BACKGROUND
cysteine side chains of cytosolic, nuclear and mitochondrial matrix proteins are maintained in their reduced state by a vigorous reductive pathway effected by a large pool of reduced thioredoxins.
by contrast, in the lumen of the endoplasmic reticulum  dithiol oxidation and disulfide reduction co-exist.
these competing reactions define the redox poise of the er, which is dominated by the twenty or so members of the protein disulfide isomerase family, the er counterparts of the thioredoxins .
oxidizing members of the protein disulfide isomerase  family, exemplified by pdi1a  <cit> , gain disulfides from upstream oxidants, such as ero <dig>  prdx <dig> and vkor1c and donate them to reduced er client proteins .
reducing members of the pdi family, exemplified by erdj <dig>   <cit>  attack misplaced disulfides on er clients, and the acquired disulfides are reduced by yet-to-be discovered mechanism, re-cycling the reducing pdi to its active state.
changes to the redox poise of the er can be tracked by suitably tuned sensors; these are proteins with a metastable disulfide that is kinetically-coupled to the lumenal pdis and whose redox status influences a measureable outcome, typically fluorescent properties  <cit> .
applying such sensors has revealed that cultured mammalian cells effectively defend their redox poise: neither imposition of unfolded protein load , nor manipulations that compromise the upstream oxidases, affect the steady-state disposition of er localized redox sensors  <cit> .
manipulations that affect er calcium stores present an intriguing contrast to this theme of stability of er redox poise.
inhibition of calcium re-uptake into the er, by targeting the smooth endoplasmic reticulum calcium atpase  pump with thapsigargin resulted in a reductive shift in er thiol redox poise.
this reductive shift is robust, as it has been observed with redox probes that have different biochemical properties: both the oxyr-yfp fusion known as hyper  <cit> , which in the er is predominantly a thiol redox probe  <cit> , and rogfpie  <cit>  and by measurements that exploit either redox-dependent shifts in excitation profiles  <cit>  or changes in fluorescence lifetime  <cit> .
in vitro measurements excluded a direct effect of calcium on the redox probe, validating the observed changes as faithful reflections of a reductive shift of the calcium-depleted er lumen.
furthermore, in a cell culture model of a professional secretory cell - the ar42j pancreatic acinar cell line - physiological mobilization of calcium stores by engagement of the cholecystokinin receptor, which couples to inositol trisphosphate receptor  release channels, promoted a similar reductive shift in the er  <cit> .
the link between calcium release and changes to er redox thus extends to the physiological range of er calcium signaling, implying that fluctuations in er redox accompany activity-dependent oscillations in calcium stores of secretory cells.
here, we report on findings that suggest a molecular mechanism for this pervasive phenomenon.
RESULTS
the core machinery for disulfide bond formation is indifferent to changes in calcium concentrations in the physiological range
to determine if the previously noted reductive shift in the steady state poise of the er affected by calcium depletion had a kinetic component, we compared the rate of recovery of the disulfide in reduced rogfpie following a dithiothreitol  pulse and its washout in untreated cells and in cells exposed to the er calcium re-uptake inhibitor thapsigargin.
the fluorescent lifetime of the probe declined in thapsigargin-treated hek 293 t cells, reflecting the reductive shift in the dithiol-disulfide steady state  <cit> .
a dtt reductive pulse further lowered the lifetime , but its rate of recovery was noticeably slower in the thapsigargin-treated cells: half time to recovery  <dig>  ±  <dig>  minutes  and  <dig>  ±  <dig>  minutes  .figure  <dig>
calcium insensitivity of the core disulfide oxidative machinery of the er.
trace of time-dependent changes in fluorescent lifetime  of er-localized rogfpie preceding and following a dithiothreitol  pulse and washout in untreated  and thapsigargin -treated  hek 293 t cells.
each data point represents the mean ± sd of fluorescence lifetime measured in ≥ <dig> cells.
note the shortening of the lifetime and slowness of its recovery following dtt reduction in the thapsigargin-treated group.
a comparison of the mean half-time to recovery in the untreated and treated cells is provided in the bar diagram to the right .
plot of the initial velocity of rogfpie oxidation in vitro  in the presence of varying concentration of pdi1a, in a glutathione redox buffer .
kinetic parameters were extracted by fitting the data to a non-linear regression.
as in ‘b’, with pdi1a held at k <dig> max  and varying concentration of ca2+.
as above, but in the presence of a saturating concentration of pdi1a  and varying concentration of ero <dig> .
as above, but in the presence of a saturating concentration of pdi1a , ero <dig> at k <dig> max  and varying concentration of ca2+.
er, endoplasmic reticulum; pdi1a, protein disulfide isomerase 1a; sd, standard deviation.
rogfpie is kinetically coupled to the major er oxidoreductase pdi1a and isolated from the small molecule redox buffer glutathione  <cit> .
to determine if the aforementioned delay in oxidation observed in vivo reflected a direct effect of calcium concentration on the relevant enzymes, we reconstructed the oxidative cascade linking molecular oxygen to reduced rogfpie in vitro, using purified ero <dig> and pdi1a, and established conditions for dependence on the two enzymes.
oxidation of reduced rogfpie by pdi1a, pre-equilibrated with a glutathione redox buffer, was half-maximal at  k <dig> max = 16 μm , but the oxidation of rogfpie was unaffected by calcium concentrations over a range of 1 μm to 10 mm .
the concentration-dependence of rogfpie oxidation on ero <dig>  was steep, but at ero <dig> concentration of  <dig>  μm, below the half-maximal concentration , oxidation of rogfpie was indifferent to calcium variation over a broad range .
pdi1a mobility is retarded by er calcium depletion in vivo
the experiments presented above suggest that the effects of calcium on the er redox poise observed in cells lack a simple in vitro counterpart in terms of the activity of the core oxidative machinery of the er.
to explore features that might be lacking from the simplified in vitro system, we compared the mobility of fluorescently-tagged pdi1a in untreated and calcium-depleted cells using fluorescent recovery after photobleaching .
to obtain suitable spatial resolution of er imaging we sought to make use of cos <dig> cells  and firstly confirmed that in these cells, too, thapsigargin-induced er calcium depletion resulted in a reductive shift of the er .
an mcherry genetically-encoded fluorescent probe was inserted into pdi1a, n-terminal to the kdel er retention signal.
when transfected into cos <dig> cells the fluorescent signal produced a typical er pattern that colocalized with the endogenous er chaperone bip .figure  <dig>
impaired pdi1a mobility in the calcium-depleted er.
fluorescent lifetime imaging of er rogfpie in untreated and thapsigargin-treated cos <dig> cells.
note the decline in mean lifetime, from  <dig>  ns to  <dig>  ns, reflecting a reductive shift in the probe.
a histogram of the distribution of lifetimes in the cells is provided  noting the mean ± sd  lifetime, on the background of the color code corresponding to the pseudocolored image.
a vertical dashed line extending through both panels is provided for alignment of the two measurements.
photomicrographs of immunostaining of endogenous bip , pdi1a-mcherry fluorescence  and an overlay of the two  in a cos <dig> cell transfected with a plasmid encoding an er-localized pdi1a-mcherry .
the purple hoechst stains the nucleus.
trace of time-dependent changes in the fluorescence intensity of pdi1a-mcherry before and after photobleaching a small patch of transfected cos <dig> cell volume.
the green trace is of an untreated cell and the orange of a cell exposed to thapsigargin 10 minutes before data acquisition.
photomicrographs of the pdi1a-mcherry fluorescence in the time-dependent series quantified in ‘c’ above.
as in ‘c’, except that cells were transfected with an expression plasmid encoding er-localized mcherry.
as in ‘d’ but from the time series of the er mcherry-expressing cells.
as in ‘c’, but cells were treated with the glycosylation inhibitor tunicamycin to promote unfolded protein stress in the er, without affecting er ca2+ levels.
time series of the sample shown in .
the mean ± sd of the half-time to recovery of the two samples is provided.
the size bar is 20 μm.
er, endoplasmic reticulum; pdi1a, protein disulfide isomerase 1a; standard deviation.
the pdi1a-mcherry fluorescence signal recovered  <dig>  times more rapidly in a photobleached patch of er from untreated cells compared with cells treated with thapsigargin .
the recovery of er localized mcherry fluorescence was unaffected by thapsigargin , as was the recovery of pdi1a-mcherry in cells treated with the glycosylation inhibitor tunicamycin, which induces high levels of er stress, without affecting calcium .
importantly, er calcium depletion retarded pdi1a mobility even in cells exposed to dtt , implying that reduced pdi1a is also subject to immobilization, a point we shall return to later.
pdi1a binds calreticulin in the calcium depleted er
the calcium depletion-dependent attenuation in pdi1a’s mobility in the er was consistent with its incorporation into a complex with other protein that were absent from the in vitro reconstituted system described in figure 1b-e. michalak and colleagues had previously noted quenching of fluorescently-labeled pdi1a by calreticulin  upon calcium chelation in an in vitro system, suggesting an interaction that could explain our in vivo observations  <cit> .
to explore this possibility further, we purified pdi1a-mcherry and crt-gfp from bacteria and measured the interaction between them by the intensity of the fluorescence resonance energy transfer  signal, as reflected in acceptor -dependent increase in fret ratio .
the fret signal decreased progressively with calcium concentration, with a midpoint,  <dig> , of 190 μm .
these -dependent changes in fret ratio were not observed with mcherry as the acceptor , attesting to the specificity of the pdi1a-crt interaction originally observed by michalak and colleagues.figure  <dig>
calcium concentration-dependent association of pdi1a and calreticulin
in vitro
.
schema of the calcium-dependent fluorescence resonance energy transfer  between a green fluorescent protein tagged calreticulin  donor and a monomeric cherry tagged pdi1a  acceptor.
photomicrograph of coomassie-stained fusion proteins, before and after tev protease cleavage of the gst tag, resolved on a 10% sds-page.
trace of calcium concentration-dependent changes in fret between the crt-gfp donor and the pdi1a-mcherry acceptor .
the half-maximal change in the fret signal is observed at 190 μm free ca2+.
shown is a typical trace of an experiment reproduced three times.
as in ‘c’ above, but substituting mcherry for pdi1a-mcherry as the fret acceptor.
pdi1a, protein disulfide isomerase 1a.
the crt tertiary structure is modulated by calcium concentration, with enhanced exposure of hydrophobic side-chains at low calcium concentrations  <cit> .
in keeping with this, we observed crt precipitation with declining concentration of calcium in the buffer.
crt precipitation was prevented by including pdi1a in the solution , supporting the fret measurements noted above and suggesting a role for the calcium-dependent association of the two proteins in maintaining crt’s solubility.
to explore the possible in vivo relevance of the pdi1a-crt interaction we exploited the superior accuracy of fluorescent lifetime imaging  measurements to detect fret  <cit> .
when applied to thapsigargin-treated cos <dig> cells, a calibrated er-localized calcium probe   <cit> , measured in flim-fret mode, revealed a decline in er  into the range at which pdi1a and crt were observed to interact in vitro .
this finding encouraged us to measure the effects of changes in er calcium on the interaction between pdi1a and crt in vivo.
the dynamic range of in vivo measurements of fret between a gfp donor and an mcherry acceptor were established by expressing gfp and mcherry separately and as a fusion protein in the er.
fusion of the two led to a drop in donor fluorescence lifetime  , setting an upper limit for fret efficiency of this pair.
when co-expressed with free mcherry, lifetime of a crt-gfp donor was unaffected by er calcium depletion ; however, a reproducible thapsigargin-dependent drop in donor lifetime was observed when a pdi1a-mcherry acceptor was introduced into the er .
fret with the pdi1a-mcherry acceptor further correlated with a two-fold attenuation in crt-gfp’s mobility as assessed by frap measurements in thapsigargin-treated cells , further suggesting the formation of a complex between the two proteins in the calcium depleted er.figure  <dig>
calcium depletion-dependent association of pdi1a and calreticulin
in vivo
.
donor  fluorescence  and donor lifetime images  of an er calcium probe, d1er cameleon, in untreated and thapsigargin-treated cos <dig> cells.
a histogram of the distribution of lifetimes in the cells is provided  noting the mean ± sd  lifetime.
also shown is a pseudo-colored image of calcium concentration derived from cameleon’s lifetime.
note the decline in mean er from approximately 3 mm to 109 μm in the treated sample.
donor  fluorescent lifetime of gfp transfected alongside mcherry, setting the lower limits of the fret for this pair.
the size bar is 20 μm.
donor  fluorescent lifetime a fusion protein of gfp and mcherry, setting an upper limit of fret for this pair.
donor  fluorescent lifetime of crt-gfp fusion protein transfected alongside an er mcherry, as a fret acceptor.
where indicated the cells were treated with thapsigargin .
donor  fluorescent lifetime of crt-gfp fusion protein transfected alongside pdi1a-mcherry, as a fret acceptor.
where indicated the cells were treated with thapsigargin .
bar diagram of fraction of gfp probe bound to mcherry probe derived from the fret measurements in b-e .
trace of time-dependent changes in the normalized intensity of crt-gfp after photobleaching a small patch of transfected cos <dig> cell volume.
the green trace is of untreated sample and the red of cells exposed to thapsigargin 10 minutes before data acquisition.
the mean ± sd of the half-time to recovery of the two samples is provided.
crt, calreticulin; er, endoplasmic reticulum; fret, fluorescence resonance imaging measurement; sd, standard deviation; sem, standard error of the mean.
to address the potential physiological relevance of the interaction between pdi1a and crt, we turned to ar42j cells, a pancreatic acinar cell line in which physiological calcium store depletion is coupled to stimulation by the hormone cholecystokinin, resulting in a reductive shift in the er  <cit> .
calcium imaging revealed a cholecystokinin dose-dependent dip in er into the range at which pdi1a and crt were observed to interact in vitro .
expectedly, gfp to mcherry fret was readily observed in these cells , as was the lack of an interaction between the crt-gfp donor and a free mcherry acceptor .
however, conspicuous time-dependent fret from the crt-gfp donor to the pdi1a-mcherry acceptor was also observed in cholecystokinin-treated cells , consistent with the formation of a complex between the two proteins upon physiological er calcium store emptying.figure  <dig>
association of pdi1a and calreticulin under physiological conditions of calcium depletion.
donor  fluorescent intensity and donor life time images  of d1er cameleon in untreated and cholecystokinin -treated  pancreatic acinar ar42j cells.
a histogram of the distribution of lifetimes is provided  noting the mean ± sd  lifetime of the cells in the sample.
further to the right is a pseudo-colored image of calcium concentration derived from cameleon’s life time and notation of the mean of the concentration in each sample.
note the decline in mean er from  <dig>  mm in the untreated to 80 μm in the sample exposed to the highest concentration of cck.
the size bar is 20 μm.
donor  fluorescent lifetime of gfp transfected alongside mcherry, setting the lower limits of the fret for this pair in ar42j cells.
donor  fluorescent lifetime of a fusion protein of gfp and mcherry, setting an upper limit of fret for this pair in ar42j cells.
donor  fluorescent lifetime of crt-gfp fusion protein transfected alongside and er mcherry, as a fret acceptor.
where indicated the cells were treated with cck .
donor  fluorescent lifetime of crt-gfp fusion protein transfected alongside pdi1a-mcherry, as a fret acceptor.
samples were treated with cck  for the indicated time.
bar diagram of fraction of gfp probe bound to mcherry probe derived from the fret measurements in b-e .
the size bar is 20 μm.
crt, calreticulin; fret, fluorescence resonance energy transfer; pdi1a, protein disulfide isomerase 1a; sd, standard deviation; sem, standard error of the mean.
association of pdi1a with calreticulin was also observed in dtt-treated cells , implying that crt-binding was not restricted to the oxidized form of pdi1a, which is noted for its higher affinity for unfolded proteins  <cit> .
furthermore, complex formation with crt did not affect pdi1a’s ability to catalyze the oxidation of reduced rogfpie in vitro .
these negative results leave altered pdi1a mobility as the notable correlate of calcium depletion.
next, we turned to erdj <dig>  a pdi family member that has been reported to play an important role in disulfide bond reduction in the er  <cit> .
as expected, erdj <dig> tagged at its c-terminus with mcherry localized to the er of transfected cos <dig> cells .
however, unlike pdi1a , mobility of erdj5-mcherry remained unaffected by er calcium depletion .
furthermore, er calcium depletion did not promote an interaction between erdj5-mcherry and crt-gfp, as the fret signal in cells co-expressing the proteins was unaltered by treatment with thapsigargin .figure  <dig>
mobility of erdj <dig>  a reducing member of the pdi family, is unaffected by er calcium depletion.
photomicrographs of immunostaining of endogenous bip , erdj5-mcherry fluorescence  and an overlay of the two  in cos <dig> cells transfected with a plasmid encoding an er-localized erdj5-mcherry .
the purple hoechst stains the nucleus.
the size bar is 20 μm.
trace of time-dependent changes in the normalized intensity of erdj5-mcherry after photobleaching a small patch of transfected cos <dig> cell volume.
the green trace is of an untreated sample and the orange of cells exposed to thapsigargin 10 minutes before data acquisition.
the mean ± sd of the half-time to recovery of the two samples is provided.
donor  fluorescent lifetime of crt-gfp fusion protein transfected alongside erdj5-mcherry, as a fret acceptor.
where indicated the sample was treated with thapsigargin.
donor  fluorescent lifetime of crt-gfp fusion protein transfected alongside er-localized mcherry, as a control fret acceptor or ferdj5-mcherry.
donor  fluorescent lifetime of a gfp-mcherry fusion protein setting the upper limit for fret.
the mean ± sd  lifetime noted on the histograms.
crt, calreticulin; er, endoplasmic reticulum; fret, fluorescence resonance energy transfer; pdi1a, protein disulfide isomerase 1a; sd, standard deviation.
the contrasting behavior of the oxidizing and reducing prototypical pdi family members, suggested a role for selective partial immobilization/inactivation of pdi1a in the altered redox poise of the calcium depleted er.
pdi1a is an abundant and long-lived protein; nonetheless, it was possible to bring about a partial transient decrease of its level by shrna transfection of hek 293 t cells.
the effects of knockdown on er redox were modest compared with those of thapsigargin treatment .
however, the reductive shift, as measured by decreased fluorescence lifetime of er-localized rogfpie, correlated well with the extent of pdi1a knockdown across the six different shrna constructs used , consistent with a measureable effect of attenuated pdi1a availability on er redox poise.figure  <dig>
pdi1a depletion correlates with a more reduced er.
bar diagram of fluorescent lifetime  of rogfpie in hek 293 t cells co-transfected with different shrnas directed to pdi1a and an empty shrna vector control.
flt of untreated  thapsigargin  and dtt treated cells is included as a reference.
immunoblot of endogenous pdi1a in the cells above, with tubulin serving as a loading control.
the pdi1a to tubulin ratio  is provided under each sample.
scatter diagram of correlation of flt to pdi1a expression in the samples above .
bar diagram of fluorescent lifetime  of rogfpie in wild type mouse embryonic fibroblasts  and in mouse embryonic fibroblasts lacking calreticulin   <cit> .
where indicated, the cells were exposed to thapsigargin  or dtt .
shown is the mean ± sd of flt .
.
anova, analysis of variance; dtt, dithiotreitol; er, endoplasmic reticulum; pdi1a, protein disulfide isomerase; sd, standard deviation.
genetic evidence for the importance of crt to the reductive shift in the calcium-depleted er was provided by comparison of rogfpie fluorescent lifetime in wild type and crt-deficient mouse embryonic fibroblasts.
the absence of crt had no effect on the basal redox state of the probe or on its redox state in cells exposed to dtt.
however, the reductive shift observed upon exposure of the wild type cells to the er calcium depleting agent thapsigargin , was blunted in the crt-deficient cells   indicating a non-redundant role for crt in promoting the reductive shift in the calcium depleted er.
discussion
the reductive shift in er protein thiol redox poise that follows lumenal calcium depletion is shown here to correlate with a kinetic defect in the rate of recovery of disulfides following a reductive pulse and its washout.
however, the core enzymatic machinery that produces disulfides in the er and conveys them to reduced protein substrates is unresponsive to calcium in vitro.
in vivo, however, the mobility of the prototypical oxidizing pdi, pdi1a, is lowered by calcium depletion and this correlates with the formation of a calcium-suppressible complex between pdi1a and the er chaperone crt, both in vitro and in vivo.
while our findings fall short of establishing causality between impaired mobility of pdi1a and the oxidative defect in the calcium-depleted er lumen, the selectivity of the immobilization for the oxidizing pdi family member, reflected in the sparing of erdj <dig> mobility, supports such a link.
variation in calcium concentrations within the physiological range regulate the formation of a previously-observed complex of pdi1a and crt  <cit> , suggesting that this complex , account for the reduced mobility of the oxidizing pdi1a and, therefore, a selectively attenuated turnover of the oxidizing limb of the er lumen .
the reductive shift noted upon er calcium depletion is significantly attenuated but not completely lost in cells lacking crt, indicating the existence of other contributory factors.figure  <dig>
schematic of the effects of lumenal calcium depletion on availability of pdi1a to fuel client protein oxidation in the er.
in the calcium replete er, pdi1a is free to engage in oxidative protein folding by shuttling electrons from reduced client protein  to terminal acceptors  and oxidation  competes favorably with reduction .
in the calcium depleted er, pdi1a is sequestered in a low mobility complex with the abundant chaperone calreticulin, compromising its availability to power the oxidative limb of the er.
the reductive limb, mediated by erdj <dig>  is unaffected by calcium depletion.
the net result is a shift in the balance of the calcium-depleted er towards a more reduced poise.
er, endoplasmic reticulum; pdi1a, protein disulfide isomerase 1a.
crt is well suited to serve as a calcium sensor in the interaction with pdi1a, as it has been noted to change its tertiary structure in response to changes in calcium concentration, with increased exposure of hydrophobic surface as calcium concentration decreases  <cit> .
pdi1a and crt are both abundant er proteins and their c-termini are rich in acidic residues that could mediate calcium responsiveness of complex formation.
however, deletion of the c-terminal regions  proved insufficient to abolish the calcium depletion-mediated interaction, frustrating our efforts at gross deletional mapping of the calcium-dependent interaction between them.
association with crt does not inactivate pdi1a in vitro, and neither complex formation, nor in vivo mobility exhibit measureable specificity for the oxidized form of pdi1a.
rather, our findings fit best with non-selective sequestration of both reduced and oxidized pdi1a in lower mobility complex in the calcium depleted er.
the frap traces of pdi1a do not resolve into a simple model of only two pools , thus the pdi1a-crt complex may be heterogeneous or pdi1a may interact with other proteins too, forming an ensemble of complexes that diffuse slowly in the calcium-depleted er.
indeed, the  <dig>  fold decrease in pdi1a upon calcium depletion fits with a complex larger than merely a pdi1a-crt dimer.
unfortunately, instability of the pdi1a-crt complex has frustrated our efforts at isolation and biochemical characterization.
despite these limitations to our understanding, the mechanism promoting a reductive shift in the calcium depleted er are defined with considerable specificity.
that a reductive shift is not observed in conditions associated with markedly elevated levels of unfolded protein stress in the er  argues that promiscuous effects of calcium depletion on the handling of secreted proteins are unlikely to contribute.
the evidence for specificity is further buttressed by the significant effacement of the reductive shift in cells lacking crt.
what might be the physiological significance of the reductive shift of the calcium-depleted er?
it is best to begin by acknowledging the possibility that redox effects may not be under positive selection but rather reflect a consequence of a role for pdi1a in maintaining crt’s solubility through physiological fluctuations in er.
however, it is also intriguing to consider this question in the context of physiological circumstances associated with calcium excursions into the range that effect pdi1a-crt association and er reduction.
such excursions are noted in secretory cells responding to secretagogues and are associated with physiological activation of the unfolded protein response  <cit> .
the reductive shift in the er may contribute to such activation, transiently inhibiting protein synthesis, through rapidly reversible protein kinase rna-like endoplasmic reticulum kinase -mediated eif <dig> phosphorylation  <cit> .
transient inhibition of energetically-costly protein synthesis may be especially important in insulin-producing beta cells, in which coupling of insulin secretion to blood glucose requires high levels of intracellular atp  <cit> .
furthermore, coupling secretory stimuli  to periodic activation of upr target genes would have enduring effects on er size and on the long-term capacity to produce secreted products.
the evolution of an er that is more reducing under conditions of er calcium store depletion would promote fitness by coupling the action of calcium-mobilizing secretagogues to a upr-mediated transient attenuation of protein synthesis followed by long-term growth of the er.
transient reductive departures from the normally oxidizing poise of the er may provide other benefits to cells.
shifting the balance from the oxidizing pdi1a to the reducing erdj <dig> is predicted to favor degradation of disulfide bonded misfolded proteins  <cit> .
thus, transient cycles of reduction may be helpful in periodically purging the er of recalcitrant misfolded proteins, while maintaining er redox at levels optimized for oxidative folding most of the time.
coupling such reductive shifts to perk-mediated attenuation of protein synthesis would lessen their impact on oxidative folding of newly-synthesized proteins, while promoting the degradation of refuse.
a functional link between er redox and calcium homeostasis is suggested by the phenotype of mice lacking ero <dig> genes.
cells explanted from such animals have no steady state defect in er thiol redox poise, but recover sluggishly from reductive excursions  <cit> .
the mice are superficially indistinguishable from wild type; however, calcium handling by cardiomyocytes is grossly impaired, supporting a role for rapid kinetics of disulfide recovery in er calcium release and re-uptake  <cit> .
calcium handling by the er is subject to redox regulation.
levels of ero <dig> activity modulate calcium fluxes in and out of the er  <cit> .
subtype  <dig> of the ip <dig> receptor, which conducts lumenal calcium to the cytosol in response to activation of membrane receptors, is inhibited by the formation of a mixed disulfide between cysteine residues on its third lumenal loop and reduced erp <dig>  <cit> , a reducing pdi family member.
by contrast, the broadly-expressed calcium reuptake pump, serca2b is inhibited by the formation of a disulfide with the oxidizing pdi family member erp <dig>  <cit> .
as disulfide exchange between pdi family members is pervasive, these observations suggest that a more reduced er poise would favor re-uptake of calcium into the er and disfavor its release.
it follows that immobilization of the oxidizing pdi1a, which can transfer disulfides to erp <dig>  <cit> , and continued mobility of the reducing erdj <dig> would favor restoration of calcium stores to the calcium-depleted er closing a homeostatic loop.
CONCLUSIONS
the reductive shift in er redox poise, observed at physiological er calcium store depletion, is an unusual exception to a landscape dominated by stability of er redox balance.
direct calcium responsiveness of the core machinery for oxidizing er client protein thiols has been dismissed by our studies.
however a remarkable correlation between lumenal calcium depletion and lowered mobility of the oxidizing pdi1a in the er lumen was observed.
this, in turn, is plausibly explained by formation of a calcium responsive complex between pdi1a and the abundant er chaperone crt with the effect of sequestering a key component of the oxidizing limb of the er .
the consequent attenuation of client protein oxidation accompanies physiological signals that specify secretion, thus delineating a hitherto unanticipated link between secretagogue action and protein folding homeostasis in the er.

 cysteine side chains of cytosolic, nuclear and mitochondrial matrix proteins are maintained in their reduced state by a vigorous reductive pathway effected by a large pool of reduced thioredoxins. by contrast, in the lumen of the endoplasmic reticulum dithiol oxidation and disulfide reduction co-exist. these competing reactions define the redox poise of the er, which is dominated by the twenty or so members of the protein disulfide isomerase family, the er counterparts of the thioredoxins. oxidizing members of the protein disulfide isomerase family, exemplified by pdi1a, gain disulfides from upstream oxidants, such as ero prdx and vkor1c and donate them to reduced er client proteins. reducing members of the pdi family, exemplified by erdj attack misplaced disulfides on er clients, and the acquired disulfides are reduced by yet-to-be discovered mechanism, re-cycling the reducing pdi to its active state. changes to the redox poise of the er can be tracked by suitably tuned sensors; these are proteins with a metastable disulfide that is kinetically-coupled to the lumenal pdis and whose redox status influences a measureable outcome, typically fluorescent properties. applying such sensors has revealed that cultured mammalian cells effectively defend their redox poise: neither imposition of unfolded protein load, nor manipulations that compromise the upstream oxidases, affect the steady-state disposition of er localized redox sensors. manipulations that affect er calcium stores present an intriguing contrast to this theme of stability of er redox poise. inhibition of calcium re-uptake into the er, by targeting the smooth endoplasmic reticulum calcium atpase pump with thapsigargin resulted in a reductive shift in er thiol redox poise. this reductive shift is robust, as it has been observed with redox probes that have different biochemical properties: both the oxyr-yfp fusion known as hyper, which in the er is predominantly a thiol redox probe, and rogfpie and by measurements that exploit either redox-dependent shifts in excitation profiles or changes in fluorescence lifetime. in vitro measurements excluded a direct effect of calcium on the redox probe, validating the observed changes as faithful reflections of a reductive shift of the calcium-depleted er lumen. furthermore, in a cell culture model of a professional secretory cell - the ar42j pancreatic acinar cell line - physiological mobilization of calcium stores by engagement of the cholecystokinin receptor, which couples to inositol trisphosphate receptor release channels, promoted a similar reductive shift in the er. the link between calcium release and changes to er redox thus extends to the physiological range of er calcium signaling, implying that fluctuations in er redox accompany activity-dependent oscillations in calcium stores of secretory cells. here, we report on findings that suggest a molecular mechanism for this pervasive phenomenon.  the core machinery for disulfide bond formation is indifferent to changes in calcium concentrations in the physiological range to determine if the previously noted reductive shift in the steady state poise of the er affected by calcium depletion had a kinetic component, we compared the rate of recovery of the disulfide in reduced rogfpie following a dithiothreitol pulse and its washout in untreated cells and in cells exposed to the er calcium re-uptake inhibitor thapsigargin. the fluorescent lifetime of the probe declined in thapsigargin-treated hek 293 t cells, reflecting the reductive shift in the dithiol-disulfide steady state. a dtt reductive pulse further lowered the lifetime, but its rate of recovery was noticeably slower in the thapsigargin-treated cells: half time to recovery  ±   minutes and  ±   minutes.figure calcium insensitivity of the core disulfide oxidative machinery of the er. trace of time-dependent changes in fluorescent lifetime of er-localized rogfpie preceding and following a dithiothreitol pulse and washout in untreated and thapsigargin -treated hek 293 t cells. each data point represents the mean ± sd of fluorescence lifetime measured in ≥ cells. note the shortening of the lifetime and slowness of its recovery following dtt reduction in the thapsigargin-treated group. a comparison of the mean half-time to recovery in the untreated and treated cells is provided in the bar diagram to the right. plot of the initial velocity of rogfpie oxidation in vitro in the presence of varying concentration of pdi1a, in a glutathione redox buffer. kinetic parameters were extracted by fitting the data to a non-linear regression. as in ‘b’, with pdi1a held at k max and varying concentration of ca2+. as above, but in the presence of a saturating concentration of pdi1a and varying concentration of ero. as above, but in the presence of a saturating concentration of pdi1a, ero at k max and varying concentration of ca2+. er, endoplasmic reticulum; pdi1a, protein disulfide isomerase 1a; sd, standard deviation. rogfpie is kinetically coupled to the major er oxidoreductase pdi1a and isolated from the small molecule redox buffer glutathione. to determine if the aforementioned delay in oxidation observed in vivo reflected a direct effect of calcium concentration on the relevant enzymes, we reconstructed the oxidative cascade linking molecular oxygen to reduced rogfpie in vitro, using purified ero and pdi1a, and established conditions for dependence on the two enzymes. oxidation of reduced rogfpie by pdi1a, pre-equilibrated with a glutathione redox buffer, was half-maximal at k max = 16 μm, but the oxidation of rogfpie was unaffected by calcium concentrations over a range of 1 μm to 10 mm. the concentration-dependence of rogfpie oxidation on ero was steep, but at ero concentration of  μm, below the half-maximal concentration, oxidation of rogfpie was indifferent to calcium variation over a broad range. pdi1a mobility is retarded by er calcium depletion in vivo the experiments presented above suggest that the effects of calcium on the er redox poise observed in cells lack a simple in vitro counterpart in terms of the activity of the core oxidative machinery of the er. to explore features that might be lacking from the simplified in vitro system, we compared the mobility of fluorescently-tagged pdi1a in untreated and calcium-depleted cells using fluorescent recovery after photobleaching. to obtain suitable spatial resolution of er imaging we sought to make use of cos cells and firstly confirmed that in these cells, too, thapsigargin-induced er calcium depletion resulted in a reductive shift of the er. an mcherry genetically-encoded fluorescent probe was inserted into pdi1a, n-terminal to the kdel er retention signal. when transfected into cos cells the fluorescent signal produced a typical er pattern that colocalized with the endogenous er chaperone bip.figure impaired pdi1a mobility in the calcium-depleted er. fluorescent lifetime imaging of er rogfpie in untreated and thapsigargin-treated cos cells. note the decline in mean lifetime, from  ns to  ns, reflecting a reductive shift in the probe. a histogram of the distribution of lifetimes in the cells is provided noting the mean ± sd lifetime, on the background of the color code corresponding to the pseudocolored image. a vertical dashed line extending through both panels is provided for alignment of the two measurements. photomicrographs of immunostaining of endogenous bip, pdi1a-mcherry fluorescence and an overlay of the two in a cos cell transfected with a plasmid encoding an er-localized pdi1a-mcherry. the purple hoechst stains the nucleus. trace of time-dependent changes in the fluorescence intensity of pdi1a-mcherry before and after photobleaching a small patch of transfected cos cell volume. the green trace is of an untreated cell and the orange of a cell exposed to thapsigargin 10 minutes before data acquisition. photomicrographs of the pdi1a-mcherry fluorescence in the time-dependent series quantified in ‘c’ above. as in ‘c’, except that cells were transfected with an expression plasmid encoding er-localized mcherry. as in ‘d’ but from the time series of the er mcherry-expressing cells. as in ‘c’, but cells were treated with the glycosylation inhibitor tunicamycin to promote unfolded protein stress in the er, without affecting er ca2+ levels. time series of the sample shown in. the mean ± sd of the half-time to recovery of the two samples is provided. the size bar is 20 μm. er, endoplasmic reticulum; pdi1a, protein disulfide isomerase 1a; standard deviation. the pdi1a-mcherry fluorescence signal recovered times more rapidly in a photobleached patch of er from untreated cells compared with cells treated with thapsigargin. the recovery of er localized mcherry fluorescence was unaffected by thapsigargin, as was the recovery of pdi1a-mcherry in cells treated with the glycosylation inhibitor tunicamycin, which induces high levels of er stress, without affecting calcium. importantly, er calcium depletion retarded pdi1a mobility even in cells exposed to dtt, implying that reduced pdi1a is also subject to immobilization, a point we shall return to later. pdi1a binds calreticulin in the calcium depleted er the calcium depletion-dependent attenuation in pdi1a’s mobility in the er was consistent with its incorporation into a complex with other protein that were absent from the in vitro reconstituted system described in figure 1b-e. michalak and colleagues had previously noted quenching of fluorescently-labeled pdi1a by calreticulin upon calcium chelation in an in vitro system, suggesting an interaction that could explain our in vivo observations. to explore this possibility further, we purified pdi1a-mcherry and crt-gfp from bacteria and measured the interaction between them by the intensity of the fluorescence resonance energy transfer signal, as reflected in acceptor -dependent increase in fret ratio. the fret signal decreased progressively with calcium concentration, with a midpoint,, of 190 μm. these -dependent changes in fret ratio were not observed with mcherry as the acceptor, attesting to the specificity of the pdi1a-crt interaction originally observed by michalak and colleagues.figure calcium concentration-dependent association of pdi1a and calreticulin in vitro. schema of the calcium-dependent fluorescence resonance energy transfer between a green fluorescent protein tagged calreticulin donor and a monomeric cherry tagged pdi1a acceptor. photomicrograph of coomassie-stained fusion proteins, before and after tev protease cleavage of the gst tag, resolved on a 10% sds-page. trace of calcium concentration-dependent changes in fret between the crt-gfp donor and the pdi1a-mcherry acceptor. the half-maximal change in the fret signal is observed at 190 μm free ca2+. shown is a typical trace of an experiment reproduced three times. as in ‘c’ above, but substituting mcherry for pdi1a-mcherry as the fret acceptor. pdi1a, protein disulfide isomerase 1a. the crt tertiary structure is modulated by calcium concentration, with enhanced exposure of hydrophobic side-chains at low calcium concentrations. in keeping with this, we observed crt precipitation with declining concentration of calcium in the buffer. crt precipitation was prevented by including pdi1a in the solution, supporting the fret measurements noted above and suggesting a role for the calcium-dependent association of the two proteins in maintaining crt’s solubility. to explore the possible in vivo relevance of the pdi1a-crt interaction we exploited the superior accuracy of fluorescent lifetime imaging measurements to detect fret. when applied to thapsigargin-treated cos cells, a calibrated er-localized calcium probe, measured in flim-fret mode, revealed a decline in er into the range at which pdi1a and crt were observed to interact in vitro. this finding encouraged us to measure the effects of changes in er calcium on the interaction between pdi1a and crt in vivo. the dynamic range of in vivo measurements of fret between a gfp donor and an mcherry acceptor were established by expressing gfp and mcherry separately and as a fusion protein in the er. fusion of the two led to a drop in donor fluorescence lifetime, setting an upper limit for fret efficiency of this pair. when co-expressed with free mcherry, lifetime of a crt-gfp donor was unaffected by er calcium depletion; however, a reproducible thapsigargin-dependent drop in donor lifetime was observed when a pdi1a-mcherry acceptor was introduced into the er. fret with the pdi1a-mcherry acceptor further correlated with a two-fold attenuation in crt-gfp’s mobility as assessed by frap measurements in thapsigargin-treated cells, further suggesting the formation of a complex between the two proteins in the calcium depleted er.figure calcium depletion-dependent association of pdi1a and calreticulin in vivo. donor fluorescence and donor lifetime images of an er calcium probe, d1er cameleon, in untreated and thapsigargin-treated cos cells. a histogram of the distribution of lifetimes in the cells is provided noting the mean ± sd lifetime. also shown is a pseudo-colored image of calcium concentration derived from cameleon’s lifetime. note the decline in mean er from approximately 3 mm to 109 μm in the treated sample. donor fluorescent lifetime of gfp transfected alongside mcherry, setting the lower limits of the fret for this pair. the size bar is 20 μm. donor fluorescent lifetime a fusion protein of gfp and mcherry, setting an upper limit of fret for this pair. donor fluorescent lifetime of crt-gfp fusion protein transfected alongside an er mcherry, as a fret acceptor. where indicated the cells were treated with thapsigargin. donor fluorescent lifetime of crt-gfp fusion protein transfected alongside pdi1a-mcherry, as a fret acceptor. where indicated the cells were treated with thapsigargin. bar diagram of fraction of gfp probe bound to mcherry probe derived from the fret measurements in b-e. trace of time-dependent changes in the normalized intensity of crt-gfp after photobleaching a small patch of transfected cos cell volume. the green trace is of untreated sample and the red of cells exposed to thapsigargin 10 minutes before data acquisition. the mean ± sd of the half-time to recovery of the two samples is provided. crt, calreticulin; er, endoplasmic reticulum; fret, fluorescence resonance imaging measurement; sd, standard deviation; sem, standard error of the mean. to address the potential physiological relevance of the interaction between pdi1a and crt, we turned to ar42j cells, a pancreatic acinar cell line in which physiological calcium store depletion is coupled to stimulation by the hormone cholecystokinin, resulting in a reductive shift in the er. calcium imaging revealed a cholecystokinin dose-dependent dip in er into the range at which pdi1a and crt were observed to interact in vitro. expectedly, gfp to mcherry fret was readily observed in these cells, as was the lack of an interaction between the crt-gfp donor and a free mcherry acceptor. however, conspicuous time-dependent fret from the crt-gfp donor to the pdi1a-mcherry acceptor was also observed in cholecystokinin-treated cells, consistent with the formation of a complex between the two proteins upon physiological er calcium store emptying.figure association of pdi1a and calreticulin under physiological conditions of calcium depletion. donor fluorescent intensity and donor life time images of d1er cameleon in untreated and cholecystokinin -treated pancreatic acinar ar42j cells. a histogram of the distribution of lifetimes is provided noting the mean ± sd lifetime of the cells in the sample. further to the right is a pseudo-colored image of calcium concentration derived from cameleon’s life time and notation of the mean of the concentration in each sample. note the decline in mean er from  mm in the untreated to 80 μm in the sample exposed to the highest concentration of cck. the size bar is 20 μm. donor fluorescent lifetime of gfp transfected alongside mcherry, setting the lower limits of the fret for this pair in ar42j cells. donor fluorescent lifetime of a fusion protein of gfp and mcherry, setting an upper limit of fret for this pair in ar42j cells. donor fluorescent lifetime of crt-gfp fusion protein transfected alongside and er mcherry, as a fret acceptor. where indicated the cells were treated with cck. donor fluorescent lifetime of crt-gfp fusion protein transfected alongside pdi1a-mcherry, as a fret acceptor. samples were treated with cck for the indicated time. bar diagram of fraction of gfp probe bound to mcherry probe derived from the fret measurements in b-e. the size bar is 20 μm. crt, calreticulin; fret, fluorescence resonance energy transfer; pdi1a, protein disulfide isomerase 1a; sd, standard deviation; sem, standard error of the mean. association of pdi1a with calreticulin was also observed in dtt-treated cells, implying that crt-binding was not restricted to the oxidized form of pdi1a, which is noted for its higher affinity for unfolded proteins. furthermore, complex formation with crt did not affect pdi1a’s ability to catalyze the oxidation of reduced rogfpie in vitro. these negative results leave altered pdi1a mobility as the notable correlate of calcium depletion. next, we turned to erdj a pdi family member that has been reported to play an important role in disulfide bond reduction in the er. as expected, erdj tagged at its c-terminus with mcherry localized to the er of transfected cos cells. however, unlike pdi1a, mobility of erdj5-mcherry remained unaffected by er calcium depletion. furthermore, er calcium depletion did not promote an interaction between erdj5-mcherry and crt-gfp, as the fret signal in cells co-expressing the proteins was unaltered by treatment with thapsigargin.figure mobility of erdj a reducing member of the pdi family, is unaffected by er calcium depletion. photomicrographs of immunostaining of endogenous bip, erdj5-mcherry fluorescence and an overlay of the two in cos cells transfected with a plasmid encoding an er-localized erdj5-mcherry. the purple hoechst stains the nucleus. the size bar is 20 μm. trace of time-dependent changes in the normalized intensity of erdj5-mcherry after photobleaching a small patch of transfected cos cell volume. the green trace is of an untreated sample and the orange of cells exposed to thapsigargin 10 minutes before data acquisition. the mean ± sd of the half-time to recovery of the two samples is provided. donor fluorescent lifetime of crt-gfp fusion protein transfected alongside erdj5-mcherry, as a fret acceptor. where indicated the sample was treated with thapsigargin. donor fluorescent lifetime of crt-gfp fusion protein transfected alongside er-localized mcherry, as a control fret acceptor or ferdj5-mcherry. donor fluorescent lifetime of a gfp-mcherry fusion protein setting the upper limit for fret. the mean ± sd lifetime noted on the histograms. crt, calreticulin; er, endoplasmic reticulum; fret, fluorescence resonance energy transfer; pdi1a, protein disulfide isomerase 1a; sd, standard deviation. the contrasting behavior of the oxidizing and reducing prototypical pdi family members, suggested a role for selective partial immobilization/inactivation of pdi1a in the altered redox poise of the calcium depleted er. pdi1a is an abundant and long-lived protein; nonetheless, it was possible to bring about a partial transient decrease of its level by shrna transfection of hek 293 t cells. the effects of knockdown on er redox were modest compared with those of thapsigargin treatment. however, the reductive shift, as measured by decreased fluorescence lifetime of er-localized rogfpie, correlated well with the extent of pdi1a knockdown across the six different shrna constructs used, consistent with a measureable effect of attenuated pdi1a availability on er redox poise.figure pdi1a depletion correlates with a more reduced er. bar diagram of fluorescent lifetime of rogfpie in hek 293 t cells co-transfected with different shrnas directed to pdi1a and an empty shrna vector control. flt of untreated thapsigargin and dtt treated cells is included as a reference. immunoblot of endogenous pdi1a in the cells above, with tubulin serving as a loading control. the pdi1a to tubulin ratio is provided under each sample. scatter diagram of correlation of flt to pdi1a expression in the samples above. bar diagram of fluorescent lifetime of rogfpie in wild type mouse embryonic fibroblasts and in mouse embryonic fibroblasts lacking calreticulin. where indicated, the cells were exposed to thapsigargin or dtt. shown is the mean ± sd of flt.. anova, analysis of variance; dtt, dithiotreitol; er, endoplasmic reticulum; pdi1a, protein disulfide isomerase; sd, standard deviation. genetic evidence for the importance of crt to the reductive shift in the calcium-depleted er was provided by comparison of rogfpie fluorescent lifetime in wild type and crt-deficient mouse embryonic fibroblasts. the absence of crt had no effect on the basal redox state of the probe or on its redox state in cells exposed to dtt. however, the reductive shift observed upon exposure of the wild type cells to the er calcium depleting agent thapsigargin, was blunted in the crt-deficient cells indicating a non-redundant role for crt in promoting the reductive shift in the calcium depleted er. discussion the reductive shift in er protein thiol redox poise that follows lumenal calcium depletion is shown here to correlate with a kinetic defect in the rate of recovery of disulfides following a reductive pulse and its washout. however, the core enzymatic machinery that produces disulfides in the er and conveys them to reduced protein substrates is unresponsive to calcium in vitro. in vivo, however, the mobility of the prototypical oxidizing pdi, pdi1a, is lowered by calcium depletion and this correlates with the formation of a calcium-suppressible complex between pdi1a and the er chaperone crt, both in vitro and in vivo. while our findings fall short of establishing causality between impaired mobility of pdi1a and the oxidative defect in the calcium-depleted er lumen, the selectivity of the immobilization for the oxidizing pdi family member, reflected in the sparing of erdj mobility, supports such a link. variation in calcium concentrations within the physiological range regulate the formation of a previously-observed complex of pdi1a and crt, suggesting that this complex, account for the reduced mobility of the oxidizing pdi1a and, therefore, a selectively attenuated turnover of the oxidizing limb of the er lumen. the reductive shift noted upon er calcium depletion is significantly attenuated but not completely lost in cells lacking crt, indicating the existence of other contributory factors.figure schematic of the effects of lumenal calcium depletion on availability of pdi1a to fuel client protein oxidation in the er. in the calcium replete er, pdi1a is free to engage in oxidative protein folding by shuttling electrons from reduced client protein to terminal acceptors and oxidation competes favorably with reduction. in the calcium depleted er, pdi1a is sequestered in a low mobility complex with the abundant chaperone calreticulin, compromising its availability to power the oxidative limb of the er. the reductive limb, mediated by erdj is unaffected by calcium depletion. the net result is a shift in the balance of the calcium-depleted er towards a more reduced poise. er, endoplasmic reticulum; pdi1a, protein disulfide isomerase 1a. crt is well suited to serve as a calcium sensor in the interaction with pdi1a, as it has been noted to change its tertiary structure in response to changes in calcium concentration, with increased exposure of hydrophobic surface as calcium concentration decreases. pdi1a and crt are both abundant er proteins and their c-termini are rich in acidic residues that could mediate calcium responsiveness of complex formation. however, deletion of the c-terminal regions proved insufficient to abolish the calcium depletion-mediated interaction, frustrating our efforts at gross deletional mapping of the calcium-dependent interaction between them. association with crt does not inactivate pdi1a in vitro, and neither complex formation, nor in vivo mobility exhibit measureable specificity for the oxidized form of pdi1a. rather, our findings fit best with non-selective sequestration of both reduced and oxidized pdi1a in lower mobility complex in the calcium depleted er. the frap traces of pdi1a do not resolve into a simple model of only two pools, thus the pdi1a-crt complex may be heterogeneous or pdi1a may interact with other proteins too, forming an ensemble of complexes that diffuse slowly in the calcium-depleted er. indeed, the fold decrease in pdi1a upon calcium depletion fits with a complex larger than merely a pdi1a-crt dimer. unfortunately, instability of the pdi1a-crt complex has frustrated our efforts at isolation and biochemical characterization. despite these limitations to our understanding, the mechanism promoting a reductive shift in the calcium depleted er are defined with considerable specificity. that a reductive shift is not observed in conditions associated with markedly elevated levels of unfolded protein stress in the er argues that promiscuous effects of calcium depletion on the handling of secreted proteins are unlikely to contribute. the evidence for specificity is further buttressed by the significant effacement of the reductive shift in cells lacking crt. what might be the physiological significance of the reductive shift of the calcium-depleted er? it is best to begin by acknowledging the possibility that redox effects may not be under positive selection but rather reflect a consequence of a role for pdi1a in maintaining crt’s solubility through physiological fluctuations in er. however, it is also intriguing to consider this question in the context of physiological circumstances associated with calcium excursions into the range that effect pdi1a-crt association and er reduction. such excursions are noted in secretory cells responding to secretagogues and are associated with physiological activation of the unfolded protein response. the reductive shift in the er may contribute to such activation, transiently inhibiting protein synthesis, through rapidly reversible protein kinase rna-like endoplasmic reticulum kinase -mediated eif phosphorylation. transient inhibition of energetically-costly protein synthesis may be especially important in insulin-producing beta cells, in which coupling of insulin secretion to blood glucose requires high levels of intracellular atp. furthermore, coupling secretory stimuli to periodic activation of upr target genes would have enduring effects on er size and on the long-term capacity to produce secreted products. the evolution of an er that is more reducing under conditions of er calcium store depletion would promote fitness by coupling the action of calcium-mobilizing secretagogues to a upr-mediated transient attenuation of protein synthesis followed by long-term growth of the er. transient reductive departures from the normally oxidizing poise of the er may provide other benefits to cells. shifting the balance from the oxidizing pdi1a to the reducing erdj is predicted to favor degradation of disulfide bonded misfolded proteins. thus, transient cycles of reduction may be helpful in periodically purging the er of recalcitrant misfolded proteins, while maintaining er redox at levels optimized for oxidative folding most of the time. coupling such reductive shifts to perk-mediated attenuation of protein synthesis would lessen their impact on oxidative folding of newly-synthesized proteins, while promoting the degradation of refuse. a functional link between er redox and calcium homeostasis is suggested by the phenotype of mice lacking ero genes. cells explanted from such animals have no steady state defect in er thiol redox poise, but recover sluggishly from reductive excursions. the mice are superficially indistinguishable from wild type; however, calcium handling by cardiomyocytes is grossly impaired, supporting a role for rapid kinetics of disulfide recovery in er calcium release and re-uptake. calcium handling by the er is subject to redox regulation. levels of ero activity modulate calcium fluxes in and out of the er. subtype of the ip receptor, which conducts lumenal calcium to the cytosol in response to activation of membrane receptors, is inhibited by the formation of a mixed disulfide between cysteine residues on its third lumenal loop and reduced erp, a reducing pdi family member. by contrast, the broadly-expressed calcium reuptake pump, serca2b is inhibited by the formation of a disulfide with the oxidizing pdi family member erp. as disulfide exchange between pdi family members is pervasive, these observations suggest that a more reduced er poise would favor re-uptake of calcium into the er and disfavor its release. it follows that immobilization of the oxidizing pdi1a, which can transfer disulfides to erp, and continued mobility of the reducing erdj would favor restoration of calcium stores to the calcium-depleted er closing a homeostatic loop.  the reductive shift in er redox poise, observed at physiological er calcium store depletion, is an unusual exception to a landscape dominated by stability of er redox balance. direct calcium responsiveness of the core machinery for oxidizing er client protein thiols has been dismissed by our studies. however a remarkable correlation between lumenal calcium depletion and lowered mobility of the oxidizing pdi1a in the er lumen was observed. this, in turn, is plausibly explained by formation of a calcium responsive complex between pdi1a and the abundant er chaperone crt with the effect of sequestering a key component of the oxidizing limb of the er. the consequent attenuation of client protein oxidation accompanies physiological signals that specify secretion, thus delineating a hitherto unanticipated link between secretagogue action and protein folding homeostasis in the er. 
lumenal calcium depletion, incurred during normal cell signaling, stands out as a notable exception to this resilience, promoting a rapid and reversible shift towards a more reducing poise. however, er calcium depletion in vivo led to a selective -fold decline in pdi1a mobility, whereas the mobility of the reducing pdi family member, erdj was unaffected. in vitro, the core machinery for generating disulfides, consisting of ero and the oxidizing protein disulfide isomerase, pdi1a, was indifferent to variation in calcium concentration within the physiological range. in vivo, fluorescence resonance energy transfer measurements revealed that declining pdi1a mobility correlated with formation of a complex with the abundant er chaperone calreticulin, whose mobility was also inhibited by calcium depletion and the calcium depletion-mediated reductive shift was attenuated in cells lacking calreticulin. fluorescence lifetime imagingprotein disulfide isomerasecalreticulinendoplasmic reticulumredoxcalciumissue-copyright-statement© the author 2015 measurements with purified proteins confirmed that the pdi1a-calreticulin complex dissociated as ca2+ concentrations approached those normally found in the er lumen. our findings suggest that selective sequestration of pdi1a in a calcium depletion-mediated complex with the abundant chaperone calreticulin attenuates the effective concentration of this major lumenal thiol oxidant, providing a plausible and simple mechanism for the observed shift in er lumenal redox poise upon physiological calcium depletion. calcium depletion induced er redox alterations are relevant to physiological conditions associated with calcium signaling, such as the response of pancreatic cells to secretagogues and neuronal activity. endoplasmic reticulum lumenal protein thiol redox balance resists dramatic variation in unfolded protein load imposed by diverse physiological challenges including compromise in the key upstream oxidases.
 endoplasmic reticulum lumenal protein thiol redox balance resists dramatic variation in unfolded protein load imposed by diverse physiological challenges including compromise in the key upstream oxidases. lumenal calcium depletion, incurred during normal cell signaling, stands out as a notable exception to this resilience, promoting a rapid and reversible shift towards a more reducing poise. calcium depletion induced er redox alterations are relevant to physiological conditions associated with calcium signaling, such as the response of pancreatic cells to secretagogues and neuronal activity. the core components of the er redox machinery are well characterized; however, the molecular basis for the calcium-depletion induced shift in redox balance is presently obscure.  in vitro, the core machinery for generating disulfides, consisting of ero and the oxidizing protein disulfide isomerase, pdi1a, was indifferent to variation in calcium concentration within the physiological range. however, er calcium depletion in vivo led to a selective -fold decline in pdi1a mobility, whereas the mobility of the reducing pdi family member, erdj was unaffected. in vivo, fluorescence resonance energy transfer measurements revealed that declining pdi1a mobility correlated with formation of a complex with the abundant er chaperone calreticulin, whose mobility was also inhibited by calcium depletion and the calcium depletion-mediated reductive shift was attenuated in cells lacking calreticulin. measurements with purified proteins confirmed that the pdi1a-calreticulin complex dissociated as ca2+ concentrations approached those normally found in the er lumen.  our findings suggest that selective sequestration of pdi1a in a calcium depletion-mediated complex with the abundant chaperone calreticulin attenuates the effective concentration of this major lumenal thiol oxidant, providing a plausible and simple mechanism for the observed shift in er lumenal redox poise upon physiological calcium depletion. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords fluorescence lifetime imagingprotein disulfide isomerasecalreticulinendoplasmic reticulumredoxcalciumissue-copyright-statement© the author 2015 