Am J Physiol Cell Physiol 311: C544 –C546, 2016;
doi:10.1152/ajpcell.00214.2016.
Editorial Focus
Nuclear V-type ATPase. Focus on “Vacuolar H⫹-ATPase in the nuclear
membranes regulates nucleo-cytosolic proton gradients”
John Cuppoletti
Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio
Address for reprint requests and other correspondence: J. Cuppoletti, Dept.
of Molecular & Cellular Physiology, Univ. of Cincinnati, Cincinnati, OH
45267-0576 (e-mail: [email protected]).
C544
as a percentage for each cell type, the majority of each cell
type showed no ⌬pH, but a larger percentage of CL-2 cells
than RWPE-1 cells had inward H⫹ gradients. The differences in ⌬pH among the cell types and between individual
cells suggest that there are unknown mechanisms for regulation of the pH of the nucleus. The heterogeneity of nuclear
pH among cells in the same cultures suggests that ⌬pH (and
⌬⌿) regulation of the nucleus is subject to molecular and
biochemical signaling.
The authors showed that the inward and outward pH gradients were sensitive to bafilomycin in all cell types in aggregate
and on a percentage basis, suggesting that V-type ATPase is
responsible for gradient formation. Similar findings were observed using concanamycin in cancer cells.
The authors used antibodies to the a1 and c subunit of V-type
ATPase and showed that it was found at the nucleus by
immunocytochemistry. Immunoblots of the V0 subunit c in
ONM with nesprin 3 as a marker and INM with lamin B as a
marker demonstrated the presence of the V-type ATPase in
both the INM and ONM membranes. Bafilomycin-sensitive
ATPase activities were higher in the ONM, consistent with
acidification of the nucleus, and the ATPase activity was
highest in both INM and ONM of CL-2.
It has been shown by subtractive proteomics that numerous
transporters, pumps, and channels exist in the nuclear envelope
(2, 9). Although only a few functional studies have appeared on
nuclear envelope transporters including patch clamp of K⫹,
Cl⫺, and Ca2⫹ channels (5), it is known that K⫹ and Na⫹
gradients across the nucleus are regulated by the Na⫹-K⫹ATPase (4), and there is evidence for regulation of nuclear
Ca2⫹ (1).
⌬pH and ⌬⌿ generated by the V-type ATPase will provide
the driving force for many of the newly identified NE transporters and channels identified in proteomic studies (2, 9). ⌬pH
and ⌬⌿ will also affect proteins and nucleic acids in the
nucleus. In addition, it is likely that the V-type ATPase will be
found to be involved in numerous other processes (6).
This is the first study to demonstrate that the V-type
ATPase subunits are in the NE, an important finding on its
own. The authors have been the first to show that the
nucleus of a subset of cells in any given cell line studied
generated a bafilomycin-sensitive inward H⫹ gradient. In all
cell lines an outward H⫹ gradient was also present in a
subset of cells. The majority of the cells in each cell line
generated no H⫹ gradient. In aggregate, all of the cell lines
studied generated an inwardly directed ⌬pH. The basis for
the heterogeneity of pH gradients has not been explained. It
would be important to know if and how the cells transit
between inwardly directed, outwardly directed, and no ⌬pH
across the NE. Antibodies to V0 c subunit and a1 subunits
were employed for immunocytochemistry, and antibodies to
V0 c subunit were used for immunoblots. The literature did
not provide a clear choice of markers for a nuclear V-type
0363-6143/16 Copyright © 2016 the American Physiological Society
http://www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on June 17, 2017
(NE) consists of two continuous membranes, with pores where the inner nuclear membrane (INM)
meets the outer nuclear membrane (ONM) (7). V-type
ATPases in the plasma membrane and intracellular membranes
alter pH (⌬pH) and generate a transmembrane electrical potential (⌬⌿) across plasma membranes and intracellular compartments (6).
In the accompanying article by Santos et al. (8) published in
this issue of the American Journal of Physiology-Cell Physiology, the authors tested the hypothesis that the V-type ATPase
was present and functional in the NE. The authors established
that the nuclear pH is acidic, pH gradients across the nuclear
membrane were bafilomycin and concanamycin sensitive and
that the V-type ATPase was present in the NE by immunolocalization, and in both the ONM and INM by Western blots
and ATPase assays.
The flow of ions across the nuclear membranes is restricted
(3). Prior studies to determine whether there was an electrochemical gradient of protons across the nuclear membrane
were contradictory and no previous studies examined whether
V-type ATPases might be involved. The authors used the
ratiometric dye SNARF-1 to determine nuclear-cytoplasmic
pH gradients. They examined RWPE-1 cells, a prostate epithelial cell line, LNCaP cells, a prostate cancer cell line, and
highly tumorigenic and invasive CL-2 cells derived from
LNCaP cells. They found that the steady-state pH of the
nucleus was more acidic than the cytosol in RWPE-1 cells and
LNCaP cells, but there was no difference in the steady-state pH
between the nucleus and cytosol in CL-2 cells. The authors
attribute the differences in their findings from those of others
who also used ratiometric approaches to the failure of others to
carry out in situ calibration for each cell type and each cell. In
the present study, the authors used individual in situ calibration
procedures for nucleus and cytosol, respectively, in contrast to
other studies, which used either a single calibration curve
obtained in vitro or in situ or the average of in situ calibration
curves for nucleus and cytosol. The authors could have made a
stronger case for their attribution of their differences from the
studies of others by documenting how the different approach
would change the measurements and by documenting that an
average calibration could recapitulate the contradictory finding
of others using SNARF-1.
They found that absolute pH values varied in the various
compartments among cells, so all values were also reported as
⌬pH. No differences were seen in the outward gradients
between RWPE-1, LNCaP, and CL-2 cells in aggregate. However, there were larger inward H⫹ gradients in CL-2 cells than
RWPE-1 or LNCaP cells, in aggregate. When ⌬pH was given
THE NUCLEAR ENVELOPE
Editorial Focus
C545
Nuclear Pore Complex
Nuclear Pore Complex
Cytoplasm
ONM
Antiport
A
Symport
B A
Ion
Channels
Pumps
Carriers
Uniport
A
Active
Transport
ADP + Pi
S1
S2
ATP
B
Side 1
Side 2
Transmembrane Electrochemical Gradients
ΔNa+, ΔK+,ΔpH, ΔCl–, ΔCa2+ and ΔΨ
Perinuclear Space
Uniport
A
Symport
A B
Antiport
A
Active
Transport
S1
ADP + Pi
S2
ATP
B
Side 1
Side 2
Transmembrane Electrochemical Gradients
ΔNa+, ΔK+,ΔpH, ΔCl–, ΔCa2+ and ΔΨ
Nucleoplasm with DNA, RNA, Protein, enzymes and
signaling systems all affected by ions and ΔΨ
INM
Perinuclear Space
Transmembrane Electrochemical Gradients
ΔNa+, ΔK+,ΔpH, ΔCl–, ΔCa2+ and ΔΨ
ONM
Cytoplasm
Fig. 1. Diagram of electrochemical gradients generated by pumps and used by carriers and ion channels of the nuclear envelope (NE). A section of the NE is
shown bounded and traversed by nuclear pore complex proteins, which may either be in the open or closed states. Na⫹-K⫹-ATPase, Ca2⫹-ATPase, and V-type
ATPase generate transmembrane gradients for Na⫹, K⫹, Ca2⫹, H⫹, Cl⫺, and electrical gradients (⌬⌿). These electrochemical gradients provide the driving force
for solute transport by carriers and ion transport by ion channels. Ion concentrations and ⌬⌿ may also play a role in regulation of the structures and functions
of the nucleus. The featured article (8) shows that the NE contains a functional V-type ATPase for generation of pH gradients and ⌬⌿. Evidence for other nuclear
pumps, carriers, and ion channels and their regulation is outlined in refs. 2, 4, and 9.
ATPase subunit in any cell type, and future studies will be
required to characterize the isoforms in membranes of
prostate epithelial and cancer cells.
Considering the key role of the highly regulated (10) V-type
ATPase in generation of ⌬pH and ⌬⌿ and its many other roles
(6), these findings will lead to greater understanding of the
regulation of nuclear physiology.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author.
AUTHOR CONTRIBUTIONS
J.C. prepared figure; drafted manuscript; edited and revised manuscript;
approved final version of manuscript.
REFERENCES
1. Badminton MN, Kendall JM, Rembold CM, Campbell AK. Current
evidence suggests independent regulation of nuclear calcium. Cell Calcium 23: 79 –86, 1998.
2. Batrakou DG, Kerr AR, Schirmer EC. Comparative proteomic analyses
of the nuclear envelope and pore complex suggests a wide range of
heretofore unexpected functions. J Proteomics 72: 56 –70, 2009.
AJP-Cell Physiol • doi:10.1152/ajpcell.00214.2016 • www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on June 17, 2017
INM
Ion
Channels
Pumps
Carriers
Editorial Focus
C546
3. Bustamante JO, Liepins A, Hanover JA. Nuclear pore complex ion
channels (review). Mol Membr Biol 11: 141–150, 1994.
4. Garner MH. Na,K-ATPase in the nuclear envelope regulates Na⫹: K⫹
gradients in hepatocyte nuclei. J Membr Biol 187: 97–115,
2002.
5. Matzke AJ, Weiger TM, Matzke M. Ion channels at the nucleus:
electrophysiology meets the genome. Mol Plant 3: 642–652,
2010.
6. Maxson ME, Grinstein S. The vacuolar-type H⫹-ATPase at a glance - more than a proton pump. J Cell Sci 127: 4987–4993,
2014.
7. Pappas GD. The fine structure of the nuclear envelope of Amoeba
proteus. J Biophys Biochem Cytol 2, 4 Suppl: 431–434, 1956.
8. Santos JM, Martinez- Zaguilán R, Façanha AR, Hussain F, Sennoune
SR. Vacular H⫹-ATPase in the nuclear membranes regulates nucleocytosolic proton gradients. Am J Physiol Cell Physiol. doi:10.1152/ajpcell.
00019.2016.
9. Schirmer EC, Florens L, Guan T, Yates JR 3rd, Gerace L. Nuclear
membrane proteins with potential disease links found by subtractive
proteomics. Science 301: 1380 –1382, 2003.
10. Toei M, Saum R, Forgac M. Regulation and isoform function of the
V-ATPases. Biochemistry 49: 4715–4723, 2010.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on June 17, 2017
AJP-Cell Physiol • doi:10.1152/ajpcell.00214.2016 • www.ajpcell.org