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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.
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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
This information is current as of November 13, 2020
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