VAPB interacts with the mitochondrial protein
PTPIP51 to regulate calcium homeostasis
Kurt J. De Vos1,2,∗, Ga ´bor M. Mo ´rotz1, Radu Stoica1, Elizabeth L. Tudor1, Kwok-Fai Lau1,3,
Steven Ackerley1, Alice Warley4, Christopher E. Shaw2and Christopher C.J. Miller1,2,∗
1Department of Neuroscience and2Department of Clinical Neurosciences, MRC Centre for Neurodegeneration
Research, Institute of Psychiatry, King’s College London, London SE5 8AF, UK,3Department of Biochemistry,
The Chinese University of Hong Kong, Shatin, NT, Hong Kong and4Centre for Ultrastructural Imaging,
King’s College London, London SE1 1UL, UK
Received August 26, 2011; Revised November 1, 2011; Accepted November 22, 2011
A proline to serine substitution at position 56 in the gene encoding vesicle-associated membrane protein-
associated protein B (VAPB) causes some dominantly inherited familial forms of motor neuron disease
including amyotrophic lateral sclerosis (ALS) type-8. VAPB is an integral endoplasmic reticulum (ER) protein
whose amino-terminus projects into the cytosol. Overexpression of ALS mutant VAPBP56S disrupts ER
structure but the mechanisms by which it induces disease are not properly understood. Here we show
that VAPB interacts with the outer mitochondrial membrane protein, protein tyrosine phosphatase-interact-
ing protein 51 (PTPIP51). ER and mitochondria are both stores for intracellular calcium (Ca21) and Ca21
exchange between these organelles occurs at regions of ER that are closely apposed to mitochondria.
These are termed mitochondria-associated membranes (MAM). We demonstrate that VAPB is a MAM protein
and that loss of either VAPB or PTPIP51 perturbs uptake of Ca21by mitochondria following release from ER
stores. Finally, we demonstrate that VAPBP56S has altered binding to PTPIP51 and increases Ca21uptake by
mitochondria following release from ER stores. Damage to ER, mitochondria and Ca21homeostasis are all
seen in ALS and we discuss the implications of our findings in this context.
Amyotrophic lateral sclerosis (ALS) is an adult onset neurode-
generative disease characterized by selective loss of motor
neurons in the spinal cord, motor cortex and brain stem,
which leads to progressive muscle atrophy and ultimately par-
alysis and death, typically within 3–5 years of onset. Most
forms of ALS are sporadic but approximately 5% are inherited
and mutations in a number of genes have now been shown to
be causative for these familial forms (1,2).
A mutation in the gene encoding vesicle-associated mem-
brane protein-associated protein B (VAPB) causes ALS
type-8 and some other related forms of motor neuron disease
including late onset spinal muscular atrophy (3). VAPB is
an integral endoplasmic reticulum (ER) membrane protein. It
contains an N-terminal domain homologous to the major
sperm protein of nematode worms, a central coiled-coil
region and a C-terminal transmembrane domain through
which it is anchored in the ER membrane; the N-terminus of
VAPB projects from the ER into the cytoplasm (4–9). The
mutation that causes ALS type-8 involves a proline to serine
substitution at position-56 (VAPBP56S) although a further
mutation (VAPBT46I) has recently been identified in a
single ALS patient and this too may cause ALS (3,10).
VAPBP56S induces the formation of abnormal ER-derived
inclusions (3,8,9,11–13) but the mechanisms by which
VAPBP56S induces disease are not clear and this is partly
because the function of VAPB is not properly understood.
∗To whom correspondence should be addressed at: MRC Centre for Neurodegeneration Research, Department of Neuroscience PO37, The Institute of
Psychiatry, King’s College London, De Crespigny Park, Denmark Hill, London SE5 8AF, UK. Tel: +44 2078480393; Fax: +44 2077080017;
Email: firstname.lastname@example.org (C.C.J.M.); MRC Centre for Neurodegeneration Research, Department of Neuroscience PO37, The Institute of
Psychiatry, King’s College London, De Crespigny Park, Denmark Hill, London SE5 8AF, UK; Email: email@example.com (K.J.D.V.)
# The Author 2011. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/
licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is
Human Molecular Genetics, 2012, Vol. 21, No. 6
Advance Access published on November 30, 2011
VAPB has been implicated in a variety of processes including
ER stress and the unfolded protein response (UPR), ER to
Golgi transport and bouton formation at the neuromuscular junc-
tion (6,14–19). There is also evidence that implicates VAPB in
microtubule organization (7,14,20) and finally, a cleaved and
secreted VAPB fragment acts as a ligand for ephrin receptors
(21). ER stress is linked to the pathogenesis of ALS (22,23)
and several studies implicate VAPBP56S in abnormal UPR but
again the mechanisms are unclear (6,10,11,18,19,21).
Here, we report that VAPB interacts with the outer mito-
chondrial membrane protein, protein tyrosine phosphatase-
interacting protein 51 (PTPIP51). ER and mitochondria are
both stores for calcium (Ca2+) and we demonstrate that the
VAPB–PTPIP51 interaction impacts on intracellular Ca2+
handling and that VAPBP56S has altered properties in this
function. Damage to Ca2+homeostasis is seen in ALS
(24,25) and as such, our results provide a novel function for
VAPB that has relevance to ALS.
VAPB interacts with PTPIP51
We screened a human brain cDNA yeast two-hybrid library
with the cytoplasmic domain of VAPB as ‘bait’ [i.e. VAPB
lacking its carboxy-terminal transmembrane domain; amino
acids (aa) 1–220]. We isolated four interacting clones
that all encoded partial PTPIP51 sequences [aa36–470,
aa84–470 (2 clones), aa75–175] (Fig. 1A). PTPIP51 is also
known as family with sequence similarity 82 member A2
(FAM82A2), human cerebral protein-10 and regulator of
microtubule dynamics protein-3 (RMD-3). PTPIP51 binds
protein tyrosine phosphatase-1B and T-cell protein tyrosine
phosphatase in yeast two-hybrid (26) and proximity ligation
assays (27), but the functional significance of these interac-
tions is unknown. RMD-3 is a putative homologue of
PTPIP51 in Caenorhabditis elegans and another RMD
family member, RMD-1 functions in chromosome segregation
(28). However, in mammalian cells, PTPIP51 is a mitochon-
drial protein of unclear function that has been implicated in
the regulation of cell morphology, motility and apoptosis
domain and a central coiled-coil domain (29,30) (Fig. 1A).
Both VAPB and PTPIP51 are ubiquitously expressed although
their expression levels vary in different tissues (7,26,29).
To confirm the interaction between VAPB and PTPIP51, we
first performed co-immunoprecipitation assays of transfected
and endogenous proteins in HEK293 cells. Hemagglutinin
(HA)-tagged PTPIP51 (HA-PTPIP51) co-immunoprecipitated
with myc-tagged VAPB (myc-VAPB) from HA-PTPIP51
and myc-VAPB co-transfected cells but not HA-PTPIP51-only
transfected cells and this interaction was not detected when the
immunoprecipitating myc antibody was replaced with non-
immune mouse antibody (Fig. 1B). Likewise, endogenous
VAPB co-immunoprecipitated with endogenous PTPIP51 but
this interaction was abrogated when pre-immune serum was
used instead of immunoprecipitating PTPIP51 antibody
(Fig. 1C). Both PTPIP51 and VAPB antibodies detected
protein species of the correct molecular masses (Supplemen-
tary Material, Fig. S1).
We also monitored the endogenous VAPB and PTPIP51
interaction in cells using an in vivo, in situ proximity ligation
assay (31). Here, conventionally fixed and permeabilized cul-
tured CV1 cells were probed with anti-VAPB and anti-
PTPIP51 primary antibodies. However, instead of fluores-
cently labelled secondary antibodies, antibodies coupled to
different oligonucleotides (one for each of the two primary
antibodies) were used as secondary antibodies. If the distance
between two antibody-coupled oligonucleotides of different
types is less than 50 nm, they can hybridize and serve as
primers for rolling-circle amplification with fluorescent
Figure 1. VAPB interacts with PTPIP51. (A) Domain structure of PTPIP51.
CC, coiled coil domain; TM, transmembrane domain. The VAPB-interacting
PTPIP51 clonesidentified byyeast
Myc-VAPB and HA-PTPIP51 co-immunoprecipitate in transfected HEK293
cells. Myc-VAPB was immunoprecipitated with anti-myc antibody from
cells transfected with myc-VAPB and/or HA-PTPIP51. Non-immune mouse
antibody was used as control (Ctrl). The immune pellets were probed for
myc-VAPB (Myc) and HA-PTPIP51 (HA) on immunoblots. The input
levels of myc-VAPB and HA-PTPIP51 in the transfected cells are shown
(input). (C) Endogenous PTPIP51 and VAPB co-immunoprecipitate in
HEK293 cells. PTPIP51 was immunoprecipitated using rat anti-PTPIP51 anti-
body (PTPIP51 IP) and the immune pellet probed for VAPB and PTPIP51 on
immunoblots with rabbit VAPB (#3504) and PTPIP51 (FAM82A2) antibodies,
respectively; immunoprecipitation with pre-immune rat serum was used as a
control (Ctrl IP). A sample of the input lysate is also shown. (D) Quantification
of in situ proximity ligation assay results (mean+SEM). Cells were probed
with no primary antibodies (no Abs) (n ¼ 10), with VAPB antibody only
(VAPB) (n ¼ 10), with PTPIP51 antibody only (PTPIP51) (n ¼ 10) and
with VAPB+PTPIP51 antibodies (n ¼ 32) and the numbers of signals/cell
determined. Representative VAPB+PTPIP51 labelling with corresponding
phase contrast image are shown (scale bar, 20 mm).
two-hybrid are indicated.(B)
1300Human Molecular Genetics, 2012, Vol. 21, No. 6
oligonucleotides. The resulting fluorescent signals can then be
imaged using by fluorescence microscopy and correspond
to interacting protein pairs. Using VAPB and PTPIP51 anti-
bodies, we observed proximity signals in all cells examined
(Fig. 1D), confirming the VAPB–PTPIP51 interaction in
intact cells. As negative controls, we omitted either or both
of the primary antibodies from the assays and these produced
only very low numbers of proximity signals. Together these
data identify PTPIP51 as a novel VAPB binding protein.
PTPIP51 is an amino-terminal anchored outer
mitochondrial membrane protein
Although PTPIP51 is a mitochondrial protein (29), its precise
sub-mitochondrial localization and membrane orientation are
not known. Our discovery that PTPIP51 interacts with VAPB,
an ER protein whose amino-terminus project into the cytosol,
suggests that PTPIP51 is an outer mitochondrial membrane
protein whose carboxy-terminus projects into the cytosol. To
begin to test this possibility, we first confirmed the mitochon-
drial localization of PTPIP51 and the role of its amino-terminal
transmembrane domain. Endogenous PTPIP51 co-localized
with the known outer mitochondrial membrane protein translo-
case of outer membrane-20 (TOM20) (Fig. 2A). Likewise,
transfected HA-PTPIP51 also localized to mitochondria
(Fig. 2B). However, HA-PTPIP51 lacking its amino-terminal
transmembrane domain (HA-PTPIP51DTM) displayed diffuse
labelling with no clear mitochondrial localization whereas an
amino-terminal PTPIP51 domain containing the transmem-
to mitochondria (Fig. 2B). Thus, sequences encompassing
the amino-terminal transmembrane domain are responsible for
Figure 2. PTPIP51 is an integral OMM protein. (A) PTPIP51 localizes to mitochondria. Endogenous PTPIP51 and the mitochondrial protein TOM20 were visua-
lized in CV1 cells by immunofluorescence microscopy. Scale bar, 20 mm. (B) Sequences spanning the amino-terminal transmembrane domain of PTPIP5 are
involved in targeting PTPIP51 to mitochondria. CV1 cells were co-transfected with either full-length HA-PTPIP51 and AcGFP-Mito (a), or HA-PTPIP51 lacking
its amino-terminal transmembrane domain (HA-PTPIP51△TM) and AcGFP-Mito (b), or the amino-terminal 72 amino acids of PTPIP51 (which contain the
transmembrane domain) fused to EGFP (EGFP-PTPIP51TM) (c). In (a) and (b), HA-PTPIP51 and HA-PTPIP51△TM were detected by immunostaining
with anti-HA antibody and mitochondria with AcGFP-mito. In (c), PTPIP51TM was detected with the EGFP tag and mitochondria by immunostaining with
antibody to MnSOD. Full-length HA-PTPIP51 and EGFP-PTPIP51TM both localized to mitochondria whereas HA-PTPIP51△TM labelling was cytosolic.
Scale bar, 20 mm. (C–E) PTPIP51 is an outer mitochondrial membrane protein. (C) Outer mitochondrial membrane (OMM) and inner mitochondrial membrane
(IMM) plus mitochondrial matrix fractions (matrix/IMM) were prepared and probed for PTPIP51, the OMM markers Miro1, VDAC and TOM20, the integral
IMM protein TIM23 and mitochondrial matrix protein HSP60. (D) Mitochondria were treated with proteinase K or alkaline as indicated; control treatments
involved incubation in appropriate buffer (Ctrl). After treatment, samples were probed for PTPIP51, HSP60 (mitochondrial matrix), TIM23 (IMM), TOM20
(OMM) and cytochrome c (peripheral IMM) on immunoblots. Input shows untreated mitochondria (left panel). (E) Isolated mitochondria labelled with Mito-
Tracker GreenFM were immunostained for PTPIP51 and the mitochondrial matrix, inner and outer mitochondrial membrane proteins TOM20, TIM23, COXIV,
MnSOD and HSP60. Antibodies were detected with Alexa633-conjugated secondary antibody and the numbers of mitochondria showing Alexa633 signal were
quantified from confocal images. As a negative control, mitochondria were incubated with secondary antibody only (CTRL). Bar graph shows percentages of
mitochondria labelled with the different antibodies. Also shown are representative images mitochondria in control (CTRL), PTPIP51 and TOM20 labelled
samples (image width ¼ 5 mm).
Human Molecular Genetics, 2012, Vol. 21, No. 61301
targeting PTPIP51 to mitochondria. These findings are in
agreement with a previous report (29).
We next determined the sub-mitochondrial localization and
membrane orientation of PTPIP51 using three different assays.
First, we separated outer from inner mitochondrial membranes
and the mitochondrial matrix by digitonin treatment of
HEK293 cell mitochondria (32) and probed these fractions
for PTPIP51 on immunoblots. As controls, we probed for well-
characterized outer mitochondrial membrane proteins TOM20,
voltage-dependent anion channel (VDAC) and mitochondrial
Rho GTPase 1 (Miro1), the inner mitochondrial membrane
proteins translocase of inner membrane-23 (TIM23) and the
mitochondrial matrix protein heat shock protein 60 (HSP60).
The known mitochondrial proteins were present in the
correct fractions and PTPIP51 was exclusively in the outer
mitochondrial membrane fraction (Fig. 2C).
Secondly, we treated mitochondria with proteinase K or
alkaline. Membrane-anchored outer mitochondrial membrane
proteins exposed to the cytosol are degraded by proteinase K
but are resistant to alkaline (33). Proteinase K but not alkaline
treatment removed PTPIP51 signal on immunoblots and this
was identical to TOM20 that was used as a positive control
(Fig. 2D). The loss of PTPIP51 signal was not due to disintegra-
tion of mitochondria because the matrix protein HSP60 and two
inner mitochondrial membrane proteins, TIM23 and cytochrome
c, resisted the proteinase K treatment. Alkaline extraction was
effective because the peripheral inner mitochondrial membrane
protein cytochrome c was removed after treatment (Fig. 2D).
Thirdly, we immunostained freshly purified mitochondria in
solution using an antibody raised against PTPIP51 that lacked
the amino-terminal transmembrane domain; only PTPIP51
exposed to the cytosol can interact with the antibody in this
assay. As a positive control, we labelled mitochondria with
an antibody for the outer mitochondrial membrane protein
TOM20, and as negative controls we labelled mitochondria
with antibodies for the inner mitochondrial membrane proteins
TIM23 and cytochrome oxidase subunit IV (COXIV), or
the matrix proteins HSP60 and Mn-superoxide dismutase
(MnSOD). Most mitochondria were labelled with PTPIP51
and positive control TOM20 antibodies but the negative
control antibodies stained only few mitochondria (Fig. 2E).
These data support the notion that PTPIP51 is an integral
outer mitochondrial membrane protein that projects into the
cytosol and complement the immunoprecipitation and proxim-
ity ligation assays, which demonstrate that VAPB and
PTPIP51 are binding partners.
VAPB is present in mitochondria-associated membranes
(MAM) and modulating PTPIP51 expression alters
the association of VAPB with mitochondria
20% of the mitochondrial surface being closely apposed to
ER membrane domains (34). These ER domains are termed
by Percoll gradient centrifugation (34–36). Our finding that
VAPB interacts with PTPIP51 suggests that VAPB is present
in MAM. To test this possibility, we prepared MAM, mitochon-
noblots. As controls, the samples were also probed for PTPIP51
and HSP60 as mitochondrial markers, fatty-acid-coenzyme A
ligase long-chain 4 (FACL4) as a MAM marker (37,38) and
protein disulfide-isomerase (PDI) as a general ER marker. All
markers were enriched in their respective fractions and a prom-
inent signal for VAPB was obtained in the MAM fraction
(Fig. 3A). We also compared the localization of endogenous
VAPB with that of the type III inositol 1,4,5-trisphosphate
receptor (IP3R3), an ER protein enriched in MAM (38), and
mitochondria by confocal laser scanning microscopy. A
proportion of VAPB and IP3R3 were present as punctate struc-
tures that aligned with mitochondria (Fig. 3B).
We next tested whether expression of PTPIP51 influences
the association of VAPB with mitochondria. To do so, we
modulated PTPIP51 expression and monitored on immuno-
blots, the presence of VAPB in a biochemical fraction that
contains both mitochondria and MAM but not ER. PTPIP51
expression was increased by transfection of HA-PTPIP51
and reduced by use of PTPIP51 siRNAs. Two different
PTPIP51 siRNAs (PTPIP51#10 and PTPIP51#11) were used
and both achieved knockdowns of over 90% without affecting
Figure 3. VAPB is a MAM protein. (A) MAM, mitochondria (Mito) and ER fractions were prepared from HEK293 cells and equal protein amounts from each
fraction probed on immunoblots for PTPIP51 and HSP60 (mitochondrial proteins) FACL4 (MAM enriched protein), PDI (ER protein) and VAPB. (B) A pro-
portion of VAPB colocalizes with IP3R3 and mitochondria. CV1 cells were immunostained for VAPB, IP3R3 (MAM enriched protein) and HSP60 (mitochon-
drial protein). Some punctate VAPB and IP3R3 staining aligned with mitochondria (Merge). Zoomed cellular areas are indicated [zoom (a), and zoom (b)].
Colocalized VAPB and IP3R3 labelling along mitochondria is indicated with arrowheads. Scale bar, 20 mm; zoom (a), 4 mm.
1302Human Molecular Genetics, 2012, Vol. 21, No. 6
VAPB expression (Fig. 4C). Transfection of HA-PTPIP51
increased whereas siRNA knockdown decreased the amount
of endogenous VAPB that was associated with mitochondria
(Fig. 4A and B). We also monitored how overexpression of
PTPIP51 influenced the cellular distribution of VAPB in rela-
tion to mitochondria by immunofluorescence microscopy. In
non-transfected control cells, VAPB displayed a vesicular
cytosolic pattern consistent with its known localization to
ER. However, transfection of HA-PTPIP51 induced a notice-
able redistribution of VAPB such that it appeared more
closely aligned with mitochondria (Fig. 4D). Thus, although
VAPB is not exclusively localized to MAM, a significant pro-
portion is present in this ER compartment and modulating
PTPIP51 expression influences the amount of VAPB that is
associated with mitochondria.
Depletion of VAPB and PTPIP51 disturbs Ca21handling
Both ER and mitochondria are stores for intracellular Ca2+.
Moreover, the ER–mitochondria interface and MAM play a
crucial role in Ca2+exchange between these organelles; dis-
ruption of ER–mitochondria connections perturbs Ca2+
uptake by mitochondria following release from ER stores
(34,36,37,39). To assess if VAPB or PTPIP51 affect Ca2+
homeostasis, we suppressed VAPB or PTPIP51 expression
using siRNAs and monitored cytosolic Ca2+levels ([Ca2+]c)
after induction of inositol 1,4,5-trisphosphate receptor (IP3R)-
mediated Ca2+release from ER stores. Again two different
siRNAs each for PTPIP51 and VAPB were used and all
achieved knockdowns of over 90%; siRNA loss of PTPIP51
did not influence expression of VAPB and loss of VAPB did
not influence expression of PTPIP51 (Figs 4C and 5A). For
these experiments, we used HEK293 cells transfected with
the M3 muscarinic-ACh-receptor (M3R) and triggered physio-
logical IP3R-mediated Ca2+release from ER stores by appli-
cation of the M3R agonist oxotremorine-M (Oxo-M). [Ca2+]c
was measured with the Ca2+indicator dye Fluo4-AM and was
calculated as relative fluorescence compared to baseline fluor-
escence at the beginning of the measurement (F/F0). In VAPB
or PTPIP51 siRNA-treated cells, the peak Oxo-M-induced
[Ca2+]c reached significantly higher levels compared to
control siRNA-treated cells (Fig. 5B). Mitochondria rapidly
take up Ca2+released from ER stores (34,36,37,39). Thus,
the increased peak [Ca2+]c observed in these cells might be
caused by reduction and/or delay in mitochondrial Ca2+
uptake. We therefore measured mitochondrial Ca2+levels
([Ca2+]m) under the same conditions using Rhod2-AM. De-
pletion of VAPB or PTPIP51 reduced the peak [Ca2+]m
Figure 4. Modulating PTPIP51 expression influences VAPB association with mitochondria. (A and B) Overexpression or siRNA knockdown of PTPIP51 influ-
ences the amount of endogenous VAPB present in a mitochondria plus MAM but not ER containing fraction. Mitochondria plus MAM fractions were prepared
from HEK293 cells transfected with control empty vector (Ctrl) or HA-PTPIP51 (A), or control siRNA (Ctrl) or PTPIP51 siRNAs (B). Samples were probed for
HA-PTPIP51 (using anti HA) in (A) or endogenous PTPIP51 in (B), and VAPB and COXIV as a mitochondrial marker. (C) shows siRNA knockdown of
PTPIP51 in cells transfected with control or PTPIP51 (PTPIP51#10, PTPIP51#11) siRNAs. Samples were also probed for VAPB and tubulin as indicated.
(D) Overexpression of PTPIP51 increases the localization of endogenous VAPB with mitochondria. CV1 cells were co-transfected with DsRed-Mito (to
label mitochondria) and either control empty vector (CRTL) or HA-PTPIP51 (+HA-PTPIP51). Cells were then immunostained for VAPB and HA-PTPIP51
(using HA antibody). Representative confocal images show DeRed-Mito (mitochondria), VAPB and HA-PTPIP51. Scale bar, 20 mm.
Human Molecular Genetics, 2012, Vol. 21, No. 6 1303
after IP3R-mediated Ca2+release from ER stores (Fig. 5C). The
magnitude of this reduction was similar to that previously
reported in mitofusin 2 knockout cells; mitofusin 2 is known
to connect and to regulate Ca2+exchange between ER and
mitochondria (37). Finally, we analysed the time lag between
the time points where the peak [Ca2+]c and [Ca2+]m were
reached in Fluo4/Rhod2 co-loaded cells. Both VAPB and
PTPIP51 knockdown caused a significant delay in mitochon-
drial Ca2+uptake (Fig. 5D). Thus, siRNA-mediated knockdown
of either VAPB or PTPIP51 increased peak [Ca2+]c following
IP3R-mediated Ca2+release from ER stores and this was asso-
ciated with a reduction and delay in mitochondrial Ca2+uptake.
VAPBP56S displays increased binding to PTPIP51 in
immunoprecipitation assays and accumulates in MAM
(3,6,8,9,19,40). We therefore examined whether VAPBP56S
had altered binding to PTPIP51 and distribution in MAM.
First, we analysed the binding of VAPBP56S to PTPIP51 in
Figure 5. siRNA knockdown of VAPB or PTPIP51 disturbs Ca2+handling. (A) shows siRNA knockdown of VAPB without influencing PTPIP51 expression.
HEK293 cells were transfected control siRNA (CTRL) or two different VAPB siRNAs (VAPB#05, VAPB#07) and the samples then probed on immunoblots for
VAPB and PTPIP51 as indicated. (B and C) siRNA loss of either VAPB or PTPIP51 increases cytosolic (B) and decreases mitochondrial (C) Ca2+levels upon
Oxo-M induced Ca2+release from ER stores. HEK293 cells were transfected with M3R and control (CTRL), VAPB#05, VAPB#07, PTPIP51#10 or PTPIP51#11
siRNAs and treated with Oxo-M. Representative traces of Fluo4 and Rhod2 fluorescence are shown on the left and normalized peak values are shown on the
right. Fluo4 and Rhod2 fluorescence show a transient increase in cytosolic (B) and mitochondrial (C) Ca2+levels upon Oxo-M induced Ca2+release from ER
stores. However, compared to control, VAPB and PTPIP51 siRNAs increased the peak [Ca2+]c levels (B, right; mean+SEM; CTRL, n ¼ 158 cells; VAPB#05,
n ¼ 255; VAPB#07, n ¼ 108; PTPIP51#10, n ¼ 149; PTPIP51#11, n ¼ 184), and decreased the peak [Ca2+]m levels (C, right; control siRNA, n ¼ 64;
VAPB#05, n ¼ 49; VAPB#07, n ¼ 55; PTPIP51#10, n ¼ 34; PTPIP51#11, n ¼ 64). (D) siRNA knockdown of VAPB or PTPIP51 increases the time lag
between peak [Ca2+]c and [Ca2+]m after Oxo-M induced release of Ca2+from ER stores. The time lag between peak [Ca2+]c and [Ca2+]m after Oxo-M
induced release of Ca2+from ER stores was measured in HEK293 cells-treated with control (CTRL), VAPB#07 or PTPIP51#10 siRNAs as indicated. Repre-
sentative traces show [Ca2+]c (full line) and [Ca2+]m (dashed line) in cells treated with control or VAPB siRNAs (left), or control or PTPIP51 siRNAs (middle).
Bar chart shows time lag between peak [Ca2+]c and [Ca2+]m (mean+SEM; control siRNA, n ¼ 59; VAPB#07, n ¼ 33; PTPIP51#10, n ¼ 20).
1304Human Molecular Genetics, 2012, Vol. 21, No. 6
immunoprecipitation assays. We co-transfected HEK293 cells
with HA-PTPIP51 and myc-VAPB or HA-PTPIP51 and
myc-VAPBP56S and compared the amounts of bound proteins
following immunoprecipitation of either myc-VAPB/myc-
VAPBP56S or HA-PTPIP51. Compared to myc-VAPB, myc-
VAPBP56S bound approximately 3–4-fold more PTPIP51 in
these assays (Fig. 6A).
Since PTPIP51 influences the amount of VAPB that is asso-
ciated with mitochondria (Fig. 4), this increase in binding of
VAPBP56S to PTPIP51 might lead to increased levels of
with myc-VAPB or myc-VAPBP56S and determined the
amount of VAPB in this fraction. Compared to myc-VAPB,
myc-VAPBP56S levels were increased almost 2-fold in the
pared to myc-VAPB, myc-VAPBP56S levels were elevated in
MAM and correspondingly decreased in non-MAM ER;
myc-VAPB and myc-VAPBP56S were not detected in pure
mitochondria (Fig. 6C). This increase in VAPBP56S in MAM
was not due to altered fractionation properties of ER, as the
levels of PDI in MAM did not change upon expression of
VAPBP56S. Thus, VAPBP56S binds more PTPIP51 than
VAPB in immunoprecipitation assays and VAPBP56S levels
are elevated in MAM.
VAPBP56S induces clustering of mitochondria
The increased interaction of VAPBP56S with PTPIP51 suggests
that VAPBP56S may influence mitochondria in some fashion.
We therefore monitored mitochondrial distribution by immunos-
myc-VAPBP56S. Transfected VAPB was detected using the
myc-tag and mitochondria visualized by staining with anti-
MnSOD. Mitochondria in cells transfected with myc-VAPB
were distributed throughout the cytoplasm in a pattern not notice-
nuclear clustering of mitochondria. This was particularly notice-
able in cells expressing higher levels of myc-VAPBP56S (as
determined by fluorescent signal intensity) (Fig. 7C and D). This
clustering of mitochondria was most marked in perinuclear
regions containing VAPBP56S aggregates.
VAPBP56S perturbs Ca21handling
Our findings that loss of VAPB and PTPIP51 disrupts Ca2+
handling following Ca2+release from ER stores and that
VAPBP56S has increased binding to PTPIP51 suggest that
VAPBP56S may influence Ca2+handling. To test this, we
monitored [Ca2+]m and [Ca2+]c upon IP3R-mediated Ca2+
release from ER stores in HEK293 cells co-transfected with
M3R and control vector, myc-VAPB or myc-VAPBP56S.
Transfection of myc-VAPBP56S but not myc-VAPB signifi-
cantly increased peak [Ca2+]m and decreased [Ca2+]c com-
pared to control (Fig. 8A and B). To gain insight into the
role of PTPIP51 in this effect, we reduced PTPIP51 expression
using siRNAs and again monitored [Ca2+]m upon IP3R-
Figure 6. Compared to VAPB, VAPBP56S levels associated with PTPIP51 are
elevated and VAPBP56S levels are increased in MAM. (A) VAPBP56S binds
more PTPIP51 than VAPB in immunoprecipitation assays. HEK293 cells were
co-transfected with either HA-PTPIP51 and myc-VAPB, or HA-PTPIP51 and
myc-VAPBP56S. myc-VAPB/VAPBP56S or HA-PTPIP51 were immunopreci-
pitated using anti-myc or anti-HA antibodies and bound HA-PTPIP51/
myc-VAPB then detected on immunoblots. Control immunoprecipitations (Ctrl)
were performed with non-immune antibody. Upper panel shows myc-VAPB/
VAPBP56S immunoprecipitations; middle panel shows HA-PTPIP51 immuno-
precipitations. Lower panel shows input levels of transfected proteins. Bar chart
shows relative amounts of co-immunoprecipitated PTPIP51 (upper) and VAPB
(middle) following densitometric quantification of signals.∗∗P , 0.01, n ¼ 3,
t-test. Error bars are mean+SEM. (B) Compared to VAPB, VAPBP56S levels
associated with mitochondria are increased. Mitochondria containing MAM
were purified from HEK293 cells transfected with empty vector (Ctrl),
myc-VAPB (VAPB) or myc-VAPBP56S (P56S). The samples were then
probed on immunoblots for myc-VAPB/VAPBP56S using anti-myc antibody,
tubulin and COXIV (as a mitochondrial marker). Bar chart shows relative
tion of signals.∗P , 0.05, n ¼ 3, t-test. Error bars are mean+SEM. (C) Com-
pared to VAPB, VAPBP56S levels are increased in MAM and decreased in
non-MAM ER. HEK293 cells transfected as in (B) were fractionated into
marker) and PDI (ER marker). Total shows immunoblot of the total cell lysates
(before fractionation); equal loading was confirmed by immunoblot for tubulin.
Human Molecular Genetics, 2012, Vol. 21, No. 61305
mediated Ca2+release from ER stores. Loss of PTPIP51 abro-
gated the effect of VAPBP56S on elevation of [Ca2+]m
(Fig. 8C). Thus, expression of VAPBP56S but not VAPB dis-
rupts mitochondrial Ca2+handling and this effect is associated
with its binding partner PTPIP51.
Finally, we monitored the effect of VAPBP56S on calcium
handling in neurons. Cultured rat cortical neurons were trans-
fected with CFP, CFP-VAPB or CFP-VAPBP56S and [Ca2+]m
and [Ca2+]c determined following depolarization of the
neurons to induce a transient increase in intracellular Ca2+.
VAPBP56S caused an increase in peak [Ca2+]m and a decrease
in peak [Ca2+]c compared to VAPB or CFP controls (Fig. 8D
Both ER and mitochondria are important stores for intracellu-
lar Ca2+and exchange of Ca2+between these organelles
Figure 7. VAPBP56S induces clustering of mitochondria. CV1 cells trans-
fected with empty vector (A), myc-VAPB (B) or myc-VAPBP56S (C and
D) were immunostained for mitochondria (red) and VAPB/VAPBP56S
(green) using MnSOD and myc antibodies, respectively. Representative
CLSM images are shown. Scale bar ¼ 20 mm.
Figure 8. VAPBP56S perturbs Ca2+handling. (A) VAPBP56S increases the
peak [Ca2+]m following Ca2+release from ER stores. HEK293 cells were
co-transfected with M3R and either empty vector (CTRL), myc-VAPB
(VAPB) or myc-VAPBP56S (P56S). Peak [Ca2+]m levels were then deter-
mined following application of Oxo-M to induce Ca2+release from ER
stores. Representative traces of Rhod2 fluorescence are shown on the left
and normalized peak values are shown on the right (mean+SEM; CTRL,
n ¼ 45; VAPB, n ¼ 46, VAPBP56S, n ¼ 79). (B) VAPBP56S decreases the
peak [Ca2+]c following Ca2+release from ER stores. HEK293 cells were
co-transfected with M3R and either empty vector (CTRL), myc-VAPB
(VAPB) or myc-VAPBP56S (P56S). Peak [Ca2+]c levels were then deter-
mined following application of Oxo-M to induce Ca2+release from ER
stores (mean+SEM; CTRL, n ¼ 47; VAPB, n ¼ 82, VAPBP56S, n ¼ 67).
(C) siRNA loss of PTPIP51 abrogates the VAPBP56S induced increase in
peak [Ca2+]m following Ca2+release from ER stores. HEK293 cells treated
with control (CTRL) or PTPIP51 siRNAs were co-transfected with M3R
and either empty vector (CTRL), or myc-VAPBP56S (P56S). Peak [Ca2+]m
were then determined following application of Oxo-M to induce Ca2+
release from ER stores. Values are mean+SEM; CTRL siRNA + CTRL,
n ¼ 43;
n ¼ 39;
VAPBP56S increases the peak [Ca2+]m following depolarization of rat cor-
CFP-VAPB (VAPB) or CFP-VAPBP56S (P56S) and [Ca2+]m determined
using Rhod2 after transient influx of Ca2+by a 2 min application of 50 mM
KCl to depolarize the neurons (mean+SEM; CTRL, n ¼ 6; VAPB, n ¼ 10;
P56S, n ¼ 4). (E) VAPBP56S decreases the peak [Ca2+]c following depolar-
ization of rat cortical neurons. Neurons were transfected with either CFP
(CTRL), CFP-VAPB (VAPB) or CFP-VAPBP56S (P56S) and [Ca2+]c deter-
mined using Fluo4 after transient influx of Ca2+by a 2 min application of
50 mM KCl to depolarize the neurons (mean+SEM; CTRL, n ¼ 4; VAPB,
n ¼ 9; P56S, n ¼ 5).
n ¼ 28;
n ¼ 40.(D)
1306Human Molecular Genetics, 2012, Vol. 21, No. 6
impacts upon a number of physiological processes. To facili-
tate this exchange, up to 20% of the mitochondrial surface is
closely apposed to ER membranes (34,36). These ER
domains associated with mitochondria are termed MAM
(41–43). Here, we report that the integral ER protein VAPB
interacts with the outer mitochondrial membrane protein
PTPIP51 and that a proportion of VAPB is present in MAM.
We also present evidence that both VAPB and PTPIP51 are
involved in the regulation of intracellular Ca2+homeostasis.
In particular, siRNA loss of VAPB or PTPIP51 reduced
[Ca2+]m following release of Ca2+from ER stores. Interest-
ingly, the magnitude of this reduction (approximately 86%
of control) was very similar to that reported previously in
mitofusin 2 knockout cells (approximately 82% of control);
mitofusin 2 is a known mediator of ER–mitochondria connec-
tions that regulates Ca2+exchange between these organelles
(37). Finally, we demonstrate that VAPBP56S, which causes
familial ALS type-8, has altered binding to PTPIP51 and
The mechanisms by which VAPBP56S induce disease are
not known although several studies implicate a role for ER
stress and the UPR in this process. However, the precise
details are controversial. In some reports, expression of
VAPB induces UPR and VAPBP56S inhibits this process
(6,10,18). In another, expression of VAPB and VAPBP56S
both inhibit UPR but VAPBP56S is more potent in this
effect (19). Finally, other studies have shown that VAPB
and/or VAPBP56S can induce ER stress and UPR (11,21).
Whatever the precise details, recent studies have shown
that the UPR can influence Ca2+release from ER stores
(44). VAPBP56S perturbation of the ER structure and/or
UPR may therefore impact on this process. Interestingly,
mutants of Cu/Zn superoxide dismutase-1 (SOD1) that
cause familial ALS type-1 have also been implicated in
ER stress (22) and elevated Ca2+levels are also seen in
mutant SOD1 expressing cells (24). Despite this, mutant
SOD1 induced alterations to mitochondria morphology and
cell toxicity can be independent of any [Ca2+]m increases
(24,45). By contrast, others have recently demonstrated
that VAPBP56S toxicity in motor neurons is linked to
defects in Ca2+handling (11). Clearly, further studies on
this topic will help resolve these issues.
Damage to both mitochondria and ER are seen in ALS
(reviewed in 23,46,47). Thus mutant ALS SOD1 selectively
associates with mitochondria and perturbs mitochondrial func-
tion (48–53). Likewise, TDP-43 that is present in ubiquiti-
nated inclusions in ALS and which when mutated can also
cause familial ALS (54–56) has been linked to mitochondrial
dysfunction (57,58). In apparent contrast, both mutant SOD1
and TDP-43 mis-metabolism have been linked to ER stress
(22,59) and VAPB functions in ER stress and the UPR
(6,10,11,18,19). Our findings that VAPB is a MAM protein
that interacts with PTPIP51 suggest that such damage to mito-
chondria and ER in ALS may involve the ER–mitochondrial
axis. Indeed, one known molecular target for damage by
ALS mutant SOD1 is the VDAC (45). VDAC is located in
the outer mitochondrial membrane and interacts with IP3Rs
in ER via glucose-regulated protein 75 (60,61). Also, muta-
tions in mitofusin 2 cause forms of Charcot-Marie-Tooth
disease (62) and mitofusin 2 has recently been shown to
tether ER to mitochondria and regulate Ca2+exchange
between these organelles (37). Future studies aimed at exam-
ining the role of MAM and the ER/mitochondria axis in ALS
may thus be warranted.
MATERIALS AND METHODS
Amino-terminal myc-tagged VAPB (myc-VAPB) was gener-
ated by polymerase chain reaction (PCR) from a human
VAPB clone purchased from Origene and cloned into
pCI-Neo (Promega). Myc-VAPBP56S was generated from
myc-VAPB with a QuikChange mutagenesis kit (Stratagene).
CFP-VAPB and CFP-VAPBP56S were produced by cloning
into pECFP-C1 (Clontech). Carboxy-terminal HA-tagged
PTPIP51 (HA-PTPIP51) was generated by PCR from a full-
Primer sequences were—myc-VAPB: ATCGGTCGACGC
GCAG and ATCGGTCGACCTACAAGGCAATCTTCCCAA
TAATTACACCAACG; myc-VAPBP56S mutagenic primer:
CATCGATGC, HA-PTPIP51: ATCGGAATTCGCCACCAT
GTCTAGACTGGGAGCCCTGGGTGGTGCCCGTG and CG
pDsRed-mito and pAcGFP1-mito were from Clontech and
muscarinic-ACh-receptor type 3 was a gift of Dr E. Seward,
University of Sheffield, UK.
VAPB antibodies were raised in rat (sk83) and rabbit
(#3504) and PTPIP51 antibodies in rat (skr9) by immuniza-
tion with glutathione S-transferase (GST)-VAPB(1–220) and
detected single species of the correct molecular mass by
immunoblotting (Supplementary Material, Fig. S1). Primary
antibodies used were: rabbit anti-PTPIP51 (FAM82A2) and
rabbit anti-Miro1 (RHOT1) (both from Atlas Antibodies);
mouse anti-TIM23 and mouse anti-TOM20 (BD Transduc-
anti-cytochrome c, rabbit anti-COXIV and rabbit anti-VDAC
(Cell Signaling Technology); rabbit anti-HA, mouse anti-
tubulin (DM1A) and mouse anti-HSP60 (Sigma); rabbit
(Stressgen); mouse anti-PDI (RL77, Affinity BioReagents);
rabbit anti-IP3R3 (Millipore); rabbit anti-ACLS4 (FACL4;
peroxidase-coupled goat anti-mouse, anti-rabbit and anti-rat
Ig (GE Healthcare or Dako), Alexa fluorophore (488, 546,
633)-coupled goat anti-mouse, anti-rabbit and anti-rat IgG
(Invitrogen), Cy3 or Cy5-coupled donkey anti-mouse, anti-
rabbit and anti-rat IgG (Jackson ImmunoResearch).
Human Molecular Genetics, 2012, Vol. 21, No. 61307
Yeast two-hybrid screen
brain cDNA library (Clontech) with VAPB ‘bait’ lacking it
carboxy-terminal transmembrane domain (VAPB1–220) in
pY1 according to the manufacturer’s instructions. Following
mating yeast underwent tryp2/leu2/his2selection and vigor-
ously growing clones were subjected to b-galactosidase
assays. Prey plasmids were rescued by transformation into
co-transformation back into yeast either alone or with
VAPB(1–220) bait as described elsewhere (63).
plasmids identified by
Cell culture and plasmid transfection
HEK293 and CV1 cells were maintained in Dulbecco’s modi-
fied Eagle’s medium (Invitrogen) containing 4.5 g/l glucose,
10% fetal bovine serum (Sera Laboratories), 2 mM L-glutamine
(Invitrogen), and 1 mM sodium pyruvate (Sigma) and were
transfected with Exgen500 according to the manufacturer’s
instructions (Fermentas). HEK293 cells were used for bio-
chemical and Ca2+measurement studies because of the high
transfection efficiencies that are achieved in this cell type.
CV1 cells were used for immunostaining experiments
because they are particularly suitable for studying intracellular
structure due to their large size and highly spread morphology.
All cells were used in experiments 16–24 h post-transfection.
Cortical neurons were isolated from embryonic day 18 rat
embryos and cultured on glass coverslips coated with
poly-L-lysine in 6 or 12-well plates in neurobasal medium
supplemented with B27 supplement (Invitrogen), 100 IU/ml
penicillin, 100 mg/ml streptomycin and 2 mM L-glutamine.
Neurons were cultured for 5 days and then transfected using
a calcium phosphate Profection kit (Promega) as previously
described (64). Neurons were used in experiments 48 h post-
siRNA sequences and transfection
VAPB and PTPIP51 siRNAs were from Dharmacon or Invi-
trogen. Non-targeting control siRNA was from Dharmacon.
siRNA sequences were: VAPB#05: UGUUACAGCCUUUC
PTPIP51#11: GAAGCUAGAUGGUGGAUGAUU. HEK293
cells were siRNA transfected with Lipofectamine 2000 (Invi-
trogen) according to the manufacturer’s instructions. The cells
were used for experiments 4 days post-transfection.
Cells were harvested in ice-cold lysis buffer [50 mM Tris–HCl
pH 7.4, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid
(EDTA), 1% Triton X-100 and complete protease inhibitors
(Roche)], and lysed for 30 min on ice. The lysate was
cleared by centrifugation at 100 000g for 30 min at 48C and
precleared with protein G sepharose beads (Sigma), followed
by incubation with antibodies for 16 h at 48C. Antibody was
captured with protein G sepharose for 2 h at 48C. The
immune pellets were washed with ice-cold lysis buffer and
analysed by SDS–PAGE and immunoblotting.
Immunostaining was performed as described previously (65).
CV1 cells on glass coverslips were fixed with 3.7% formalde-
hyde in phosphate buffered saline (PBS) for 20 min at room
temperature. After washing with PBS, residual formaldehyde
was quenched by incubation with 50 mM NH4Cl in PBS for
15 min at room temperature, followed by a second round of
washing with PBS. Subsequently, the cells were permeabilized
with 0.2% Triton X-100 in PBS for 3 min. Alternatively,
cells were fixed and permeabilized with 2208C MeOH for
20 min (Figs 3B and 7), and MeOH was removed by
washing with PBS.
After fixing, the cells were incubated with PBS containing
0.2% fish gelatin (PBS/F) for 30 min at room temperature
and then with the primary antibody in PBS/F for 1 h. After
washing with PBS/F, the cells were incubated with secondary
antibody in PBS/F for 45 min at room temperature. After a
final wash, the samples were mounted in Moviol (Calbiochem)
containing 1% DABCO (Sigma). Immunostaining of isolated
mitochondria was performed as described previously (66).
In situ proximity ligation assays
In situ proximity ligation assays were performed with rabbit
VAPB and rat PTPIP51 antibodies using a Duolink kit follow-
ing the manufacturer’s protocol (Olink Bioscience). Proximity
signals were quantified using the Particle Analysis function of
Confocal images were recorded with an LSM510Meta con-
focal microscope equipped with a 63×/1.4NA Plan-Apochro-
mat objective using a pinhole size of one Airy unit
(Carl Zeiss). Conventional immunofluorescence microscopy
was done with a Leica DM5000B microscope equipped
with 63×/1.25NA and 40×/0.75NA HCX-PL-FLUOTAR
objectives and appropriate filtersets (Leica).
as described elsewhere (35). Briefly, cells were harvested and
washed by centrifugation at 13 000g for 30 s once with PBS
and once with isolation buffer (250 mM mannitol, 5 mM 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH
7.4, 0.5 mM ethylene glycol tetraacetic acid (EGTA), 0.1%
bovine serum albumin (BSA) and complete protease inhibitors)
at 48C. The cell pellet was resuspended in isolation buffer and
homogenized using a teflon/glass dounce homogenizer (100
strokes; Kontes Pestle 19; Kimble Chase). The homogenate
was centrifuged twice at 600g for 5 min to remove nuclei and
unbroken cells. The MAM-enriched mitochondrial fraction
was pelleted by centrifugation at 10 300g for 10 min. The
supernatants were centrifuged at 100 000g for 30 min to
1308 Human Molecular Genetics, 2012, Vol. 21, No. 6
mitochondria, the MAM-enriched mitochondrial pellet was
resuspended in isolation buffer and layered on top of a self-
forming 30% Percoll gradient (225 mM mannitol, 1 mM
EGTA, 0.05% BSA, 30% Percoll, 25 mM Na-HEPES pH 7.4).
After centrifugation at 95 000g for 30 min, a dense band con-
dient; the MAM-containing band was retrieved above the
mitochondrial band. To remove residual Percoll, the mitochon-
drial band was diluted in isolation medium and mitochondria
were washed twice by centrifugation at 6300g for 10 min.
The MAM band was diluted with isolation buffer and centri-
fuged once at 6300g for 10 min to remove contaminating mito-
chondria. MAM was pelleted from the resulting supernatant by
centrifugation at 100 000g for 1 h. All final organelle pellets
were resuspended and lysed in radio-immunoprecipitation
assay (RIPA) buffer (50 mM Tris–HCl pH 6.8, 150 mM NaCl,
1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.5% deoxycho-
late, 0.1% SDS, complete protease inhibitors) and the protein
For sub-fractionation of mitochondria, mitochondria were
isolated from HEK293 cells on a discontinuous sucrose gradi-
ent as described elsewhere (67). Proteinase K and alkaline
treatments were essentially as described previously (33). For
proteinase K treatment, 0.5 ml 2.5 mg/ml proteinase K was
added to 100 ml mitochondria in isolation buffer (10 mM
K-HEPES pH 7.4, 220 mM mannitol, 70 mM sucrose, 1 mM
EDTA, 1 mM dithiothreitol). After 10 min incubation, 1 ml
200 mM phenylmethanesulfonylfluoride was added to inhibit
proteinase K and the mitochondria were pelleted by centrifu-
gation at 10 300g for 10 min and washed once with isolation
buffer. For alkaline extraction, mitochondria were resus-
pended in 100 mM Na2CO3 pH 11.5, and incubated for
30 min on ice. To remove the alkaline solution, mitochondria
were pelleted by centrifugation at 10 300g for 10 min and
washed once with isolation buffer.
Mitochondriaweresub-fractionated intoouter mitochondrial
membrane and inner mitochondrial membrane/matrix fractions
as described previously (32). Briefly, purified mitochondria
were incubated with 3 mg/ml digitonin (Calbiochem) in
250 mM sucrose, 10 mM K-MOPS pH 7.2 for 20 min at 48C.
After incubation, an inner mitochondrial membrane/matrix
pellet was recovered by centrifugation (9500g, 15 min, 48C).
An outer mitochondrial membrane pellet was recovered from
the supernatant by centrifugation at 100 000g, for 1 h at 48C.
Both pellets were resuspended in isolation buffer and loaded
on top of sucrose gradients (51.3, 37.4 and 23.2% sucrose in
10 mM KH2PO4pH 7). Pure inner mitochondrial membrane/
matrix and outer mitochondrial membrane fractions were
recovered and concentrated by centrifugation (100 000g, 1 h,
48C). All fractions were solubilized in RIPA buffer and
protein concentrations determined by Bradford assay.
SDS–PAGE and immunoblotting
Protein samples were separated by SDS–PAGE and trans-
ferred to nitrocellulose membranes (Schleicher & Schuell)
by wet electroblotting (BioRad). After transfer, membranes
were blocked with Tris–HCl-buffered saline (TBS) containing
5% milk, and 0.1% Tween-20 for 1 h at room temperature, and
then incubated with primary antibodies in blocking buffer for
1 h at room temperature. After washing with wash buffer
(TBS, 0.1% Tween-20), the membranes were incubated with
secondary antibodies in wash buffer for 1 h at room tempera-
ture. After washing, the membranes were processed for
chemiluminescent detection with the Pierce SuperSignal
West Pico Chemiluminescent Substrate System according to
the manufacturer’s instructions. Signal intensities on immuno-
blots were quantified with ImageJ after scanning with an
Epson Precision V700 Photo scanner as described (68).
HEK293 cells and cortical neurons were loaded with 2 mM
Fluo4-AM and/or Rhod2-AM dye (Invitrogen) in external solu-
tion (145 mM NaCl, 2 mM KCl, 5 mM NaHCO3, 1 mM MgCl2,
2.5 mMCaCl2,10 mMglucose,10 mMNa-HEPESpH7.25)con-
378C with MetaMorph (Molecular Dynamics) on an Axiovert
S100 microscope (Zeiss) equipped with YFP (Fluo4) and
DsRed (Rhod2) filtersets (Chroma Technology), a 40×/
1.3NA Plan-Neofluar objective (Zeiss) and a Photometrics
Cascade-II 512B EMCCD. The cells were kept under constant
perfusion with external solution (0.5 ml/min). IP3R-mediated
Ca2+release from ER stores was triggered by application of
100 mMOxo-Mfor2 min.Neuronsweredepolarizedbyapplica-
tion of 50 mM KCl for 2 min. Ca2+levels were calculated as
relative Fluo4 or Rhod2 fluorescence compared to baseline
fluorescence at the start of the measurement.
All experiments were repeated at least three times. Statistical
analysis was performed with Prism 5.0d (GraphPad Software).
Unless stated otherwise, statistical significance was deter-
mined by one-way ANOVA followed by Bonferroni’s mul-
tiple comparison test;∗P , 0.05,∗∗P , 0.01,∗∗∗P , 0.001,
∗∗∗∗P , 0.0001.
Supplementary Material is available at HMG online.
We thank C. Bauer, K. Brady, A. Brain and E. Gray for tech-
nical assistance, and E. Seward for providing reagents.
Conflict of Interest statement. None declared.
This work was supported by the Medical Research Council
C.C.J.M. and C.E.S.), by the Wellcome Trust (http://www.
wellcome.ac.uk/) (078662 to C.C.J.M.), the Motor Neurone
Disease Association (MNDA; http://www.mndassociation.
Human Molecular Genetics, 2012, Vol. 21, No. 61309
org/) (Miller6231 to C.C.J.M.) and the European Union 7th
Framework Programme for RTD (http://ec.europa.eu/resea
rch/fp7) (Project MitoTarget—Grant Agreement HEALTH-
F2-2008-223388 to C.C.J.M. and K.J.D.V.). Funding to pay
the Open Access publication charges for this article was pro-
vided by the Wellcome Trust.
1. Lill, C.M., Abel, O., Bertram, L. and Al-Chalabi, A. (2011) Keeping up
with genetic discoveries in amyotrophic lateral sclerosis: The ALSoD and
ALSGene databases. Amyotroph. Lateral Scler., 12, 238–249.
2. Byrne, S., Walsh, C., Lynch, C., Bede, P., Elamin, M., Kenna, K.,
McLaughlin, R. and Hardiman, O. (2011) Rate of familial amyotrophic
lateral sclerosis: a systematic review and meta-analysis. J. Neurol.
Neurosurg. Psychiatry, 82, 623–627.
3. Nishimura, A.L., Mitne-Neto, M., Silva, H.C., Richieri-Costa, A.,
Middleton, S., Cascio, D., Kok, F., Oliveira, J.R., Gillingwater, T., Webb,
J. et al. (2004) A mutation in the vesicle-trafficking protein VAPB causes
late-onset spinal muscular atrophy and amyotrophic lateral sclerosis.
Am. J. Hum. Genet., 75, 822–831.
4. Kagiwada, S., Hosaka, K., Murata, M., Nikawa, J. and Takatsuki, A.
(1998) The Saccharomyces cerevisiae SCS2 gene product, a homolog of a
synaptobrevin-associated protein, is an integral membrane protein of the
endoplasmic reticulum and is required for inositol metabolism.
J. Bacteriol., 180, 1700–1708.
5. Kaiser, S.E., Brickner, J.H., Reilein, A.R., Fenn, T.D., Walter, P. and
Brunger, A.T. (2005) Structural basis of FFAT motif-mediated ER
targeting. Structure, 13, 1035–1045.
6. Kanekura, K., Nishimoto, I., Aiso, S. and Matsuoka, M. (2006)
Characterization of amyotrophic lateral sclerosis-linked P56S mutation of
vesicle-associated membrane protein-associated protein B (VAPB/ALS8).
J. Biol. Chem., 28, 30223–30232.
7. Skehel, P.A., Fabian-Fine, R. and Kandel, E.R. (2000) Mouse VAP33 is
associated with the endoplasmic reticulum and microtubules. Proc. Natl
Acad. Sci. USA, 97, 1101–1106.
8. Teuling, E., Ahmed, S., Haasdijk, E., Demmers, J., Steinmetz, M.O.,
Akhmanova, A., Jaarsma, D. and Hoogenraad, C.C. (2007) Motor neuron
disease-associated mutant vesicle-associated membrane
protein-associated protein (VAP) B recruits wild-type VAPs into
endoplasmic reticulum-derived tubular aggregates. J. Neurosci., 27,
9. Fasana, E., Fossati, M., Ruggiano, A., Brambillasca, S., Hoogenraad,
C.C., Navone, F., Francolini, M. and Borgese, N. (2010) A VAPB mutant
linked to amyotrophic lateral sclerosis generates a novel form of
organized smooth endoplasmic reticulum. FASEB J., 24, 1419–1430.
10. Chen, H.J., Anagnostou, G., Chai, A., Withers, J., Morris, A., Adhikaree,
J., Pennetta, G. and de Belleroche, J.S. (2010) Characterisation of the
properties of a novel mutation in VAPB in familial ALS. J. Biol. Chem.,
11. Langou, K., Moumen, A., Pellegrino, C., Aebischer, J., Medina, I.,
Aebischer, P. and Raoul, C. (2010) AAV-mediated expression of wildtype
and ALS-linked mutant VAPB selectively triggers death of motoneurons
through a Ca-dependent ER-associated pathway. J. Neurochem., 114,
12. Kim, S., Leal, S.S., Ben Halevy, D., Gomes, C.M. and Lev, S. (2010)
Structural requirements for VAP-B oligomerization and their implication
in amyotrophic lateral sclerosis-associated VAP-B(P56S) neurotoxicity.
J. Biol. Chem., 285, 13839–13849.
13. Tudor, E.L., Galtrey, C.M., Perkinton, M.S., Lau, K.F., De Vos, K.J.,
Mitchell, J.C., Ackerley, S., Hortobagyi, T., Vamos, E., Leigh, P.N. et al.
(2010) Amyotrophic lateral sclerosis mutant VAPB transgenic mice
develop TDP-43 pathology. Neuroscience, 167, 774–785.
14. Pennetta, G., Hiesinger, P., Fabian-Fine, R., Meinertzhagen, I. and Bellen,
H. (2002) Drosophila VAP-33A directs bouton formation at
neuromuscular junctions in a dosage-dependent manner. Neuron, 35,
15. Ratnaparkhi, A., Lawless, G.M., Schweizer, F.E., Golshani, P. and
Jackson, G.R. (2008) A Drosophila model of ALS: human ALS-associated
mutation in VAP33A suggests a dominant negative mechanism. PLoS
ONE, 3, e2334.
16. Prosser, D.C., Tran, D., Gougeon, P.Y., Verly, C. and Ngsee, J.K. (2008)
FFAT rescues VAPA-mediated inhibition of ER-to-Golgi transport and
VAPB-mediated ER aggregation. J. Cell Sci., 121, 3052–3061.
17. Peretti, D., Dahan, N., Shimoni, E., Hirschberg, K. and Lev, S. (2008)
Coordinated lipid transfer between the endoplasmic reticulum and the
Golgi complex requires the VAP Proteins and is essential for
Golgi-mediated transport. Mol. Biol. Cell, 19, 3871–3884.
18. Suzuki, H., Kanekura, K., Levine, T.P., Kohno, K., Olkkonen, V.M., Aiso,
S. and Matsuoka, M. (2009) ALS-linked P56S-VAPB, an aggregated
loss-of-function mutant of VAPB, predisposes motor neurons to ER
stress-related death by inducing aggregation of co-expressed wild-type
VAPB. J. Neurochem., 108, 973–985.
19. Gkogkas, C., Middleton, S., Kremer, A.M., Wardrope, C., Hannah, M.,
Gillingwater, T.H. and Skehel, P. (2008) VAPB interacts with and
modulates the activity of ATF6. Hum. Mol. Genet., 17, 1517–1526.
20. Amarilio, R., Ramachandran, S., Sabanay, H. and Lev, S. (2005)
Differential regulation of endoplasmic reticulum structure through
VAP-Nir protein interaction. J. Biol. Chem., 280, 5934–5944.
21. Tsuda, H., Han, S.M., Yang, Y., Tong, C., Lin, Y.Q., Mohan, K., Haueter,
C., Zoghbi, A., Harati, Y., Kwan, J. et al. (2008) The amyotrophic lateral
sclerosis 8 protein VAPB is cleaved, secreted, and acts as a ligand for Eph
receptors. Cell, 133, 963–977.
22. Saxena, S., Cabuy, E. and Caroni, P. (2009) A role for motoneuron
subtype-selective ER stress in disease manifestations of FALS mice. Nat.
Neurosci., 12, 627–636.
23. Kanekura, K., Suzuki, H., Aiso, S. and Matsuoka, M. (2009) ER Stress
and unfolded protein response in amyotrophic lateral sclerosis. Mol.
Neurobiol., 39, 81–89.
24. Tradewell, M.L., Cooper, L.A., Minotti, S. and Durham, H.D. (2011)
Calcium dysregulation, mitochondrial pathology and protein aggregation
in a culture model of amyotrophic lateral sclerosis: mechanistic
relationship and differential sensitivity to intervention. Neurobiol. Dis.,
25. Grosskreutz, J., Van Den Bosch, L. and Keller, B.U. (2010) Calcium
dysregulation in amyotrophic lateral sclerosis. Cell Calcium, 47,
26. Stenzinger, A., Kajosch, T., Tag, C., Porsche, A., Welte, I., Hofer, H.W.,
Steger, K. and Wimmer, M. (2005) The novel protein PTPIP51 exhibits
tissue- and cell-specific expression. Histochem. Cell Biol., 123, 19–28.
27. Brobeil, A., Graf, M., Oeschger, S., Steger, K. and Wimmer, M. (2010)
PTPIP51-a myeloid lineage specific protein interacts with PTP1B in
neutrophil granulocytes. Blood Cells Mol. Dis., 45, 159–168.
28. Oishi, K., Okano, H. and Sawa, H. (2007) RMD-1, a novel
microtubule-associated protein, functions in chromosome segregation in
Caenorhabditis elegans. J. Cell Biol., 179, 1149–1162.
29. Lv, B.F., Yu, C.F., Chen, Y.Y., Lu, Y., Guo, J.H., Song, Q.S., Ma, D.L.,
Shi, T.P. and Wang, L. (2006) Protein tyrosine phosphatase interacting
protein 51 (PTPIP51) is a novel mitochondria protein with an N-terminal
mitochondrial targeting sequence and induces apoptosis. Apoptosis, 11,
30. Yu, C., Han, W., Shi, T., Lv, B., He, Q., Zhang, Y., Li, T., Song, Q.,
Wang, L. and Ma, D. (2008) PTPIP51, a novel 14-3-3 binding protein,
regulates cell morphology and motility via Raf-ERK pathway. Cell
Signal., 20, 2208–2220.
31. Soderberg, O., Gullberg, M., Jarvius, M., Ridderstrale, K., Leuchowius,
K.J., Jarvius, J., Wester, K., Hydbring, P., Bahram, F., Larsson, L.G. et al.
(2006) Direct observation of individual endogenous protein complexes in
situ by proximity ligation. Nat. Methods, 3, 995–1000.
32. Benga, G., Hodarnau, A., Tilinca, R., Porutiu, D., Dancea, S., Pop, V. and
Wrigglesworth, J. (1979) Fractionation of human liver mitochondria:
enzymic and morphological characterization of the inner and outer
membranes as compared to rat liver mitochondria. J. Cell Sci., 35,
33. Ryan, M.T., Voos, W. and Pfanner, N. (2001) Assaying protein import
into mitochondria. Methods Cell Biol., 65, 189–215.
34. Rizzuto, R., Pinton, P., Carrington, W., Fay, F.S., Fogarty, K.E., Lifshitz,
L.M., Tuft, R.A. and Pozzan, T. (1998) Close contacts with the
endoplasmic reticulum as determinants of mitochondrial Ca2+ responses.
Science, 280, 1763–1766.
35. Vance, J.E. (1990) Phospholipid synthesis in a membrane fraction
associated with mitochondria. J. Biol. Chem., 265, 7248–7256.
36. Csordas, G., Renken, C., Varnai, P., Walter, L., Weaver, D., Buttle, K.F.,
Balla, T., Mannella, C.A. and Hajnoczky, G. (2006) Structural and
1310Human Molecular Genetics, 2012, Vol. 21, No. 6
functional features and significance of the physical linkage between ER Download full-text
and mitochondria. J. Cell Biol., 174, 915–921.
37. de Brito, O.M. and Scorrano, L. (2008) Mitofusin 2 tethers endoplasmic
reticulum to mitochondria. Nature, 456, 605–610.
38. Mendes, C.C., Gomes, D.A., Thompson, M., Souto, N.C., Goes, T.S.,
Goes, A.M., Rodrigues, M.A., Gomez, M.V., Nathanson, M.H. and Leite,
M.F. (2005) The type III inositol 1,4,5-trisphosphate receptor
preferentially transmits apoptotic Ca2+ signals into mitochondria. J. Biol.
Chem., 280, 40892–40900.
39. Csordas, G. and Hajnoczky, G. (2009) SR/ER-mitochondrial local
communication: calcium and ROS. Biochim. Biophys. Acta, 1787,
40. Chai, A., Withers, J., Koh, Y.H., Parry, K., Bao, H., Zhang, B., Budnik, V.
and Pennetta, G. (2008) hVAPB, the causative gene of a heterogeneous
group of motor neuron diseases in humans, is functionally interchangeable
with its Drosophila homologue DVAP-33A at the neuromuscular junction.
Hum. Mol. Genet., 17, 266–280.
41. Rusinol, A.E., Cui, Z., Chen, M.H. and Vance, J.E. (1994) A unique
mitochondria-associated membrane fraction from rat liver has a high
capacity for lipid synthesis and contains pre-Golgi secretory proteins
including nascent lipoproteins. J. Biol. Chem., 269, 27494–27502.
42. Simmen, T., Lynes, E.M., Gesson, K. and Thomas, G. (2010) Oxidative
protein folding in the endoplasmic reticulum: tight links to the
mitochondria-associated membrane (MAM). Biochim. Biophys. Acta,
43. Hayashi, T., Rizzuto, R., Hajnoczky, G. and Su, T.P. (2009) MAM: more
than just a housekeeper. Trends Cell Biol., 19, 81–88.
44. Li, G., Mongillo, M., Chin, K.T., Harding, H., Ron, D., Marks, A.R. and
Tabas, I. (2009) Role of ERO1-alpha-mediated stimulation of inositol
1,4,5-triphosphate receptor activity in endoplasmic reticulum
stress-induced apoptosis. J. Cell Biol., 186, 783–792.
45. Israelson, A., Arbel, N., Da Cruz, S., Ilieva, H., Yamanaka, K.,
Shoshan-Barmatz, V. and Cleveland, D.W. (2010) Misfolded mutant
SOD1 directly inhibits VDAC1 conductance in a mouse model of
inherited ALS. Neuron, 67, 575–587.
46. Cozzolino, M. and Carri, M.T. (2011) Mitochondrial dysfunction in ALS.
Prog. Neurobiol., in press.
47. Duffy, L.M., Chapman, A.L., Shaw, P.J. and Grierson, A.J. (2011) The
role of mitochondria in the pathogenesis of Amyotrophic Lateral
Sclerosis. Neuropathol. Appl. Neurobiol., 37, 336–352.
48. Higgins, C.M., Jung, C., Ding, H. and Xu, Z. (2002) Mutant Cu, Zn
superoxide dismutase that causes motoneuron degeneration is present in
mitochondria in the CNS. J. Neurosci., 22, RC215.
49. Ferri, A., Cozzolino, M., Crosio, C., Nencini, M., Casciati, A., Gralla,
E.B., Rotilio, G., Valentine, J.S. and Carri, M.T. (2006) Familial
ALS-superoxide dismutases associate with mitochondria and shift their
redox potentials. Proc. Natl Acad. Sci. USA, 103, 13860–13865.
50. Mattiazzi, M., D’Aurelio, M., Gajewski, C.D., Martushova, K., Kiaei, M.,
Beal, M.F. and Manfredi, G. (2002) Mutated human SOD1 causes
dysfunction of oxidative phosphorylation in mitochondria of transgenic
mice. J. Biol. Chem., 277, 29626–29633.
51. Pasinelli, P., Belford, M.E., Lennon, N., Bacskai, B.J., Hyman, B.T.,
Trotti, D. and Brown, R.H. Jr. (2004) Amyotrophic lateral
sclerosis-associated SOD1 mutant proteins bind and aggregate with Bcl-2
in spinal cord mitochondria. Neuron, 43, 19–30.
52. Liu, J., Lillo, C., Jonsson, P.A., Velde, C.V., Ward, C.M., Miller, T.M.,
Subramaniam, J.R., Rothstein, J.D., Marklund, S., Andersen, P.M. et al.
(2004) Toxicity of familial ALS-linked SOD1 mutants from selective
recruitment to spinal mitochondria. Neuron, 43, 5–17.
53. De Vos, K.J., Chapman, A.L., Tennant, M.E., Manser, C., Tudor, E.L.,
Lau, K.F., Brownlees, J., Ackerley, S., Shaw, P.J., McLoughlin, D.M.
et al. (2007) Familial amyotrophic lateral sclerosis-linked SOD1 mutants
perturb fast axonal transport to reduce axonal mitochondria content. Hum.
Mol. Genet., 16, 2720–2728.
54. Arai, T., Hasegawa, M., Akiyama, H., Ikeda, K., Nonaka, T., Mori, H.,
Mann, D., Tsuchiya, K., Yoshida, M., Hashizume, Y. et al. (2006)
TDP-43 is a component of ubiquitin-positive tau-negative inclusions in
frontotemporal lobar degeneration and amyotrophic lateral sclerosis.
Biochem. Biophys. Res. Commun., 351, 602–611.
55. Neumann, M., Sampathu, D.M., Kwong, L.K., Truax, A.C., Micsenyi,
M.C., Chou, T.T., Bruce, J., Schuck, T., Grossman, M., Clark, C.M. et al.
(2006) Ubiquitinated TDP-43 in frontotemporal lobar degeneration and
amyotrophic lateral sclerosis. Science, 314, 130–133.
56. Sreedharan, J., Blair, I.P., Tripathi, V.B., Hu, X., Vance, C., Rogelj, B.,
Ackerley, S., Durnall, J.C., Williams, K.L., Buratti, E. et al. (2008)
TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis.
Science, 319, 1668–1672.
57. Shan, X., Chiang, P.M., Price, D.L. and Wong, P.C. (2010) Altered
distributions of Gemini of coiled bodies and mitochondria in motor
neurons of TDP-43 transgenic mice. Proc. Natl Acad. Sci. USA, 107,
58. Xu, Y.F., Gendron, T.F., Zhang, Y.J., Lin, W.L., D’Alton, S., Sheng, H.,
Casey, M.C., Tong, J., Knight, J., Yu, X. et al. (2010) Wild-type human
TDP-43 expression causes TDP-43 phosphorylation, mitochondrial
aggregation, motor deficits, and early mortality in transgenic mice.
J. Neurosci., 30, 10851–10859.
59. Suzuki, H., Lee, K. and Matsuoka, M. (2011) TDP-43-induced death is
associated with altered regulation of BIM and BCL-XL and attenuated by
caspase-mediated TDP-43 cleavage. J. Biol. Chem., 286, 13171–13183.
60. Szabadkai, G., Bianchi, K., Varnai, P., De Stefani, D., Wieckowski, M.R.,
Cavagna, D., Nagy, A.I., Balla, T. and Rizzuto, R. (2006)
Chaperone-mediated coupling of endoplasmic reticulum and
mitochondrial Ca2+ channels. J. Cell Biol., 175, 901–911.
61. Rapizzi, E., Pinton, P., Szabadkai, G., Wieckowski, M.R., Vandecasteele,
G., Baird, G., Tuft, R.A., Fogarty, K.E. and Rizzuto, R. (2002)
Recombinant expression of the voltage-dependent anion channel enhances
the transfer of Ca2+ microdomains to mitochondria. J. Cell Biol., 159,
62. Zuchner, S., Mersiyanova, I.V., Muglia, M., Bissar-Tadmouri, N.,
Rochelle, J., Dadali, E.L., Zappia, M., Nelis, E., Patitucci, A., Senderek, J.
et al. (2004) Mutations in the mitochondrial GTPase mitofusin 2 cause
Charcot-Marie-Tooth neuropathy type 2A. Nat. Genet., 36, 449–451.
63. McLoughlin, D.M. and Miller, C.C.J. (1996) The intracellular
cytoplasmic domain of the Alzheimer’s disease amyloid precursor protein
interacts with phosphotyrosine binding domain proteins in the yeast
two-hybrid system. FEBS Lett., 397, 197–200.
64. Ackerley, S., Grierson, A.J., Brownlees, J., Thornhill, P., Anderton, B.H.,
Leigh, P.N., Shaw, C.E. and Miller, C.C.J. (2000) Glutamate slows axonal
transport of neurofilaments in transfected neurons. J. Cell Biol., 150,
65. De Vos, K.J., Allan, V.J., Grierson, A.J. and Sheetz, M.P. (2005)
Mitochondrial function and actin regulate dynamin-related protein
1-dependent mitochondrial fission. Curr. Biol., 15, 678–683.
66. De Vos, K.J., Sable, J., Miller, K.E. and Sheetz, M.P. (2003) Expression
of phosphatidylinositol (4,5) bisphosphate-specific pleckstrin homology
domains alters direction but not the level of axonal transport of
mitochondria. Mol. Biol. Cell, 14, 3636–3649.
67. De Vos, K., Severin, F., Van Herreweghe, F., Vancompernolle, K.,
Goossens, V., Hyman, A. and Grooten, J. (2000) Tumor necrosis factor
induces hyperphosphorylation of kinesin light chain and inhibits
kinesin-mediated transport of mitochondria. J. Cell Biol., 149,
68. Vagnoni, A., Rodriguez, L., Manser, C., De Vos, K.J. and Miller, C.C.J.
(2011) Phosphorylation of kinesin light chain-1 at serine-460 modulates
binding and trafficking of calsyntenin-1. J. Cell Sci., 124, 1032–1042.
Human Molecular Genetics, 2012, Vol. 21, No. 61311