Arabidopsis Intracellular NHX-Type Sodium-Proton
Antiporters are Required for Seed Storage Protein Processing
Joanne R. Ashnest1, Dung L. Huynh1, Jonathan M. Dragwidge1, Brett A. Ford1,2 and Anthony R. Gendall1,*
1
Regular Paper
Department of Animal, Plant and Soil Sciences, AgriBio, Centre for AgriBiosciences, 5 Ring Road, La Trobe University, Bundoora, VIC 3086, Australia
Present address: Commonwealth Scientific and Industrial Research Organization Agriculture Flagship, Clunies Ross Street, Acton, ACT 2601, Australia
2
*Corresponding author: E-mail, [email protected]; Fax, +61 3 9032 7501.
(Received March 31, 2015; Accepted September 18, 2015)
The Arabidopsis intracellular sodium–proton exchanger
(NHX) proteins AtNHX5 and AtNHX6 have a welldocumented role in plant development, and have been
used to improve salt tolerance in a variety of species.
Despite evidence that intracellular NHX proteins are important in vacuolar trafficking, the mechanism of this role is
poorly understood. Here we show that NHX5 and NHX6 are
necessary for processing of the predominant seed storage proteins, and also influence the processing and activity of a vacuolar processing enzyme. Furthermore, we show by yeast twohybrid and bimolecular fluorescence complementation
(BiFC) technology that the C-terminal tail of NHX6 interacts
with a component of Retromer, another component of the
cell sorting machinery, and that this tail is critical for NHX6
activity. These findings demonstrate that NHX5 and NHX6 are
important in processing and activity of vacuolar cargo, and
suggest a mechanism by which NHX intracellular (IC)-II antiporters may be involved in subcellular trafficking.
Keywords: Arabidopsis thaliana Protein processing Protein
storage vacuole Sorting nexin Vacuolar processing enzyme.
Abbreviations: BFA, brefeldin A; BiFC, bimolecular fluorescent complementation; C/G/RFP, cyan/green/red fluorescent
protein; CPA1, cation–proton antiporter; CPY, carboxypeptidase Y; CRU, CRUCIFERIN; ER, endoplasmic reticulum; IC,
intracellular; MVB, multivesicular body; NhaP/NHE/NHX,
sodium–hydrogen antiporter/exchanger; PSV, protein storage
vacuole; SNX1, sorting nexin 1; SOS1, salt-overly-sensitive 1;
SSP, seed storage protein; TGN, trans-Golgi network; VPE,
vacuolar processing enzyme; VPS, vacuolar protein sorting;
VSR, vacuolar sorting receptor.
Introduction
Maintenance of cellular ion homeostasis and pH is important in
the regulation of metabolic processes and cellular activity. The
cation/proton (CPA1) class of transmembrane antiporters facilitates this cellular homeostasis at the plasma membrane and
at intracellular organelles through the exchange of an Na+ or K+
ion for a H+ ion across a membrane (Chanroj et al. 2012). The
CPA1 family is conserved in bacteria, plants, fungi and animals,
and, apart from the yeast Saccharomyces cerevisiae, all
eukaryotes sequenced to date contain multiple isoforms
(Brett et al. 2005a, Chanroj et al. 2012, Ford et al. 2012). In
addition to roles in pH and cation homeostasis, CPA1 proteins
are important in cellular processes such as endosomal protein
transport and multivesicular body (MVB) formation in yeast
(Bowers et al. 2000, Brett et al. 2005b, Qiu and Fratti 2010), and
the regulation of cell shape and organelle morphology in animal
cells (reviewed in Orlowski and Grinstein 2007). In plants, these
antiporters have roles in the regulation of cell volume, expansion and differentiation (Pardo et al. 2006, Rodrı́guez-Rosales
et al. 2008).
In plants, members of the CPA1 gene family are assigned to
either the NhaP (Na+/H+ antiporter) or the NHX clade (Brett
et al. 2005a, Chanroj et al. 2012). The NhaP clade clusters with
the ancestral prokaryotic NhaP genes, and includes Arabidopsis
NHX7 (salt-overly-sensitive 1; SOS1) and NHX8. These proteins
localize to the plasma membrane and show similarity to human
isoforms NHE1–NHE5 (Qiu et al. 2002, Chanroj et al. 2012).
Members of the remaining plant NHX clade localize to intracellular (IC) membranes, and are distinguished as either tonoplast localized (IC-I) or endosome localized (IC-II). The IC-I
clade includes AtNHX1–AtNHX4, and is specific to plants; however, the IC-II clade includes AtNHX5 and AtNHX6, the yeast
ScNHX1, as well as the human isoforms NHE6 and NHE7
(Bowers et al. 2000, Brett et al. 2005a, Chanroj et al. 2012).
The C-terminal tails of both NHX1 and SOS1 are essential for
mediation of protein–protein interactions, and subsequent activation and selectivity of these antiporters (Yamaguchi et al.
2005, Katiyar-Agarwal et al. 2006, Quintero et al. 2011).
However, a similar examination of the NHX5/NHX6 C-terminus
has not yet been performed.
Overexpression of the Arabidopsis IC-II member AtNHX5
improved salt tolerance in the ornamental plant Torenia fournieri (Shi et al. 2008), the paper mulberry Broussonetia papyrifera (Li et al. 2011a) and rice Oryza sativa (Li et al. 2011b).
T-DNA knockout mutant studies have shown that NHX5 and
the paralogous NHX6 are redundantly important for plant
growth, in particular cellular expansion and polarity, and root
growth (Bassil et al. 2011). Their co-localization to the Golgi and
trans-Golgi network (TGN; Bassil et al. 2011) suggests a possible
role in subcellular sorting and trafficking. Bassil et al. (2011)
showed that a transiently expressed fragment of S. cerevisiae
carboxypeptidase Y (ScCPY), containing an N-terminal vacuolar
Plant Cell Physiol. 56(11): 2220–2233 (2015) doi:10.1093/pcp/pcv138, Advance Access publication on 28 September 2015,
available online at www.pcp.oxfordjournals.org
! The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Plant Cell Physiol. 56(11): 2220–2233 (2015) doi:10.1093/pcp/pcv138
targeting motif fused to green fluorescent protein (GFP), was
mistrafficked to the apoplast in the nhx5 nhx6 double mutant.
This sorting defect is reminiscent of the trafficking phenotype
observed in the homologous yeast nhx1 mutant (Bowers et al.
2000).
In the classical model of Golgi-dependent trafficking to the
vacuole, it is believed that the family of vacuolar sorting receptors (VSRs) recognize and bind soluble cargo at the TGN, and
redirect this cargo to the MVB (also known as the pre-vacuolar
compartment). The MVBs then merge to form the vacuole
(Otegui et al. 2006, Scheuring et al. 2011). In this model, VSRs
release their cargo ligand at the MVB, and are then recycled
back to the TGN by the activity of Retromer, a protein complex
composed of a ‘core’ of vacuolar protein sorting (VPS) proteins
(VPS26, VPS29 and VPS35 in Arabidopsis) and the peripheral
sorting nexins (SNX1, SNX2a and SNX2b) (Oliviusson et al.
2006, Pourcher et al. 2010). However, an alternative model for
Golgi-dependent trafficking has recently been proposed
(Robinson and Pimpl 2014) in which VSRs bind and sort
cargo at the endoplasmic reticulum (ER), and are recycled
from the TGN by Retromer. This model considers recent evidence that the SNX and Retromer core proteins reside on the
TGN, rather than the MVB (Niemes et al. 2010), and that lumenal conditions of these organelles are more suitable for ligand
binding and release, respectively (Robinson and Pimpl 2014).
Seed storage proteins (SSPs) are the predominant proteins
found in mature seeds, and undergo post-translational cleavage
en route to specialized protein storage vacuoles (PSVs; HaraNishimura et al. 1998, Gruis et al. 2004, Otegui et al. 2006). At
present, it is thought that there are two routes for SSP trafficking to the vacuole (Vitale and Hinz 2005). The first is receptor
mediated and Golgi dependent, requiring VSR and Retromer for
complete vacuolar trafficking and processing of SSPs, and for
normal PSV formation (Shimada et al. 2003a, Shimada et al.
2006, Yamazaki et al. 2008, Pourcher et al. 2010). Additionally,
VSR1 binds the C-terminus of the Arabidopsis 12S globulin
in vitro (Shimada et al. 2003a, Maruyama et al. 2015), and
p12S and VSR1 associate in the same 480 kDa complex
(Reguera et al. 2015). Interestingly, many genes involved in
SSP processing and PSV formation have also been shown to
be developmentally important, as mutants often have defects
in growth and leaf senescence (Shimada et al. 2003a, Shimada
et al. 2003b, Li et al. 2006, Shimada et al. 2006, Takahashi et al.
2010). A proportion of correctly processed, mature SSPs is still
found in vsr mutant seed, even when multiple vsr isoforms are
silenced (Zouhar et al. 2010). The interpretation of these data is
that VSR acts as the sorting receptor for 12S and 2S proteins via
the Golgi-dependent route, while a proportion of 12S and 2S
aggregates are trafficked to the PSVs in a Golgi-independent
‘bulk flow’ process via precursor accumulating (PAC) vesicles.
PAC vesicles are SSP-rich vesicles which bud directly from the
ER, and were first identified in castor bean (Hiraiwa et al. 1993)
and subsequently found in pumpkin seeds, soybean and
rice (Hara-Nishimura et al. 1998, Mitsuhashi et al. 2001,
Mori et al. 2004, Takahashi et al. 2005). Factors influencing
the segregation of SSPs into these two pathways remain largely
unknown.
Luminal conditions including pH and Ca2+ concentrations
are likely to be important at several stages in SSP trafficking,
particularly VSR–ligand binding and release (Watanabe et al.
2002). Likewise, cleavage of the SSPs by the vacuolar processing
enzymes (VPEs) is pH dependent (Gruis et al. 2004). However,
evidence regarding the luminal pH changes of organelle compartments on endosomal maturation to the vacuole is conflicting (Otegui et al. 2006, Shen et al. 2013, Martinière et al. 2013). It
has been suggested that NHX5 and NHX6 affect vacuolar
trafficking by alkalinization of secretory compartments during
maturation, and it had previously been shown that overexpressed NHX5 localizes to alkalinized compartments, and partially co-localizes with VSR (23%; Martinière et al. 2013). Recent
evidence has revealed that in nhx5 nhx6 cells, the Golgi, TGN
and MVB are hyperacidified (pH = 0.25–0.4) compared with
the wild type, suggesting that plant IC-II NHXs are required for
pH homeostasis within the secretory pathway (Reguera et al.
2015). In this study, cells of nhx5 nhx6 embryos were found to
mislocalize SSP precursors to the apoplast in mature and
developing seeds. Furthermore, compartments containing
VSR1 were found to be acidified by 0.6 pH units, and VSR–
cargo interactions were found to be compromised in nhx5
nhx6 protoplasts. Interestingly, this defect in pH homeostasis
did not result in significant mistrafficking of VSR or its soluble
cargo, aleurain, in protoplasts (Reguera et al. 2015).
In this study, we examined the processing of vacuolar proteins in nhx5 nhx6 embryos, and discovered that similarly to
VSR1, NHX5 and NHX6 is required for normal PSV formation
and for the complete processing of the major SSPs. Additional
analysis revealed that nhx5 nhx6 mutants exhibit altered VPE
activity, processing and localization in mature seeds. We examined the role of the C-terminal region of NHX6 for potential
interactions by which NHX5 and NHX6 may impact VPE and
SSP processing. Deletion mutants of NHX6 lacking this tail were
correctly localized, but were unable to rescue the nhx5 nhx6
mutant phenotype. Protein–protein interaction experiments
revealed that the C-terminal tail of NHX6 interacts with
SNX1, a component of Retromer, which is important for retrograde receptor trafficking and indirectly affects storage protein
processing. These observations suggest that NHX IC-II antiporters may be important in Retromer-dependant vacuolar
trafficking.
Results
NHX5, NHX6 and SNX1 are expressed
during seed development
Seeds provide a useful tissue to study trafficking and processing
of vacuolar cargo, due to their relatively simple proteome, consisting primarily of a few isoforms of SSPs, and well characterized trafficking pathways. However, clearly this approach can
only be useful in cases where gene expression is high in developing seeds during seed protein accumulation, such as for VSR or
SNX1 (Fig. 1A; Shimada et al. 2003a, Pourcher et al. 2010). To
determine the suitability of this approach to study the role of
NHX5 and NHX6 in subcellular trafficking, we examined the
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expression profiles of NHX5 and NHX6 in reproductive tissues
and developing seeds. Analysis of microarray expression data
from the Bio-Analytic Resource for Plant Biology (http://bar.
utoronto.ca/efp/cgi-bin/efpWeb.cgi; Winter et al. 2007)
revealed that NHX6 expression peaks in the mid to late stages
of seed development, while NHX5 expression throughout seed
development remains approximately consistent with levels in
vegetative tissues (Fig. 1A). We confirmed this expression pattern of NHX6 in developing embryos using a 3 kb NHX6 promoter fused to the b-glucuronidase (GUS) reporter gene.
Consistent with microarray data, NHX6 promoter activity was
observed in mature and developing embryos at early to late
torpedo and bent-cotyledon stages (corresponding to stages
5–7; Winter et al. 2007) and 1–3 d after germination (Fig.
1B–G). This expression pattern indicates that further investigation of the role of NHX5 and NHX6 in seed protein trafficking
was warranted.
Storage protein processing and protein
storage vacuole formation are altered in nhx5
nhx6 embryos
Very recently, the PSVs and storage protein profile of nhx5-1
nhx6-1 seeds were described (Reguera et al. 2015). Here, we
present an analysis of a different set of mutant alleles; nhx5-2
and nhx6-3 (Supplementary Fig. S1). We examined the PSVs of
mature nhx5-2, nhx6-3 and nhx5-2 nhx6-3 embryos using confocal microscopy. Hypocotyl cells of nhx5 nxh6 embryos were
found to have an increased number of smaller PSVs (Fig. 1H).
Quantification showed that these PSVs were approximately
one-third the size of those in wild-type cells, but almost three
times more numerous; thus the overall area of PSV per cell was
not significantly different (Fig. 1I). Notably, the PSV phenotype
of the nhx5 nhx6 double mutant was strikingly similar to that of
the known trafficking mutant, vsr1.
As many mutants with abnormal PSVs accumulate abnormally processed precursors of the 12S and 2S storage proteins,
we examined the protein profiles of dry nhx5, nhx6 and nhx5
nhx6 seeds by one-dimensional gel electrophoresis. Consistent
with protein accumulation in vsr1 (Shimada et al. 2003a), this
analysis revealed that the nhx5 nhx6 seeds accumulated
increased amounts of three 49–52 kDa proteins (Fig. 2A). The
identity of these accumulated proteins was confirmed by subjecting the isolated bands to mass spectrometry analysis, which
determined that they were derived from the p12S globulin precursors CRUCIFERIN1 (CRU1), CRU2 and CRU3 (Fig. 2B;
Supplementary Fig. S2). For each band, the primary CRU identified comprised between 47% and 58% of all peptide hits. Some
non-CRU peptide hits were also identified, comprising 1–6% of
Fig. 1 Continued
Fig. 1 The nhx5 nhx6 double mutant accumulates smaller, more numerous PSVs (A) Expression of NHX5, NHX6, SNX1 and VSR1 sourced
from the Arabidopsis eFP browser (http://bar.utoronto.ca/efp/cgibin/efpWeb.cgi; Winter et al. 2007). (B–G) GUS expression indicating
NHX6 promoter activity in early (B) and late (C) torpedo, walkingstick (D) and mature (E) stage embryos, and 1 (F) and 3 d (G) after
2222
germination. Scale bars represent 200 mm (B–D) or 0.5 mm (E–G). (H)
Autofluorescent PSVs in wild-type, nhx5, nhx6, nhx5 nhx6 double
mutant and vsr1 embryos. Scale bars = 10 mm (I) PSV size and number.
Asterisks indicate P < 0.01 compared with the wild type. Data are means
and SEs calculated from 10 individual cells from each of three biological
replicates.
Plant Cell Physiol. 56(11): 2220–2233 (2015) doi:10.1093/pcp/pcv138
is similar to the phenotype observed in vsr1 and other mutants
with PSV trafficking defects.
As most mutants with PSV trafficking defects also misprocess
the 2S storage proteins, we performed immunoblot analysis of
dry seeds using anti-12S and anti-2S antibodies (Fig. 2C, D;
Shimada et al. 2003b). This analysis confirmed the accumulation
of p12S in the nhx5 nhx6 mutants, and also revealed a slight
accumulation of the p2S albumin; however, the accumulation
of both precursors in nhx5 nhx6 was less severe than in vsr1
(Supplementary Fig. S3B). Since internal cleavage of the SSP
precursors usually begins when SSP precursors meet the VPE
enzyme at the MVB (Otegui et al. 2006), aberrant processing
suggests a failure in the delivery of SSP precursors to the
MVBs, or a reduction in VPE activity. Interestingly, nhx5 nhx6
seeds contained a substantial amount of fully processed 12S
globulin and 2S albumin subunits, indicating that the majority
of 12S and 2S processing still occurs normally in the nhx5 nhx6
double mutant. These data are consistent with the phenotype
described by Reguera et al. (2015).
Misprocessed bVPE is found in nhx5 nhx6 seeds
Fig. 2 nhx5 nhx6 seeds accumulate precursors of the SSPs. (A) Protein
profiles of dry seeds (10 seed per lane) of the wild type and nhx5, nhx6,
nhx5 nhx6 and vsr1 mutants. The arrow and arrowheads indicate
differently accumulated bands. (B) The indicated bands from (A)
(arrowheads) were identified by LC-MS. The cartoons indicate the
wild-type processing for CRU1 (Gruis et al. 2004). Black boxes indicate
the relative position of mass spectrometry peptide hits compared
with the wild-type 12S globulin. Filled arrowheads indicate a VPE
cleavage site; the open arrowhead indicates the signal peptide cleavage site. (C, D) Immunoblot analysis of the dry seeds of the wild type,
nhx5, nhx6, nhx5 nhx6, and vsr1 with (C) anti-12S (5 seeds perlane)
and (D) anti-2S (10 seeds per lane) antibodies.
peptide hits (Supplementary Table S2). This analysis suggested that the signal peptide was removed normally
(Fig. 2B), indicating that the ER processing of the signal peptide
is not affected in nhx5 nhx6 seeds. However, in all CRU proteins
identified, an unbroken peptide fragment was detected which
spanned the normal VPE cleavage site (Fig. 2B). This fingerprint, together with the band size indicated by Coomassie
SDS–PAGE, indicated that the accumulated p12S proteins
were not cleaved normally in nhx5 nhx6 seeds (Fig. 2B). This
We hypothesized that the defective SSP precursor processing in
nhx5 nhx6 seeds may be due to a reduction in activity of the
major cleavage enzyme, VPE. To investigate this, we first examined the PSV size and morphology in vpe null (a quadruple
mutant for a-, b-, g- and dVPE) mutant embryos, and found
that vpe null PSVs were similar in size and morphology to nhx5
nhx6 PSVs (Fig. 3A, B). This supports the possibility that the
nhx5 nhx6 PSV phenotype may be a result of altered VPE localization or activity.
To determine the subcellular localization of VPE, we performed in vivo examination of VPE activity in developing embryos using JOPD1, an improved VPE activity sensor with
similar properties to AMS101 (Misas-Villamil et al. 2013, Lu
et al. 2015). VPE is autocatalytically activated upon maturation,
which occurs in three stages (Kuroyanagi et al. 2002). For bVPE,
removal of a 21 amino acid signal peptide from the pre-protein
precursor (ppbVPE; 54 kDa) produces proVPE (pbVPE; 52 kDa).
ppVPE and pVPE are inactive forms (Kuroyanagi et al. 2002,
Misas-Villamil et al. 2013). VPE activation occurs with the cleavage of a C-terminal autoinhibitory domain, resulting in the
intermediate VPE (ibVPE; 37 kDa), followed by removal of
the N-terminal propeptide to produce mature VPE
(mbVPE; 27 kDa). iVPE and mVPE both contribute to total
VPE activity. Active VPE catalyzes the highly specific cleavage
of JOPD1 and results in a covalent, irreversible bond between
VPE protein and the fluorescent probe, which is detectable
by confocal microscopy. In wild-type late cotyledon hypocotyl
cells, active VPE was detected in large vacuoles, which appear to
be PSVs (filled arrowheads, Fig. 3C). This observation is
consistent with previous immunogold labeling data which
showed that VPE localizes to PSVs in mature embryos and
the lytic vacuole in mesophyll cells (Otegui et al. 2006, MisasVillamil et al. 2013). In nhx5 nhx6 embryos but not in wild-type
embryos, VPE activity was detected in the apoplast (inset,
Fig. 3C; Supplementary Fig. S3C), indicating that a proportion
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J. R. Ashnest et al. | NHX antiporters are required for SSP processing
Fig. 3 VPE activity and accumulation is affected in nhx5 nhx6 seeds (A) Autofluorescent PSVs in wild-type, nhx5 nhx6 double mutant and vpe
null mutant embryos. Scale bars represent 10 mm. (B) PSV size and number. Asterisks indicate P < 0.05 compared with the wild type, except
where indicated. Data are means and SEs calculated from 10 individual cells from each of three biological replicates. (C) In situ VPE activity
labeling of radical cells of late bent-cotyledon stage embryos. At 12 d after anthesis, liberated wild-type, nhx5 nhx6 and vpe null embryos were
incubated in liquid GM containing 2 mM JOPD1 for 20 h. Arrowheads indicate PSVs labeled with the VPE activity probe. The inset shows a 2
(continued)
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of VPE is mislocalized. As expected, vpe null embryos had no
VPE activity signal, confirming the specificity of the VPE activity
probe and imaging (Fig. 3C).
As previous use of JOPD1/AMS101 for in vivo imaging of
VPE activity has only demonstrated VPE presence or absence
(Misas-Villamil et al. 2013), this assay may lack the sensitivity to
assess subtle changes in VPE activity. Thus to quantify total VPE
activity in mature seeds of nhx5 nhx6 mutants, total seed protein extracts were incubated with JOPD1, then separated by
one-dimensional SDS–PAGE. VPE labeling resulted in major
bands at 37 and 27 kDa (Fig. 3D), which is consistent with
the size of ibVPE and mbVPE, respectively (Shimada et al.
2003b, Misas-Villamil et al. 2013). Fainter bands of approximately 42 and 34 kDa may be the immature and mature
forms, respectively, of aVPE or gVPE, both of which are expressed at low levels in seeds (Gruis et al. 2004). There were
reduced activities of both mbVPE and ibVPE in nhx5 nhx6 seeds
(50% and 25% of wild-type levels, respectively; Fig. 3H). We also
performed this experiment on developing embryos (late cotyledon stage) and found that this trend was consistent
(Supplementary Fig. S3F, G).
It is possible that VPE is regulated by a feedback mechanism,
and thus VPE gene expression may be altered in the event that
VPE activity or localization is affected. However, a search of the
literature did not find any evidence that the VPEs, or similar
enzymes, are subject to feedback regulation. To investigate the
processing and accumulation of bVPE, we performed an immunoblot analysis of dry seeds using an anti-bVPE antibody
(Shimada et al. 2003b). The level of the 52 kDa precursor
pbVPE was proportionally much higher in nhx5 nhx6 seeds
than in the wild type (Fig. 3E, I), while levels of ibVPE in
nhx5 nhx6 were reduced. Total VPE levels were similar between
the two genotypes, suggesting that NHX5 and NHX6 are not
required for the normal accumulation of bVPE protein.
Interestingly, when VPE activity was compared relative to the
amount of ibVPE, mbVPE and total VPE, the activity of the VPE
present was still lower in nhx5 nhx6 seeds (Fig. 3J). As expected,
the vpe null mutant was negative for both VPE activity and VPE
protein. Total SSP abundance was measured as the sum of the
precursor and mature forms in wild-type and nhx5 nhx6 seeds.
Despite the major SSPs being less abundant in nhx5 nhx6 seeds,
the total amount of protein per seed in nhx5 nhx6 was
equivalent to that in the wild type (Supplementary Fig.
S3A). Hence for both VPE activity and anti-bVPE immunoblots,
lanes were loaded with protein from equivalent numbers of
seeds, and bands of interest were normalized to an unknown
40 kDa band visualized by Coomassie staining, which appeared
unaffected in all genotypes (open arrowheads, Fig. 3F, G;
Supplementary Fig. S3D–F).
NHX6 is predicted to have a conserved
cytoplasmic C-terminal domain
To investigate the mechanism by which intracellular antiporters may be involved in SSP processing and regulation of VPE
activity, and interact with components of the TGN and other
sorting machinery, we sought to identify proteins that interact
with possible lumenal or cytoplasmic regions of NHX6 using a
yeast two-hybrid approach. The predicted amino acid sequences of NHX6 and NHX5 and 22 putative orthologs from
other plants were aligned (Supplementary Fig. S4), which
identified a conserved antiporting domain, consisting largely
of hydrophobic regions (amino acids 26–432 of AtNHX6),
and a putative cytoplasmic or luminal domain (amino acids
433–535) (Fig. 4A). Consensus prediction determined that
the transmembrane domains of both NHX5 and NXH6 were
in similar positions. We then aligned the amino acid sequences
of NHX5 and NXH6 with those of NhaP from Methanococcus
jannaschii (MjNhaP) and Escherichia coli NhaA (EcNhaA)
using a similar approach to that previously described
(Supplementary Fig. S5; Goswami et al. 2011). MjNhaP is the
most closely related protein to NHX6 for which a detailed structure has been determined, and is more closely related to the
intracellular NHXs/NHEs than is the more distantly related
NhaA. Superimposing the predicted putative transmembrane
domains of NHX5 and NXH6 (Fig. 4A) onto this alignment
revealed close agreement of 12 of the 13 predicted transmembrane domains of NHX5 and NHX6 with the predicted transmembrane domains of MjNhaP (Supplementary Fig. S5). This
comparative approach produced a putative topology for NHX6
(Fig. 4A) which placed the C-terminus of NHX6 in the cytosol.
Interestingly, we identified a conserved domain within this
region (amino acids 467–512), containing several phosphorylated amino acids (T470, S486 and S489; Fig. 4B) identified in
recent phosphoproteomic approaches.
Fig. 3 Continued
magnification of the apoplastic region indicated by an open arrowhead; p, PSV; a, apoplast. Scale bars represent 10 mm (main) or 2 mm (inset); chloroplast
autofluorescence is shown in white. Images are representative of six biological replicates from two independent experiments. (D) SDS–PAGE of mature
seed protein extracts (20 seeds per lane) of the wild type, nhx5 nhx6 and the quadruple VPE knockout line (vpe null) labeled with 1 mM JOPD1. (E)
Immunoblot analysis of protein extracts (15 seeds per lane) from dry seeds of the wild type, nhx5 nhx6 and vpe null mutants with anti-bVPE antibodies.
(F) Coomassie stain of the polyacrylamide gel in (D). An unknown 40 kDa band (open arrowhead) is unaffected in either mutant and was therefore used
as a loading control; relative densities of this band are shown in Supplementary Fig. S3D, and were used to normalize data in (H). (G) Coomassie stain of
a duplicate polyacrylamide gel of (E). The same 40 kDa band (open arrowhead) was used as a loading control; relative densities of this band are shown in
Supplementary Fig. S3D, and were used to normalize data in (I). (H) Relative contribution of bands from (D) to total VPE activity in the wild type or nhx5
nhx6 by densitometry. Data are means and SEs of three biological replicates; asterisks indicate P < 0.05. (I) Relative intensities of each band, and total VPE,
in wild-type and nhx5 nhx6 seed extracts, based on densitometry scans of (E). Data are means and SEs of three biological replicates; asterisks indicate
P < 0.05. (J) VPE activity relative to total VPE for ibVPE and mbVPE forms, calculated from densitometry data from (H) and (I). Data are means and SEs of
two biological replicates; asterisks indicate P < 0.05.
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Fig. 4 The C-terminal domain of NHX6 is conserved. (A) The topology of NHX was predicted using a consensus approach, and the relative
sequence conservation of each amino acid of AtNHX6 compared with 23 other plant IC-II NHXs. The C-terminal conserved domain is shown in a
dashed box. Vertical lines indicate boundaries of various constructs described in the text. Phosphorylated residues Thr470, Ser486 and Ser489 are
indicated. (B) The highly conserved region of the IC-II C-terminal domain is shown, with identical and conserved amino acids shaded in black and
gray, respectively. Full alignment, species names abbreviations and sequence accession numbers are described in Supplementary Fig. S4.
The cytosolic C-terminal domain is required for
NHX6 function
Having established the boundaries of the C-terminal region of
NHX6 and before proceeding with a two-hybrid screen, we
determined if this region is required for NHX6 function.
A truncated version of NHX6 lacking the C-terminus fused to
cyan fluorescent protein (NHX61–449–CFP) localized normally
in nhx5 nhx6 root cells, and showed a similar response to fulllength NHX5 to the fungal toxin brefeldin A (BFA; Fig. 5A, B). In
contrast to full-length NHX5–CFP or NHX6–YFP (yellow fluorescent protein), however, none of the 10 transgenic lines analyzed was able to rescue the nhx5 nhx6 mutant phenotype,
suggesting that amino acids 450–535 of NHX6 are critical for
2226
function or protein stability (Fig. 5C, D; Supplementary Fig.
S1). NHX5–CFP was used for localization experiments due to a
stronger fluorescence signal than observed in complementing
NHX6–YFP lines; mRNA expression levels in all lines were comparable with that of NHX5 in NHX5–CFP, and greater than wildtype expression levels (Supplementary Fig. S1).
NHX6 interacts with Retromer components
To identify proteins interacting with the NHX6 C-terminal
domain, we used this region as bait in a yeast two-hybrid
screen with an Arabidopsis cDNA library. A full-length
NHX6433–535 bait and a shorter truncation lacking the 28
most distal amino acids (NHX6433–507) both caused
Plant Cell Physiol. 56(11): 2220–2233 (2015) doi:10.1093/pcp/pcv138
Fig. 5 The C-terminal tail is required for NHX6 function, but not
subcellular localization. (A, B) Localization of NHX5–CFP and
NHX61–449–CFP (line 25) fusion proteins in nhx5 nhx6 root cap cells
counterstained with FM5-95, in the absence (A) or presence (B) of
BFA. The scale bar represents 5 mm. (C) Aerial photographs of 37-day
old wild-type or nhx5 nhx6 mutant plants on soil, stably transformed
with either full-length NHX5–CFP or the C-terminal deletion
NHX61–449–CFP. (D) Plant size was quantified by measuring rosette
diameter. Five independent lines of NHX61–449–CFP are shown.
autoactivation of multiple reporter genes; we therefore generated a truncated NHX6459–535 bait that did not autoactivate
(Fig. 6A). We screened approximately 2 106 library clones
and identified two interacting clones encoding SNX1.
Interestingly, both SNX1 clones were truncated, encoding
only the first 128 amino acids of the full-length SNX1 protein,
with both clones having a Q129STOP mutation; the resulting
SNX1 clones therefore only encompass the PX phosphatidylinositide-binding domain. A shorter NHX6 truncation,
NHX6459–507, did not interact with SNX1 (Fig. 6A), suggesting
that the NHX6–SNX1 interaction is mediated in part by the last
28 amino acids of the NHX6 protein. Co-localization analysis
supported this interaction; SNX1 has previously been described
to co-localize weakly with NHX6 in root hair cells (Bassil et al.
2011), and we found that NHX5–CFP appeared to co-localize
with SNX1–monomeric red fluorescent protein (mRFP) in wildtype root cells (Fig. 6B–D), with a high correlation (rp = 0.62;
Fig. 6E), and overlapping intensity (Fig. 6F, G). This co-localization was also stable during TGN movement/maturation,
as time-lapse confocal microscopy of live cells expressing
SNX1–mRFP and NHX5–CFP revealed the presence of colabeled structures during ‘vesicle budding’ (Fig. 6N–P;
Supplementary Movie S1).
The interaction between full-length NHX6 and SNX1 proteins was further confirmed in planta using the split bimolecular fluorescence complementation (BiFC) system (Fig. 7A;
Gehl et al. 2009). Two fusion constructs, 35S::NHX6–VenusN
and 35S::SNX1–VenusC, were expressed transiently by
Agrobacterium-mediated co-infiltration of tobacco (Nicotiana
benthamiana) leaves. The fluorescence of reconstituted Venus
was only detectable when both SNX1 and NHX6 were
co-expressed, confirming the direct interaction between
NHX6 and SNX1 in tobacco cells (Fig. 7A); a similar result
was obtained for NHX5–SNX1 interaction (Supplementary
Fig. S1E). NHX6–VenusN and NHX6–VenusC cotransformations served as negative controls as this combination
never produced any detectable fluorescence (n = 6 independent experiments; Fig. 7A). This indicates that there is no dimerization of NHX6 proteins.
To determine whether the interaction between NHX6 and
SNX1 is important for NHX or SNX1 localization, an SNX1–
mRFP fusion protein was stably expressed in an nhx5 nhx6
background. Similarly, we expressed NHX5–CFP in an snx1
mutant background. In the absence of the interacting partner,
neither NHX5 nor SNX1 misaccumulates at the vacuole or
plasma membrane (Fig. 7B–E). Taken together, the storage
protein phenotype, localization and interaction data may indicate the involvement of NHX6 in Retromer-mediated recycling
within the secretory pathway.
Discussion
To understand the role of NHX5 and NHX6 in trafficking to the
vacuole, the processing of SSPs and associated PSV formation
was investigated. Mature nhx5-2 nhx6-3 seeds contained PSVs
which were similar in size and number to those of the vsr1
mutant, although the defect in SSP processing was less severe
2227
J. R. Ashnest et al. | NHX antiporters are required for SSP processing
than that in vsr1 seeds. While this paper was under review,
Reguera et al. (2015) showed that seeds of a different allelic
combination, nhx5-1 nhx6-1, also exhibit PSVs which are smaller
and more numerous than in the wild type, and accumulate SSP
precursors. This accumulation was attributed to a defect in SSP
trafficking to the MVB, leading to an accumulation of mislocalized SSP precursors, as occurs in vsr1 seeds. Notably, both our
data and those of Reguera et al. (2015) suggest that only a
proportion of SSPs in nhx5 nhx6 are misprocessed. This is in
contrast to previous data from Bassil et al. (2011), which
showed that a transiently expressed exogenous carboxypeptidase (CPY) fragment fused to GFP was completely mislocalized
to the apoplast in nhx5 nhx6 mesophyll cells, with no GFP signal
observed in the vacuole. It may be that in nhx5 nhx6 mutants
the Golgi-independent ‘bulk flow’ of SSPs to the PSVs is still
occurring normally, while Golgi-dependent SSP traffic is
affected. Alternatively, the CPY trafficking route in leaf mesophyll cells is more sensitive to a loss of NHX5 and NHX6 activity
than those carrying the SSPs in seeds, particularly as mechanisms for trafficking to the lytic vacuole may be distinct from
those to the PSV. Interestingly, data from Reguera et al. (2015)
showed that some pS2 signal was found in the nhx5 nhx6 PSV,
which did not occur in the wild type, indicating that some SSPs
are not cleaved, despite being correctly localized. This raises a
third possibility-that the reduction in processing of the SSPs
may be an indirect result due to the reduction in activity of the
major cleavage enzymes, the VPEs.
The primary cleavage enzymes of the SSPs are the family of
VPEs, which are also trafficked to the PSV in a Golgi-dependent
manner, and are autocatalytically processed to their mature,
active forms en route (Kuroyanagi et al. 2002, Otegui et al.
2006). Protoplasts of nhx5 nhx6 knockout mutants have a
reduced pH in the MVB (pH = –0.4), where VPE cleavage of
SSPs occurs, and in the Golgi and TGN (pH = –0.25), where
VPE autocatalytic cleavage and activation is likely to take place
(Kuroyanagi et al. 2002, Otegui et al. 2006, Reguera et al. 2015).
As both VPE cleavage and VPE activity are pH-sensitive processes (Kuroyanagi et al. 2002, Misas-Villamil et al. 2013), alterations in the lumenal pH in nhx5 nhx6 knockout mutants may
inhibit VPE activity and processing. If this is the case, nhx5 nhx6
seed may exhibit alternative processing by aspartic proteases,
which have a lower optimal pH (3–4; D’Hondt et al. 1993). This
alternative processing has only been identified in the complete absence of VPE in the vpe quadruple mutant (Gruis
et al. 2004). Interestingly, the nhx5 nhx6 SSP phenotype is reminiscent of the storage protein phenotype of the bvpe single
mutant, in which the majority of SSP processing still occurs, but
there is an accumulation of storage protein precursors.
Fig. 6 Continued
Fig. 6 NHX6 interacts with SNX1. (A) Two-hybrid interaction between AtNHX6459–535 and SNX11–128. Triplicate co-transformants
were applied on double dropout media (SD–Trp/–Leu) and quadruple dropout media (SD–Ade/–His/–Leu/–Trp,+X-a-gal); genuine
interactions were confirmed by growth and galactosidase activity.
2228
(B–E) Co-localization of NHX5–CFP and SNX1–mRFP in A. thaliana root
cells. Strong co-localization was observed; rp = 0.62 (E; French et al. 2008).
Scale bars = 5 mm. (F–G) Corresponding intensity plot profile of lines from
(D). (H–P) Time-lapse imaging of NHX5–CFP and SNX1–mRFP shows colocalization through possible vesicle budding events (arrowheads). Scale
bars = 2 mm.
Plant Cell Physiol. 56(11): 2220–2233 (2015) doi:10.1093/pcp/pcv138
Fig. 7 Bimolecular fluorescent complementation association between
NHX6 and SNX1. (A) Expression of Venus as the result of the association of NHX6–VenusN and SNX1–VenusC in N. benthamiana epidermal cells; BF, bright field; Chl, Chl auto-fluorescence; the negative
interaction between NHX6–VenusN and NHX6–VenusC is used as a
negative control. Scale bars = 20 mm. (B, C) Subcellular localization of
SNX1–mRFP in stably transformed wild-type (B) and nhx5-2 nhx6-3
plants (C). (D, E) Subcellular localization of NHX5–CFP in stably transformed wild-type (D) and snx1-1 plants (E). Scale bars = 5 mm.
Alternatively, trafficking defects previously seen in nhx5 nhx6
cells (Bassil et al. 2011), probably also a result of altered pH, may
impact VPE delivery to the MVB, and thus processing and
activity.
Although previous experiments using light microscopy did
not report a change in PSV morphology in vpe null mutants
(Gruis et al. 2004), confocal microscopy showed that vpe null
embryos had smaller and more numerous PSVs than the wild
type, similar to those of nhx5 nhx6 and vsr1 seeds. Despite
intensive interest over many years, it remains unclear why
changes to the processing and trafficking of SSPs lead to a
defect in PSV morphology (Shimada et al. 2003a, Li et al.
2006, Shimada et al. 2006, Yamazaki et al. 2008, Pourcher
et al. 2010, Takahashi et al. 2010). However, correct SSP cleavage
may be necessary for optimal structure and high density filling,
which may be important for PSV formation.
The partial mislocalization of VPE to the apoplast in developing nhx5 nhx6 embryos suggests that, like the SSPs, vacuolar trafficking of VPE is disrupted in these cells, affecting VPE
delivery to the MVB/PSV and resulting in reduced SSP cleavage.
Although the mechanisms which deliver VPE to the vacuole are
unknown, trafficking via the secretory system is likely to be
regulated by sorting receptors, the activity of which may be
pH dependent. In our VPE in-gel activity assay, cells were homogenized before VPE activity was assessed, thus any difference in
the pH of the TGN or MVB compartments are unlikely to have
any effect on liberated VPE enzyme activity. However, the reduction in VPE activity observed may be due to inhibition of
pH-sensitive, autocatalytic processing of the inactive preVPE to
the active iVPE or mVPE forms. Our immunoblot data indicate
that although relative levels of mbVPE in wild-type and nhx5
nhx6 seeds are approximately equal, prebVPE levels in nhx5
nhx6 were approximately 1.5 times higher than those of the
wild type, while ibVPE levels were reduced by approximately
half. This suggests that the processing of immature VPE in
mature seeds is being inhibited in nhx5 nhx6 mutants, impacting the activity of VPE and leading to an inhibition of SSP processing. Furthermore, when VPE activity was calculated relative
to VPE protein levels, activity was still reduced in nhx5 nhx6
seeds. Together, these data indicate that both localization and
activity of VPE are altered in the nhx5 nhx6 mutant, and that
changes in VPE processing alone do not account for the reduction in VPE activity. However, both defects may be caused by
changes in lumenal pH (Reguera et al. 2015).
By analogy with NHX1 and SOS1, it is likely that the Cterminus of NHX6 mediates some protein–protein interactions
(Yamaguchi et al. 2005, Katiyar-Agarwal et al. 2006, Quintero
et al. 2011). The C-terminal tail of AtSOS1 is required for interaction with the protein kinase SOS2, leading to phosphorylation and activation of SOS1 (Quintero et al. 2011), and the
C-terminal region of SOS1 has also been shown to interact with
RCD1, a regulator of the oxidative stress response (KatiyarAgarwal et al. 2006). Likewise, the C-terminal tail of AtNHX1
is required for antiporter cation selectivity via its interaction
with calmodulin-like protein AtCaM15 in the vacuole
(Yamaguchi et al. 2005). In this study, we identified and investigated a conserved domain within the cytosolic NHX6
2229
J. R. Ashnest et al. | NHX antiporters are required for SSP processing
C-terminal tail. The localization pattern of the truncated form
of NHX6 lacking this tail was not different from that of the fulllength NHX6 protein, indicating that the C-terminal tail is not
required for NHX6 localization. Despite this, the truncated
NHX6 protein was not able to rescue the nhx5 nhx6 phenotype,
demonstrating that the C-terminal tail is essential for activity.
These data suggest that the C-terminal tail may be important in
protein–protein interactions with cytosolic components, and
may mediate the activation, regulation or function of NHX6.
We have identified that the cytosolic tail of NHX6 interacts
with SNX1, a component of Retromer and an important member
of the cell sorting machinery. SNX1 (with SNX2a and 2b) constitutes the peripheral component of plant Retromer which, in
conjunction with the core sorting proteins VPS26, VPS29 and
VPS35, is important for membrane receptor recycling, including
the family of VSRs (Oliviusson et al. 2006, Niemes et al. 2010). As
with the various vsr mutants (Shimada et al. 2003a, Zouhar et al.
2010, Li et al. 2013), T-DNA knockout of the various Retromer
components leads to partial mistrafficking of lytic- and PSVdestined soluble cargo, presumably as an indirect result of disrupted VSR recycling and thus inefficient sorting (Shimada et al.
2006, Yamazaki et al. 2008). We thus propose a mechanism by
which NHX6 might be involved in subcellular trafficking.
In addition to demonstrating an intimate interaction
between NHX6 and SNX1 in transient assays, we found that
NHX6 and SNX1 strongly co-localized in stably transformed
Arabidopsis root cortex cells (rp = 0.62), including during
TGN/vesicle budding or maturation. Previous evidence suggested that NHX6 weakly co-localizes with SNX1 in root hair
cells (Intensity Correlation Quotient (ICQ) = 0.24 ± 0.06; Bassil
et al. 2011). Recent data suggest that SNX1 localizes to the TGN
in planta (Niemes et al. 2010, Stierhof et al. 2013), not to the
MVB as previously described (Jaillais et al. 2008), which is consistent with the proposed localization of NHX6 at the Golgi/
TGN (Bassil et al. 2011). Interestingly, neither SNX1 nor NHX6
localization diverted to the plasma membrane or tonoplast
after loss of function of the interacting partner, indicating
that this interaction is not the only factor in determining
the localization of SNX1 and NHX6. Further investigation of
the interaction between NHX6 and other components of
Retromer may shed light on this process.
We have described that IC-II NHX proteins have a role in the
processing of the major SSPs and regulation of normal PSV
morphology, and interact with a component of Retromer.
Based on this evidence, two models can be proposed. The
first, based on the observation that NHX5 and NHX6 are
important for the processing and activity of the major SSP
cleavage enzyme bVPE, is that NHX antiporters maintain the
pH within the endomembrane system, such that bVPE is correctly processed and activated at the MVB. Altered lumenal pH
in organelles of nhx5 nhx6 cells has recently been described
(Reguera et al. 2015), and may be responsible for defects in
VPE processing and activity. A second model, based on the
interaction of NHX5 and NHX6 with SNX1, suggests that IC-II
NHXs may influence the function of Retromer, and the associated VSRs. In this model, loss of function of NHX5 and NHX6
would cause defects in VSR recycling, subsequently impacting
2230
SSP trafficking and SSP processing. This model is consistent
with previous evidence which showed that nhx5 nhx6 cells
mis-secrete the SSPs, similarly to vsr1 (Shimada et al. 2003a,
Reguera et al. 2015). NHX5 and NHX6 may impact the trafficking, activity and processing of a broad range of soluble cargos
which includes both the VPEs and SSPs. Investigation of the
suite of other endogenous proteins which are potentially mistrafficked in various nhx5 nhx6 tissues may clarify this.
Materials and Methods
Plant material and growth conditions
All Arabidopsis thaliana lines used in this study were in the Columbia background. Seeds of the previously described nhx5-2 mutant (GABI_094H09; Bassil
et al. 2011) were provided by GABI-KAT (http://www.gabi-kat.de). Seeds of the
nhx6-3 mutant (SALK_145125) were provided by the SALK Institute and obtained from the Arabidopsis Biological Resource Centre (ARBC) at Ohio State
University (http://www.arabidopsis.org). Genomic DNA was extracted, and
homozygous single mutants were identified by PCR genotyping of T4
plants. Single mutant lines were crossed, and homozygous F2 nhx5-2 nhx6-3
lines were identified by PCR genotyping (primer details are given in
Supplementary Table S1).
Arabidopsis thaliana seeds were sown on GM medium [1 Murashige–
Skoog (Sigma), 0.05% MES, 1% sucrose, 0.8% agarose], stratified at 4 C for 2 d,
then transferred to long-day conditions (16 h light, 8 h dark at 22 C). Seedlings
were transferred to soil (Debco seed raising mix : vermiculite, 3 : 1) 2–3 weeks
after germination. Tobacco (N. benthamiana) plants were grown in 200 mm
diameter pots containing soil mixture under long-day conditions for 4–6 weeks
before infiltration.
Seed protein analysis
Total protein was extracted from dry seeds in 2 Laemmli sample buffer and
separated by 12% SDS–PAGE. Immunoblotting was carried out on PVDF (polyvinylidene difluoride) membranes with primary antibodies against 12S globulin
(1/5,000), 2S albumin (1/3,000) or bVPE (1/5,000; Shimada et al. 2003b). Bands
of interest on Coomassie-stained gels were excised and subject to in-gel tryptic
digestion, and tryptic peptides were identified by MALDI-TOF-ESI (matrixassisted laser deionization-time of flight-electrospary ionization) and LC-MS/
MS liquid chromatograpy–tandem mass spectrometry) at the La Trobe
Institute for Molecular Sciences Mass Spectrometry and Proteomic Facility.
HPLC was carried out on an UltiMate3000 RSLC nano System, at a flow rate
of 0.3 ml min–1 with a gradient of 2–98% acetonitrile over 70 min. The digest was
desalted and concentrated on a Dionex Acclaim PepMap 100 nanotrap
(100 mm 2 cm, 18C 5 mm, 100 Å), and peptides were separated on a
DionexAclaimPepMax RSLC (75 mm 15 cm, C18 2 mm, 100 Å). Mass spectrometry was carried out by electrospray ionization on a BrukerMicroTofQ.
Data were subjected to a SwissProt database search of the Arabidopsis
genome (version 10) using MASCOT software (Matrix Science). Total protein
amounts were quantified from a pool of 10 seeds using the GE Healthcare life
sciences 2-D Quant Kit.
Total VPE activity was assayed using 1 mM JOPD1 as described previously
(Lu et al. 2015). Labeled proteins were visualized in-gel using a Bio-Rad
TM
ChemiDoc MP Imaging System with 605/650 nm green epi-illumination. All
densitometry was conducted using ImageJ. In situ VPE activity was performed
on late cotyledon stage embryos, 12 d post-anthesis. Embryos were removed
from seed coats then incubated in 2 mM JOPD1 as described (Misas-Villamil
et al. 2013). Cells were imaged using a Zeiss LMS 510 confocal microscope with
BODIPY filters (excitation/emission 514 nm/long pass 530 nm).
Expression analysis
Publically available microarray data were sourced from the Arabidopsis eFP
browser (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi; Winter et al. 2007).
Plant Cell Physiol. 56(11): 2220–2233 (2015) doi:10.1093/pcp/pcv138
For promoter activity, an approximately 3 kb region immediately upstream
of the NHX6 start codon was cloned into pKGWFS7 (primers described in
Õ
Supplementary Table S1) using Gateway technology and transformed into
wild-type Arabidopsis. Tissues were collected, stained and cleared as previously
described (Stangeland and Salehian 2002), and visualized using a Leica MF304
(Leica Microsystems).
Protein structure prediction
Consensus prediction of a-helical transmembrane regions was performed using
TOPCONS (http://topcons.cbr.su.se/) (Hennerdal and Elofsson 2011). The relative conservation at each amino acid position was determined using CONSURF
(Ashkenazy et al. 2010). The putative transmembrane regions of AtNHX5 and
AtNXH6 were then aligned to the sequences used in the resolution of a 7 Å
MjNhaP crystal (Goswami et al. 2011).
Yeast two-hybrid screening
Screening was conducted using a GAL4-based system (Matchmaker Gold Yeast
Two-Hybrid System, Clontech) according to the manufacturer’s instructions.
Three truncations of the C-terminal region of NHX6 were generated and used
as bait in yeast two-hybid screening: NHX6433–507, NHX6459–535 and
NHX6459–507 (Supplementary Table S1). Positive interactions were identified
on restrictive SD medium (–Trp/–Leu) supplemented with 20 mg ml–1 X-a-gal
(5-bromo-4-chloro-3-indolyl-a-D-galactopyranoside) and 125 ng ml–1 aureobasidin A. Genuine interactions were confirmed on quadruple dropout medium
(SD/–Ade/–His/–Leu/–Trp), supplemented with 20 mg ml–1 X-a-gal and 150 ng
ml–1 aureobasidin A.
Funding
This work was supported by the Grains Research and
Development Corporation, Australia [through Graduate
Research Scholarships to J.A. (GRS 179) and B.F. (GRS 161)];
the Government of Vietnam [through a MOET AgriBiotechnology scholarship Scheme to H.L.D.]; the Australian
Research Council [Linkage Infrastructure, Equipment and
Facilities (LIEF) grant LE0989920].
Acknowledgements
We thank Ikuko Hara-Nishimura (Kyoto University, Japan) for
a-2S albumin, a-12S globulin and a-bVPE antibodies and vsr1
mutant seeds, Josh Mylne (University of Western Australia) for
the quadruple vpe mutant seed, Thierry Gaude (CNRS and
École Normale Supérieure de Lyon, France) for SNX1–mRFP
and snx1-1 seed, and Renier van der Hoorn (University of
Oxford, UK) for the JOPD1 VPE activity probe. We also thank
Gert Talbo and Pierre Faou (La Trobe Institute of Molecular
Science, Australia) for assistance with mass spectrometry and
proteomics, and Peter Lock (La Trobe Institute of Molecular
Science, Australia) for assistance with confocal microscopy.
Microscopy
PSV autofluorescence was imaged in mature embryos. Seeds were imbibed
overnight to soften the seed coat, then pressed between a glass slide and
cover slip to release the embryo. Images of the hypocotyl were collected
using a Leica TPS SP2 confocal microscope (Leica Microsystems) with GFP filters
(excitation/emission 488/505–53 nm). PSV quantification was carried out using
ImageJ.
Localization analysis was performed on T3 seedlings stably expressing the
NHX5–CFP or NHX61–449–CFP fusion proteins. Roots of 7- to 10-day-old seedlings
were incubated in half-strenth Murashige and Skoog medium (1/2 MS)containing
50 mM BFA for 2 h, or 3.5 mM FM5-95 (Invitrogen) for 2 min at room temperature,
and rinsed three times in 1/2 MS before observation; at least three plants were
examined for each experiment. Microscopic analysis was performed using a Zeiss
LSM 510 confocal laser scanning microscope (Carl Zeiss), with a40/1.2 water
immersion objective. Excitation/emission wavelengths were 458/470–495 nm for
CFP, 488/505–530 nm for GFP and 561/650 nm long pass for mRFP or FM5-95.
Post-processing of images was performed with Zeiss ZEN Black (v8.0) and ImageJ.
Co-localization and time-lapse imaging of NHX5–CFP and SNX1–mRFP were
acquired using line by line sequential scanning with high sensitivity avalanche
photodiode detectors. The PSC co-localization plug-in (French et al. 2008) in
ImageJ was used to calculate the Pearson correlation coefficient (rp) with a
noise threshold of 10 pixels.
For BiFC (Gehl et al. 2009), fusion proteins 35S:NHX6–VenusN and
35S:SNX1–VenusC were constructed with the open reading frames of NHX6
and SNX1 (At5g06140) amplified from cDNA of A. thaliana. VenusN–CNX6 and
VenusC-pEXP-VYCE–CNX5 were used as the interaction control. BiFC
assays were performed transiently using Agrobacterium-mediated co-infiltration of 4- to 6-week-old N. benthamiana leaves and Agrobacterium strain
GV3103. Interactions in lower epidermal cells were visualized 2–5 d after infiltration, on a Leica TCS SP2 system (Leica Microsystems using the 40/1.2 water
immersion objectives. Excitation/emission of 514 nm/525–600 nm (Venus
fluorescence; Gehl et al. 2009) or 633/650–750 nm (Chl autofluorescence)
was used for detection.
Supplementary data
Supplementary data are available at PCP online.
Disclosures
The authors have no conflicts of interest to declare.
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