strands and small PSV-like vesicles.

(C) Brefeldin A treatment of rice protoplasts does not affect the localization of TPKb to PSVs.

(D) and (E) EYFP distribution between LV and PSV-like compartments for control and brefeldin A–treated rice protoplasts (D) and barley protoplasts (E).

Protoplasts were treated with 0.1% DMSO (control) or brefeldin A (10 mg/mL) 1 h after transformation. EYFP fluorescence was observed 12 h after

transformation. Bars = 5 mm. Error bars are standard deviations (n = 3).

762 The Plant Cell

Figure 6. Subcellular Localization and Distribution of OsTPKa:OsTPKb Chimeras in Rice Protoplasts.

Rice Two-Pore K+ Channels 763

This route may also include so-called dark intrinsic protein

organelles, vesicles that contain TIP-related dark intrinsic protein

and the cargo receptor protein RMR1 (receptor homology-

transmembrane-RING H2 domain protein) in Arabidopsis (Jiang

et al., 2000; Park et al., 2005). Storage proteins, such as globulins

and Cys proteinase, can also arrive at PSVs in a Golgi-indepen-

dent way via precursor-accumulating compartment vesicles or

KDEL vesicles (Galili et al., 1993; Hara-Nishimura et al., 1998;

Jiang and Rogers, 1998; Toyooka et al., 2000; Park et al., 2004).

Although it has been suggested that storage proteins contain

specific sorting signals (Jiang and Rogers, 1998; Shimada et al.,

2002; Park et al., 2005), this remains unclear; however, like

soluble proteins, sorting of storage proteins may also rely on

selective aggregation in peripheral Golgi cisternae (Hinz et al.,

2007).

Far less is known about trafficking of integral membrane

proteins to either LVs or PSVs. For LV-destined membrane

proteins, diacidic motifs can be present that promote interaction

with COPII proteins tomediate transfer from ER to Golgi (Zelazny

et al., 2009). Masking of ER retention motifs could similarly

promote ER export (Dunkel et al., 2008). Downstream trafficking

from Golgi to LVs appears to involve Golgi-based sorting by

proteins such as BP80 (Jiang and Rogers, 1998). PSV markers,

such as a-TIPs, are transported to PSVs in a Golgi-independent

manner. However, a more recent study suggests that a-TIP

trafficking via the Golgi can also occur (Park et al., 2004). Hofte

and Chrispeels (1992) showed that the C-terminal transmem-

brane domain plus a portion of the cytoplasmic tail were suffi-

cient to direct a-TIP to the PSV tonoplast; however, themolecular

determinants that govern traffic through these diverse pathways

are unknown.

The large degree of amino acid sequence similarity between

TPKa and TPKb coupled with their different vacuole destinations

provides an ideal tool to investigate targeting to different organ-

elles. We therefore generated various chimeras between TPKa

and TPKb to identify protein domains that potentially impact on

trafficking and targeting. Partial and complete substitution of the

TPKa N terminus with the corresponding parts of TPKb caused

ER retention of the chimeric proteins. Both TPKa and TPKb

contain an active 14-3-3 domain in the N terminus, and some

earlier reports suggested participation of 14-3-3 proteins in

the regulation of ion channel trafficking (O’Kelly and Goldstein,

2008). For example, 14-3-3 binding decreased ER retention

of various proteins, leading to higher densities in the plasma

membrane (Coblitz et al., 2005; Shikano et al., 2005). Work in

Arabidopsis suggested no role for the TPK1 14-3-3 domain in ER

export and vacuolar trafficking (Dunkel et al., 2008). Swapping

the 14-3-3 binding domains between TPKa and TPKb caused

significant accumulation of chimeric protein in the ER and

prevented migration to the default organelle. Our data therefore

suggest that, in rice, 14-3-3 activity may affect ER release;

however, 14-3-3 is not a key element in differential vacuolar

targeting.

Recently, a study on the stress-inducible ERD-six-like trans-

porter of monosaccharides (ESL1) from Arabidopsis reported

that an N-terminal LXXXLL motif was essential for its localization

Figure 6. (continued).

For each panel on the left-hand side, the secondary structure denotes TPKa domains in blue and TPKb domains in green. Bright-field images and

representative confocal fluorescence patterns are shown in the middle panels. Right-hand side panels show the relative distribution of protoplasts with

fluorescence in LVs, PSVs, and ER using 100 to 150 protoplasts observed 18 to 24 h after transformation.

(A) A chimera derived from 50% of TPKa and 50% of TPKb showed ER retention, as is apparent from the overlap of fluorescence patterns derived from

the chimera-YFP and ERmarker line RFP. Chimeras containing the fourth transmembrane domain and C terminus of TPKb (B) or only the C terminus (C)

were mainly localized to PSVs.

(D) Chimeras where the TPKb-derived C terminus was further reduced but still contained two putative EF motifs exhibited TPKb-like PSV localization.

(E) The substitution of the C terminus after the first EF hand with the corresponding region of TPKb exhibited partial ER retention and PSV localization

after 12 h of incubation, but 24 h after transformation, EYFP fluorescence was observed mainly in PSVs.

(F) Chimeras containing only the second EF motif from TPKb showed equal distribution between LVs and PSVs.

(G) Substitution of the last eight residues from TPKa with those from TPKb led to a TPKa-like LV localization. Bars = 5 mm.

Figure 7. Alignment of Os TPKa, Os TPKb, and Hv TPK1 C Termini.

Amino acid targets for site-directed mutagenesis are indicated by lightning bolts. Putative ER export diacidic motifs are marked with asterisks. Putative

core EF hand motifs are enclosed by gray boxes. The following single and double residue alterations were made in TPKa: D292G, V303L, N313S,

K326N, D330H, G333A, Y338A, and the double mutation D330H Y338A. The D295E mutation was introduced in TPKb and the K328N mutation in Hv

TPK1.

764 The Plant Cell

at the LV tonoplast (Yamada et al., 2010). Sequence analysis of

TPKs from different plant species shows the presence of this

motif in the N termini of TPKa and TPKb but not in TPK1 from

Arabidopsis, in spite of the clear LV localization of the latter. This

suggests that N-terminal LXXXLL motifs are not important for

TPK destination.

The TPK C Terminus Affects TPKMembrane Localization

In contrast with N-terminal substitutions, exchanging the

C-terminal region of TPKa with the corresponding part of TPKb

led to a radical shift in observed fluorescence from LVs to PSVs.

This phenomenon occurred irrespective of the presence of TPKb

transmembrane domains. These findings therefore suggest an

important role of the cytosolic C terminus inmembrane targeting,

but leave unanswered questions regarding mechanistic details.

Dunkel et al. (2008) identified several putative diacidic motifs in

this part of At TPK1 (e.g., 296-DLE-298 in helix H1 of the first EF

hand) that had an impact on ER release. They also demonstrated

the importance of the C terminus of At TPK1 in its targeting to the

LV and suggested this region may contain an LV sorting motif.

Our results for TPKa confirm this notion. Our data for TPKb fit in

with previous models of a-TIP trafficking. According to Rogers

and colleagues (Jiang and Rogers, 1998; Oufattole et al., 2005),

the a-TIP cytoplasmic C-tail is a key element in the recognition

of a-TIP by the cytoplasmic machinery that excludes vesicle-

mediated trafficking from ER to Golgi. This is likely to occur via a

specific C-terminal domain (PIEPPPHH) that interacts with the

soybean-regulated-by-cold trafficking protein SRC2 (Oufattole

et al., 2005).

The cytosolic C termini of both TPKa and TPKb almost entirely

consist of two consecutive predicted EF domains. EF hands in K+

channel-interacting proteins have been shown to be essential for

correct trafficking ofmammalian K+ channel proteins (Flowerdew

and Burgoyne, 2009). A role for Ca2+ in the recognition and

vacuolar sorting of the ER-like Ca/Mn-ATPase ECA3 and Ca2+-

dependent recognition of cargo proteins has also been inferred

(Li et al., 2008, Mills et al., 2008). Patch clamp recordings show

little effect of cytoplasmic Ca2+ on the activity of either TPKa or

TPKb, indicating that the putative EF domains in these proteins

may not be functional. Nevertheless, we tested whether the Ca2+

binding capacity of the rice TPK EF motifs altered trafficking and

membrane localization. Mutations that altered residues in the

core loop sequence of either the first or second EF hand had no

Figure 8. Subcellular Localization of TPKa Containing Mutations in the C Terminus.

At the left, mutations are denoted with TPKa-derived residues in blue and TPKb-derived residues in green. The approximate location of the mutated

residue in the secondary structure is indicated by a lightning bolt. Representative fluorescence patterns are shown in the middle panels using bright-

field and confocal imaging. Right-hand side panels show the relative distribution of fluorescence in the LV, PSV, and ER using 100 to 150 protoplasts.

(A) The V303L mutation shifts TPKa expression toward PSVs, with around 30% PSV- and around 65% LV-based fluorescence.

(B) The K326N substitution leads to roughly equal TPKa expression in LVs and PSVs.

(C) The N313S substitution creates a larger shift of TPKa expression toward PSVs, with around 65% localization in PSVs.

(D) The double mutation (V303L N313S) leads to TPKa expression being observed almost exclusively in PSVs. Bars = 5 mm. Error bars are standard

deviations (n = 3).

Rice Two-Pore K+ Channels 765

effect on the ultimate membrane localization of TPKa but did

increase ER retention. These results argue against a prominent

role of EF-mediated Ca2+ binding in determining membrane

localization; however, Ca2+ may be required for trafficking out of

the ER.

Since Ca2+ binding appears to have little impact onmembrane

localization, we focused on residues in the C terminus that are

different between TPKa and TPKb. Among these, three residues

were identified that substantially altered the targeting to LVs and

PSVs; Val to Leu (position 303), Asn to Ser (313), and Lys to Asn

(326) mutations all shifted fluorescence away from the LV toward

PSVs. A combination of V303L and N313S shifted virtually all

TPKa- or TPKb-dependent fluorescence from LVs to PSVs.

These mutations affect side chain size, charge, and phosphor-

ylation potential of the C terminus. The strongest effect derived

from the N313S change, which affects a residue between the

two putative EF motifs. Modeling of this stretch of sequence

predicts the S at this position is very likely a phosphorylation site

with a score of 0.992 (InterPro, http://www.ebi.ac.uk/Tools/

InterProScan/). There are several reports that implicate phos-

phorylation as an essential step in targeting proteins to PSVs.

Wortmannin, a fungal toxin that inhibits several types of kinases,

stopped C-terminal vacuolar sorting signal–mediated transport

to the PSVs but did not have any effect on traffic to the LV

(Matsuoka et al., 1995). The same inhibitor also prevented chiti-

nase deposition in PSVs of tobacco (Neuhaus and Rogers, 1998).

In conclusion, our data show distinct vacuolar localizations of

two very similar tonoplast K+ channels. Domain swapping ex-

periments show that both N and C termini have an impact on

trafficking, particularly on export from the ER. Site-directed

mutagenesis identified three C-terminal residues that are heavily

implicated in directing proteins to either LVs or PSVs, one of

which is a Ser residue that possibly requires phosphorylation for

PSV targeting.

METHODS

Plant Materials

Nicotiana tabacum plants and Arabidopsis thaliana tpc1 tpk1 knockout

lines and various marker lines were grown from seeds and maintained at

228C in a glasshouse with daylengths of 14 h, supplemented with artificial

light. Six- to eight-week-old plants were used for transient expression.

Rice plants (Oryza sativa japonica Nipponbare) were grown in plastic

boxes filled with vermiculate and water in the dark at 288C.

Construction of OsTPK-FP Fusions, Chimeras, and

Site-Directed Mutagenesis

mEYFP and mRFP cDNAs were amplified using primers containing the

BamHI site and inserted into pART7 (Gleave, 1992) to create pART7-

EYFP and pART7-RFP vectors. cDNAs for TPKa and TPKbwere obtained

from the Rice Genome Resource Center (Japan). Full-length coding

sequences of TPKa and TPKbminus stop codons were amplified by PCR

using forward and reverse primers containing XhoI and SmaI sites (see

Supplemental Table 1 online), respectively, and inserted into pART7-

EYFP/RFP to produce pART7-OsTPK vectors encoding either N-terminal

or C-terminal fusions of Os TPKs with EYFP or RFP under control of a

cauliflower mosaic virus 35S promoter. g-TIP was PCR amplified from

cDNAofArabidopsiswith forward and reverse primers (see Supplemental

Table 1 online). The resulting PCR fragment was inserted into a pART7-

EYFP vector to generate pART7- At-g-TIP-EYFP C-terminal fusions.

To generate chimeric constructs, phosphorylation by T4 polynucleo-

tide kinase (Promega) of primers 6, 8, 10, 12, 14, 16, 18, 20, 21, and 22

(see Supplemental Table 1 online) was performed with subsequent

differential PCR amplification of fragments corresponding to different

parts of TPKa or TPKb sequences. The corresponding PCR-amplified

fragments of TPKa and TPKbwere ligated with T4 DNA ligase (Promega).

The ligationmixtures were used for the final PCR amplification of chimeric

sequenceswith pairs of primers 1 and 4 or 3 and 2 carrying XhoI andSmaI

restriction sites (see Supplemental Table 1 online). The chimeric se-

quences were inserted into pART7-EYFP as described previously.

Figure 9. Simplified Diagram Showing Trafficking Pathways to Different

Vacuoles.

Proteins destined for the LV are sorted into clathrin-coated vesicles

(CCVs) at the Golgi (1). Proteins destined for the PSV can be transported

through the Golgi via dense vesicles (DVs) and subsequently via multi-

vesicular prevacuolar compartments (PVCs) (2a) or traffic from ER to PSV

in a Golgi-independent way via intermediate compartments such the

precursor accumulating compartment (PAC), ER dense vesicle (ERdv), or

dark intrinsic protein (DIP) (2b). Trafficking of native TPKa occurs via the

Golgi machinery (1) and is destined for the LV. By contrast, trafficking of

native TPKb is Golgi independent (2b), and expression is predominantly

in PSVs. Changing key residues in the C terminus of TPKa shifts the

expression from predominantly LVs to predominantly PSVs.

766 The Plant Cell

To generate point mutations in pART7-OsTPKa -EYFP and pART7-

TPKb-EYFP, constructs were subjected to Quickchange mutagenesis

(Stratagene) using primers 25 to 38 (see Supplemental Table 1 online).

Transient Expression and Imaging of Os TPK Fusion Proteins

Rice aleurone protoplasts were isolated from ripening seeds as described

by Hay and Spanswick (2007). The rice and barley (Hordeum vulgare)

protoplasts from 1-week-old etiolated seedlings were prepared and

transformed with pART7 EYFP-based constructs according to Bart et al.

(2006).Arabidopsismesophyll protoplasts were isolated and transformed

with pART7-OsTPKa/OsTPKb-EYFP plasmids according to Abel and

Theologis (1994). To transform epidermal tobacco cells, the pART7

expression cartridge of TPKa was inserted into the binary vector pATT27

using NotI restriction sites. The resulting construct was introduced into

theGV3101 Agrobacterium tumefaciens strain, and infiltration of tobacco

leaves was performed as described previously (Reisen and Hanson,

2007).

Imaging of protoplasts that expressed various fluorescent reporters

was performed using confocal laser scanningmicroscopy (Zeiss LSM510

Meta). Colocalization images were obtained in the lambda microscope

mode, and signals were spectrally unmixed using presaved spectra of

GFP, EYFP, RFP, and autofluorescence.

To determine the frequency with which rice protoplasts were assigned

to categories that show LV, PSV, or ER expression, the following was

performed. Fluorescence was observed using epifluorescence micros-

copy 18 to 24 h after transformation (unless indicated otherwise). Proto-

plasts were lysed by perfusionwith a solution containing 10mMEGTA, 10

mM EDTA, pH 8.0, and 180 mM sorbitol. Fluorescent, lysed protoplasts

were categorized on the basis of vacuolar and nonvacuolar fluorescence

as follows: cells were counted as LV if vacuolar fluorescence was

restricted to the large main vacuole (e.g., Figure 1A, top right panel), as

PSV if fluorescence was restricted to multiple (n$ 2) small vacuoles (e.g.,

Figure 1A, bottom right panel), and ER if fluorescencewas evident but did

not derived from either LVs or PSVs. Thus, both the LV and the PSV

populations subsumed a large number of protoplasts that showed ER

fluorescence in addition to vacuolar fluorescence. In total, between 40

and 50 protoplasts were imaged to determine the proportion of proto-

plasts showing fluorescence in LV, PSV, and ER, and this was repeated at

least three times with protoplasts from independent transformation

events.

Treatment with Brefeldin A

A stock of brefeldin A (Sigma-Aldrich) was made at 10 mg/mL in DMSO

and diluted to 10 mg/mL for use in experiments. Protoplasts were treated

with brefeldin A by adding the stock solution to the medium 1 h after

transformation. Plant leaves were infiltrated on the abaxial surface with

brefeldin A solution 2 d after inoculation. In each experiment, treatment

with 0.1% DMSO was used as a control.

Electrophysiology

Arabidopsis mesophyll protoplasts were isolated as described (White

et al., 1999) from tpk1 tpc1 double knockout mutant lines. Vacuoles were

released from protoplasts by washing protoplasts with a solution con-

taining 10 mM EGTA, 10 mM EDTA, pH 8, with an osmolarity of 350

mOsM. General patch-clamp methodology was as described by Gobert

et al. (2007) and Maathuis et al. (1998) with luminal and cytoplasmic

solutions containing (in mM) 0.1 CaCl2, 100 KCl, 5 MES/Tris, pH 6.5, and

200 sorbitol. Rice was grown as described above, and protoplasts were

isolated as for Arabidopsis (White et al., 1999), with the addition of

hemicellulase (0.5%) to the cell wall digestion protocol.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data

libraries under the following accession numbers: TPKa (Os03g54100),

NM_001057833; and TPKb (Os07g01810), NM_001065254.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Disrupted Rice Protoplast Showing Acidic,

Neutral Red LV, and Neutral pH PSVs with TPKb Expression.

Supplemental Figure 2. Rice Protoplast Showing OsTPKa and

OsTPKb Localization with N-Terminal FP Fusions.

Supplemental Figure 3. Both TPKa and TPKb Activity Is Enhanced in

the Presence of 14-3-3.

Supplemental Figure 4. TPK Current Recordings from Rice LV and

PSV-Type Compartments.

Supplemental Figure 5. Rice Protoplast Subcellular Localization and

Distribution of N-terminal OsTPKa:OsTPKb Chimeras.

Supplemental Figure 6. Rice Protoplast Subcellular Localization of

OsTPKa Containing Point Mutations in the C Terminus.

Supplemental Figure 7. Point Mutation in Barley HvTPK1 That

Affects Membrane Localization.

Supplemental Table 1. List of Primers.

ACKNOWLEDGMENTS

We thank Jean-Marc Neuhaus (University of Neuchatel, Switzerland) for

providing us with d-TIP marker lines. This work was funded by Biotech-

nology and Biological Science Research Council Grant BB/E527155.

The 14-3-3 protein was a kind gift from Bert de Boer (Vrije Universiteit

Amsterdam, The Netherlands).

Received November 17, 2010; revised November 30, 2010; accepted

December 17, 2010; published January 11, 2011.

REFERENCES

Abel, S., and Theologis, A. (1994). Transient transformation of Arabi-

dopsis leaf protoplasts: A versatile experimental system to study gene

expression. Plant J. 5: 421–427.

Bart, R., Chern, M., Park, C.J., Bartley, L., and Ronald, P.C. (2006). A

novel system for gene silencing using siRNAs in rice leaf and stem-

derived protoplasts. Plant Methods 2: 13.

Bihler, H., Eing, C., Hebeisen, S., Roller, A., Czempinski, K., and

Bertl, A. (2005). TPK1 is a vacuolar ion channel different from the

slow-vacuolar cation channel. Plant Physiol. 139: 417–424.

Coblitz, B., Shikano, S., Wu, M., Gabelli, S.B., Cockrell, L.M.,

Spieker, M., Hanyu, Y., Fu, H., Amzel, L.M., and Li, M. (2005).

C-terminal recognition by 14-3-3 proteins for surface expression of

membrane receptors. J. Biol. Chem. 280: 36263–36272.

Czempinski, K., Frachisse, J.M., Maurel, C., Barbier-Brygoo, H., and

Mueller-Roeber, B. (2002). Vacuolar membrane localization of the

Arabidopsis ‘two-pore’ K+ channel KCO1. Plant J. 29: 809–820.

Dunkel, M., Latz, A., Schumacher, K., Muller, T., Becker, D., and

Hedrich, R. (2008). Targeting of vacuolar membrane localized mem-

bers of the TPK channel family. Mol. Plant 1: 938–949.

Rice Two-Pore K+ Channels 767

Flowerdew, S.E., and Burgoyne, R.D. (2009). A VAMP7/Vti1a SNARE

complex distinguishes a non-conventional traffic route to the cell surface

used by KChIP1 and Kv4 potassium channels. Biochem. J. 418: 529–540.

Galili, G., Altschuler, Y., and Levanony, H. (1993). Assembly and

transport of seed storage proteins. Trends Cell Biol. 3: 437–442.

Gifford, J.L., Walsh, M.P., and Vogel, H.J. (2007). Structures and

metal-ion-binding properties of the Ca2+-binding helix-loop-helix EF-

hand motifs. Biochem. J. 405: 199–221.

Gleave, A.P. (1992). A versatile binary vector system with a T-DNA

organisational structure conducive to efficient integration of cloned

DNA into the plant genome. Plant Mol. Biol. 20: 1203–1207.

Gobert, A., Isayenkov, S., Voelker, C., Czempinski, K., and Maathuis,

F.J.M. (2007). The two-pore channel TPK1 gene encodes the vacuolar

K+ conductance and plays a role in K+ homeostasis. Proc. Natl. Acad.

Sci. USA 104: 10726 –10731.

Hamamoto, S., et al. (2008). Characterization of a tobacco TPK-type K+

channel as a novel tonoplast K+ channel using yeast tonoplasts. J.

Biol. Chem. 283: 1911–1920.

Hara-Nishimura I, I., Shimada, T., Hatano, K., Takeuchi, Y., and

Nishimura, M. (1998). Transport of storage proteins to protein stor-

age vacuoles is mediated by large precursor-accumulating vesicles.

Plant Cell 10: 825–836.

Hay, J.O., and Spanswick, R.M. (2007). Isolation of ripening rice (Oryza

sativa L.) aleurone protoplasts for uptake studies. Plant Sci. 172: 659–664.

Hinz, G., Colanesi, S., Hillmer, S., Rogers, J.C., and Robinson, D.G. (2007).

Localization of vacuolar transport receptors and cargo proteins in the Golgi

apparatus of developing Arabidopsis embryos. Traffic 8: 1452–1464.

Hofte, H., and Chrispeels, M.J. (1992). Protein sorting to the vacuolar

membrane. Plant Cell 4: 995–1004.

Hunter, P.R., Craddock, C.P., Di Benedetto, S., Roberts, L.M., and

Frigerio, L. (2007). Fluorescent reporter proteins for the tonoplast and

the vacuolar lumen identify a single vacuolar compartment in Arabi-

dopsis cells. Plant Physiol. 145: 1371–1382.

Isayenkov, S.V., Isner, J.C., and Maathuis, F.J.M. (2010). Vacuolar ion

channels: Roles in plant nutrition and signalling. FEBS Lett. 584: 1982–1988.

Jauh, G.-Y., Fischer, A.M., Grimes, H.D., Ryan, C.A., Jr., and Rogers,

J.C. (1998). delta-Tonoplast intrinsic protein defines unique plant

vacuole functions. Proc. Natl. Acad. Sci. USA 95: 12995–12999.

Jauh, G.Y., Phillips, T.E., and Rogers, J.C. (1999). Tonoplast intrinsic protein

isoforms as markers for vacuolar functions. Plant Cell 11: 1867–1882.

Jiang, L., Phillips, T.E., Rogers, S.W., and Rogers, J.C. (2000). Biogen-

esis of the protein storage vacuole crystalloid. J. Cell Biol. 150: 755–770.

Jiang, L., and Rogers, J.C. (1998). Integral membrane protein sorting to

vacuoles in plant cells: Evidence for two pathways. J. Cell Biol. 143:

1183–1199.

Jiang, L., and Rogers, J.C. (1999). Functional analysis of a Golgi-localized

Kex2p-like protease in tobacco suspension culture cells. Plant J. 18: 23–32.

Jurgens, G. (2004). Membrane trafficking in plants. Annu. Rev. Cell Dev.

Biol. 20: 481–504.

Kim, K.S., Hwang, H.G., Kang, H.J., Hwang, I.K., Lee, Y.T., and Choi,

H.C. (2004). Comparative ultrastructure of Ilpumbyeo, a high-quality

japonica rice, and its mutant, Suweon 464: Scanning and transmission

electron microscopy studies. J. Agric. Food Chem. 52: 3876–3883.

Latz, A., Becker, D., Hekman, M., Muller, T., Beyhl, D., Marten, I., Eing,

C., Fischer, A., Dunkel, M., Bertl, A., Rapp, U.R., and Hedrich, R.

(2007). TPK1, a Ca2+-regulated Arabidopsis vacuole two-pore K+ chan-

nel is activated by 14-3-3 proteins. Plant J. 52: 449–459.

Levanony, H., Rubin, R., Altschuler, Y., and Galili, G. (1992). Evidence

for a novel route of wheat storage proteins to vacuoles. J. Cell Biol.

119: 1117–1128.

Li, X., Chanroj, S., Wu, Z., Romanowsky, S.M., Harper, J.F., and Sze,

H. (2008). A distinct endosomal Ca2+/Mn2+ pump affects root growth

through the secretory process. Plant Physiol. 147: 1675–1689.

Maathuis, F.J.M., May, S.T., Graham, N.S., Bowen, H.C., Jelitto,

T.C., Trimmer, P., Bennett, M.J., Sanders, D., and White, P.J.

(1998). Cell marking in Arabidopsis thaliana and its application to

patch-clamp studies. Plant J. 15: 843–851.

Marty, F. (1999). Plant vacuoles. Plant Cell 11: 587–600.

Matsuoka, K., Bassham, D.C., Raikhel, N.V., and Nakamura, K.

(1995). Different sensitivity to wortmannin of two vacuolar sorting

signals indicates the presence of distinct sorting machineries in

tobacco cells. J. Cell Biol. 130: 1307–1318.

Matsuoka, K., and Bednarek, S.Y. (1998). Protein transport within the plant

cell endomembrane system: An update. Curr. Opin. Plant Biol. 1: 463–469.

Mills, R.F., Doherty, M.L., Lopez-Marques, R.L., Weimar, T., Dupree,

P., Palmgren, M.G., Pittman, J.K., and Williams, L.E. (2008). ECA3,

a Golgi-localized P2A-type ATPase, plays a crucial role in manganese

nutrition in Arabidopsis. Plant Physiol. 146: 116–128.

Nebenfuhr, A., Ritzenthaler, C., and Robinson, D.G. (2002). Brefeldin A:

Deciphering an enigmatic inhibitor of secretion. Plant Physiol. 130: 1102–1108.

Neuhaus, J.M., and Rogers, J.C. (1998). Sorting of proteins to vacu-

oles in plant cells. Plant Mol. Biol. 38: 127–144.

O’Kelly, I., and Goldstein, S.A.N. (2008). Forward transport of K2p3.1:

Mediation by 14-3-3 and COPI, modulation by p111. Traffic 1: 72–78.

Oufattole, M., Park, J.H., Poxleitner, M., Jiang, L.W., and Rogers,

J.C. (2005). Selective membrane protein internalization accompanies

movement from the endoplasmic reticulum to the protein storage

vacuole pathway in Arabidopsis. Plant Cell 17: 3066–3080.

Park, M., Kim, S.J., Vitale, A., and Hwang, I. (2004). Identification of

the protein storage vacuole and protein targeting to the vacuole in leaf

cells of three plant species. Plant Physiol. 134: 625–639.

Park, M., Lee, D., Lee, G.J., and Hwang, I. (2005). AtRMR1 functions

as a cargo receptor for protein trafficking to the protein storage

vacuole. J. Cell Biol. 170: 757–767.

Reisen, D., and Hanson, M.R. (2007). Association of six YFP-myosin

XI-tail fusions with mobile plant cell organelles. BMC Plant Biol. 7: 6.

Shikano, S., Coblitz, B., Sun, H., and Li, M. (2005). Genetic isolation of

transport signals directing cell surface expression. Nat. Cell Biol. 7: 985–992.

Shimada, T., Watanabe, E., Tamura, K., Hayashi, Y., Nishimura, M.,

and Hara-Nishimura, I. (2002). A vacuolar sorting receptor PV72 on

the membrane of vesicles that accumulate precursors of seed storage

proteins (PAC vesicles). Plant Cell Physiol. 43: 1086–1095.

Schonknecht, G., Spoormaker, P., Steinmeyer, R., Bruggeman, L.,

Ache, P., Dutta, R., Reintanz, B., Godde, M., Hedrich, R., and

Palme, K. (2002). KCO1 is a component of the slow-vacuolar (SV) ion

channel. FEBS Lett. 511: 28–32.

Toyooka, K., Okamoto, T., and Minamikawa, T. (2000). Mass transport

of proform of a KDEL-tailed cysteine proteinase (SH-EP) to protein

storage vacuoles by endoplasmic reticulum-derived vesicle is involved

in protein mobilization in germinating seeds. J. Cell Biol. 148: 453–464.

Vitale, A., and Hinz, G. (2005). Sorting of proteins to storage vacuoles:

How many mechanisms? Trends Plant Sci. 10: 316–323.

Voelker, C., Schmidt, D., Mueller-Roeber, B., and Czempinski, K.

(2006). Members of the Arabidopsis AtTPK/KCO family form homo-

meric vacuolar channels in planta. Plant J. 48: 296–306.

White, P.J., Biskup, B., Elzenga, J.T.M., Homann, U., Thiel, G.,

Wissink, F., and Maathuis, F.J.M. (1999). Advanced patch-clamp

techniques and single channel analysis. J. Exp. Bot. 50: 1037–1054.

Yamada, K., Osakabe, Y., Mizoi, J., Nakashima, K., Fujita, Y.,

Shinozaki, K., and Yamaguchi-Shinozaki, K. (2010). Functional anal-

ysis of an Arabidopsis thaliana abiotic stress-inducible facilitated diffu-

sion transporter for monosaccharides. J. Biol. Chem. 285: 1138–1146.

Zelazny, E., Miecielica, U., Borst, J.W., Hemminga, M.A., and

Chaumont, F. (2009). An N-terminal diacidic motif is required for

the trafficking of maize aquaporins ZmPIP2;4 and ZmPIP2;5 to the

plasma membrane. Plant J. 57: 346–355.

768 The Plant Cell

DOI 10.1105/tpc.110.081463; originally published online January 11, 2011; 2011;23;756-768Plant Cell

Stanislav Isayenkov, Jean-Charles Isner and Frans J.M. Maathuis Channels Are Expressed in Different Types of Vacuoles+Rice Two-Pore K

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