pH regulation by NHX-type antiporters is required for receptormediated protein trafficking to the vacuole
Reguera M.*, Bassil E., Tajima H., Wimmer M., Chanoca A., Otegui M., Paris N. and Blumwald E.
Department of Plant Sciences, University of California, Davis, CA 95616. *[email protected]
Introduction
The Na+/H+ (NHX-type) antiporters NHX5 and NHX6 are endosomal members of the intracellular NHX family which play essential roles in pH and ion homeostasis and
localize to the Golgi and trans-Golgi network (Bassil et al. 2011). The nhx5nhx6 double knockout exhibited significantly reduced cell expansion and growth, severe
sensitivity to salt and displayed defects in trafficking to the vacuole (Bassil et al. 2011). We hypothesize that NHX5 and NHX6 control endosomal ion and pH
homeostasis regulating vesicle trafficking. In an effort to further understand how ion and pH homeostasis affect trafficking of cellular cargo, we investigate further the
effects of NHX5 and NHX6 on protein processing and trafficking to storage vacuoles in Arabidopsis seeds.
Results and discussion
Results and discussion
Protein processing is altered in the nhx5nhx6 knockout
nhx5nhx6
R
Q
We used a ratiometric, genetically encoded pHluorin-based pH sensor in a
translational fusion with VSR2;1 (pH-VSR2;1) in which pHluorin was placed in the
luminal domain of VSR2;1 (Q and R) (Martinière et al., 2013). The luminal pH of
VSR2;1 nhx5nhx6 compartments containing aleurain is significantly more acidic
(pH 6.10 ± 0.12, n>150 vesicles from at least 20 protoplasts) than comparable
WT VSR-aleurain compartments (pH 6.70 ± 0.09; n>150 vesicles from at least 20
protoplasts) (S). These results indicate that the luminal pH of VSR2;1 compartments
containing aleurain is aberrantly acidic in nhx5nhx6.
S
Seed storage protein profile at different developmental stages. A, Total protein (Coomassie gel) of
immature, dry, germinating and 6 days old seedlings (left to right) suggest that there is an accumulation
of storage proteins in the nhx5nhx6 double knockout (F37) seeds. B and C, Immunoblots of protein
extracts using 12S globulin and 2S albumin antibodies confirm that during seed development, an
accumulation of non-processed forms of both 12S globulin and 2S albumin occur. These results suggest
that NHX5 and NHX6 are required for storage protein processing in seeds.
Missorting of storage proteins in the nhx5nhx6 knockout
nhx5 nhx6
Transmission electron micrographs of dry seeds
(D-G) indicate the accumulation of electron-dense
material in the extracellular space of nhx5nhx6
(G). Immunogold labeling (10 nm gold particle)
localize 12S globulin (D-G) and 2S albumin (data
not shown) in the apoplast of nhx5nhx6
(indicated with red dots, G).
Wild Type
Anti-12S globulin (anti-12S)
PSV
CW
D
E
Intracellular alkalinization improves the in vivo interaction of VSR with aleurain
BiFC
in nhx5nhx6
X
SCYCE-VSR2;1
We incubated WT protoplasts with a buffer at pH=5 (T and U).
WT protoplast at pH=5 do not differ in the number of BiFC
bodies nor their signal intensity (T) as well as in the number and
intensity of VSR1-mRFP bodies (U) compare to I and J,
respectively. On the contrary, nhx5nhx6 protoplasts are
alkalinized with pH=7 buffer and the number of BiFC bodies
increase by 60% (V) with no changes in the number or intensity
of VSR1-mRFP bodies (W) compare to M and N (V, W, X and Y).
(Shaded bars in X and Y are the control values from
unequlibrated WT and nhx5nhx6 protoplasts for comparison).
PSV
CW
Z
H
In vivo interaction between the VSR
receptor and its cargo protein aleurain.
Interaction is visualized as bimolecular
fluorescence complementation (BiFC)
between aleurain (Aleu-VYNE) and
VSR2;1 (SCYCE-VSR2;1) in isolated
mesophyll WT and nhx5nhx6 protoplasts
(I and M, respectively).
A positive transformation control, expressing a VSR1-mRFP that properly localized but did not
competitively bind to its cargo was used as control (J and N). In nhx5nhx6 the number and intensity of
green fluorescent organelles are significantly reduced. The number of VSR1-mRFP bodies is not
significantly different between nhx5nhx6 and WT protoplasts
U
V
W
Y
In order to test whether the reduced BiFC signal is due to fluorescence quenching at acidic pH, we
generated a chimeric protein between SCYCE and VYNE (called chimera). Fluorescence intensity
of the SCYCE-VYNE chimeric protein in acidified protoplasts does not differ appreciably from the
fluorescence of unequlibrated protoplasts (Z). Unlike the chimeric SCYCE-VYNE protein,
acidification of PIP2;1-GFP expressing protoplasts results in a marked reduction in PIP2;1-GFP
relative fluorescence.
nhx5nhx6
Identification of receptor-cargo VSR-p12S complexes
using Blue Native gel electrophoresis followed by
immunobloting suggest that receptor cargo interaction
might be compromised in plants lacking NHX5 and
NHX6 antiporters (H). Proteins complexes appear
vertically (indicated by red lines).
T
Quantitative comparison of pH dependent fluorescence in protoplasts
F
G
Vacuolar sorting receptor (VSR)-cargo interaction is compromised in
the nhx5nhx6 knockout
WT
VSR1-mRFP
Aleu-VYNE
WT
A
WT
C
nhx5 nhx6
B
Luminal pH of VSR compartments is more acidic in the nhx5nhx6 mutant
Conclusions
Our results indicate that the endosomal antiporters NHX5 and NHX6 are required for the
efficient processing and sorting of storage proteins to PSVs, both essential processes that
ensure normal seed development and subsequent germination. Luminal pH of compartments
containing the VSR receptor and its cargo, depend on both antiporters to maintain pH (and
ion) homeostasis for the association (or dissociation) of the receptor and cargo. The roles of
pH regulation in vesicular trafficking are not adequately understood and these knockouts
offer the possibility to study this critical process further.
References
Bassil et al. 2011, Plant Cell 23(1):224-39. Martinière et al. 2013, Plant Cell 25(10):4028-43.
Acknowledgments
We are grateful to Professor Ikuko-Hara Nishimura for providing VSR, 2S albumin and 12S globulin antibodies.