BACKGROUND
zinc, an essential micronutrient, serves as a structural, catalytic, or regulatory component of many zinc-containing proteins, and is important in almost all aspects of biology  <cit> .
zinc deficiency is a worldwide health problem, and it is estimated that at least 25% of the population is at risk  <cit> .
low zinc can cause pleiotropic problems, such as abnormal morphogenesis, growth retardation, and dysfunction of the reproductive and immune systems  <cit> .
too much zinc accumulation is also harmful to cells and organisms.
maintenance of zinc homeostasis is therefore critical, and multiple mechanisms participate in ensuring the proper uptake, storage, and efflux of zinc.
zinc dyshomeostasis has been shown to cause or influence many common diseases including diabetes, neurodegeneration, and cancer  <cit> .
given the multi-faceted roles of zinc in biology and the alarmingly high deficiency rate observed in human populations, the importance of research into zinc is starting to be appreciated.
recent research indicates that zinc transport across membranes is mediated by two subfamilies of mammalian zinc transporters, znt  and zip   <cit> .
members of the zip protein family function in zinc influx from the extracellular medium or vesicular organelles into the cytoplasm, and those of the znt family mediate zinc efflux or compartmentalization.
systemically, dietary zinc is absorbed through the enterocyte and transported into the circulation and then to the tissues where it is needed.
adjustments in zinc absorption and excretion are the primary means of maintaining zinc homeostasis  <cit> .
human zip <dig> has been identified as the gene responsible for acrodermatitis enteropathica, a disease caused by impaired absorption of dietary zinc in the intestine  <cit> .
the zip <dig> protein has been proposed to absorb zinc from the lumen, a role which is supported by its localization in the apical membrane of the enterocyte and its functionality in the mouse  <cit> .
expression of zip <dig> was found to be strongly responsive to dietary zinc concentrations, exhibiting upregulation with zinc limitation and downregulation with zinc excess, and thus indicating a mechanism by which the absorptive rate of dietary zinc can be beneficially regulated  <cit> .
mutations in zip <dig>  zip <dig>  or zip <dig> also confer on mice a decreased ability to survive under dietary zinc limitation, particularly during pregnancy, when zinc absorption is normally increased  <cit> .
in vitro studies additionally showed that human zip <dig> can regulate zinc homeostasis in intestinal epithelial caco- <dig> cells  <cit> .
however, direct supporting evidence for the involvement of zip <dig>  zip <dig>  and zip <dig> in mammalian dietary zinc absorption is still lacking.
it has been suggested that znt <dig> on the basal membrane is involved in pumping of zinc from the cytosol of enterocytes into the circulation, and this was functionally confirmed in drosophila <cit> .
despite this progress, our knowledge of zinc absorption is still fragmented and limited, and mostly derived from studies in mammalian organisms, some of which were not functional studies.
targeted mutagenesis of many zinc transporters in mice have not yet been performed and for those that have been targeted, mutations were usually generated ubiquitously instead of in a tissue-specific manner, making analysis of their specific role in dietary zinc absorption difficult.
for example, znt <dig> knockout mice die at the embryonic stage, precluding further functional analysis of znt <dig> in dietary zinc absorption  <cit> .
znt <dig> mutant mice have an overall lower bodily zinc level, suggesting that znt <dig> might be a player in dietary zinc absorption, but it can be argued that this is a secondary effect due to the intracellular zinc dyshomeostasis in znt7-mutant cells.
indeed, znt7-mutant mice do not display typical zinc deficiency symptoms, and their phenotypes cannot be rescued by zinc supplementation  <cit> .
as a result of these limits, after years of studies we still do not have a complete picture of dietary zinc utilization in a single platform.
it remains unknown exactly how many zinc transporters are involved in dietary zinc utilization, how they are regulated, and while western and immunohistochemical evidence has shown that some intracellular zinc transporters are expressed in the mouse gastrointestinal tract  <cit> , it is not known whether those transporters found on membranes of the intracellular exocytosis pathway are important for dietary zinc absorption.
to gain a more comprehensive picture of zinc absorption, we took advantage of the powerful genetics of drosophila and systematically dissected the specific involvement of all potential dzips and dznts in gut zinc absorption.
prior to this study, our understanding of dietary zinc absorption in drosophila was extremely limited.
although some analyses of dzips and dznts have been undertaken  <cit> , none of these transporters, except for dznt <dig>  <cit> , have been studied for their involvement or regulation in the process of dietary zinc absorption.
RESULTS
identification of two close homologs, dzip <dig> and dzip <dig>  as specific zinc transporters involved in dietary zinc absorption
we previously demonstrated that dznt <dig> is involved in the efflux of zinc from the midgut enterocytes for systemic use.
however, it was not known which zip is responsible for zinc uptake into the enterocytes.
according to blastp searches for drosophila homologs of mammalian zip family members, the drosophila genome encodes  <dig> putative zip proteins   <cit> .
notably, the d. melanogaster genome lacks a close homolog of zip <dig>  a key player in mammalian absorption of dietary zinc.
this was further confirmed when hzip <dig> and its closest d. melanogaster homolog cg <dig> or foi were used as queries to blast across all genomes of various drosophila species  <cit> , suggesting that the role of zip <dig> is executed by some other zip homologs in the fly.
to identify the zip protein that mediates zinc uptake, we knocked down individually each of these putative zinc transporters, both ubiquitously  and gut-specifically , and tested the sensitivity of the larvae to zinc depletion.
a dramatic effect was observed with cg <dig>  when cg <dig> expression was knocked down, either ubiquitously or gut-specifically, only around  <dig> to 15% of the larvae survived to adulthood on a zinc-limited diet  whereas the eclosion of the control flies was only slightly affected under the same conditions , suggesting that cg <dig> is indispensable for proper zinc uptake.
the protein encoded by cg <dig> shows high homology  to human zip <dig> and zip <dig> .
interestingly, an immediately adjacent gene in this genomic region, cg <dig>  is very closely related to cg <dig>  .
compared with the cg <dig> protein, the cg <dig> protein displays slightly better homology to hzip <dig>  and is in fact the closest homolog of hzip <dig> in the fly genome .
we have therefore named cg <dig> and cg <dig> as drosophila zip <dig>  and dzip <dig>  respectively, hereafter.
both dzip <dig> and dzip <dig> are predicted to have the typical features of zip family members, including eight transmembrane domains , and extracellular amino and carboxyl termini.
the high similarity between dzip <dig> and dzip <dig> and their adjacent locations on the genome prompted us to further investigate whether dzip <dig> also participates in gut zinc uptake.
consistent with this notion, these two genes are the most highly expressed zip genes in the gut, according to the fly atlas  <cit> .
because ubiquitous knockdown of dzip <dig> did not cause significant aberrance in viability, morphology, or fertility in flies fed on either normal or edta food, we decided to use a more sensitive assay to measure the effect on zinc absorption.
activity of the secretory enzyme alkaline phosphatase  is very sensitive to zinc deficiency  <cit> .
indeed, ubiquitous or gut-specific rna interference  of either dzip <dig> or dzip <dig> significantly reduced the activity of alp, but had no effect on the activity of the iron-dependent enzyme aconitase   <cit> .
when we performed rnai of both dzip <dig> and dzip <dig>  and examined the additive effect when both genes were suppressed, virtually no larvae survived to adulthood, whereas a few larvae  survived to adulthood when dzip <dig> alone was knocked down .
adding zinc back into the edta-containing food was able to restore the survival rates of dzip2-rnai and dzip <dig>  dzip2-rnai flies to nearly normal level and to about 50% of normal level respectively, but other metals, including copper, manganese, and iron, did not have any ameliorating effect .
these results suggest that dzip <dig> and dzip <dig> are zinc-specific transporters, and while both are required for dietary zinc uptake, they are functionally partially redundant as well.
dzip <dig> and dzip <dig> are plasma membrane-resident zinc transporters responsible for zinc uptake into midgut enterocytes
during larval development, dzip <dig> is mainly expressed in the midgut, and is also present in trachea and testis, according to flyatlas expression data  <cit> .
to examine the endogenous expression pattern of dzip <dig> at the protein level, we raised a polyclonal antibody against dzip <dig>  and performed immunofluorescence staining on dissected larval gut.
intensive expression of dzip <dig> can be detected in the midgut constriction .
under higher magnification, the endogenous dzip <dig> was found to be localized to the plasma membrane of the enterocytes , and interestingly, it was mainly restricted to the apical membrane  of the enterocytes, which lines the lumen of the midgut.
this apical localization is consistent with the role of dzip <dig> in dietary zinc uptake.
it has been reported that dzip <dig>  when fused to enhanced green fluorescent protein , presents a somewhat basolateral expression in the salivary glands  <cit> .
we therefore investigated its location, particularly in the midgut.
because there is no dzip <dig> antibody currently available, we fused egfp in frame to the c terminal of dzip <dig>  and expressed the fusion protein in human caco- <dig> cells.
as predicted, dzip2-egfp was found to be located on the plasma membrane of the caco- <dig> cells .
to examine whether dzip <dig> localizes to the apical side of midgut cells, we generated a dzip2-ha transgenic fly by fusing an hemagglutinin  tag to the c terminal of dzip <dig>  and expressed it in the midgut.
a clear signal was observed on the apical membrane of the midgut .
these results thus suggest that both dzip <dig> and dzip <dig> mediate the absorption of dietary zinc from lumen into the cytosol of the enterocyte.
to confirm that dzip <dig> and dzip <dig> function as zinc importers, we monitored cytoplasmic zinc levels when they were overexpressed.
we used the zinc-activating reporter mtnb-eyfp for this purpose.
mtnb-eyfp comprises the regulatory sequence of the zinc-responsive gene metallothionein b , an intracellular zinc binding protein, fused to an enhanced yellow fluorescent protein   <cit> .
the mtnb-eyfp fluorescence signal was enhanced in dzip1-overexpressing larvae at the midgut constriction , indicating excessive zinc accumulation in the cytosol of these cells.
consistent with the mtnb-eyfp fluorescence result, semi-quantitative reverse transcriptase -pcr also showed that both mtnb and mtnc were induced in flies with ubiquitous dzip <dig> overexpression   <cit> , and these flies displayed specific sensitivity to dietary overload of zinc, but not to dietary overload of copper or iron .
zinc accumulation, as detected by the zinc indicator zinpyr- <dig>  was also evident when dzip <dig> was expressed in chinese hamster ovary  cells .
these observations confirmed that overexpression of dzip <dig> leads to zinc accumulation in the cytosol of cells.
ubiquitous dzip <dig> overexpression leads to embryonic or first-instar larval lethality.
this happened even when we used the gut-specific gal <dig> driver.
however, we identified a comparably weaker dzip <dig> line, in which ubiquitous expression still resulted in early larval lethality but gut-specific activation did not.
this line, when gut-activated, exhibited zinc-specific sensitivity .
we then used caco- <dig> and cho cells to study the zinc influx function of dzip <dig>  consistently, in dzip2-expressing cells, the mt2a transcriptional level, a reflection of cytoplasmic zinc level, was much higher than that of the control .
this zinc elevation was also evident when zinpyr- <dig> was used as the zinc indicator .
these results indicate that dzip <dig> also transports zinc into the cytoplasm.
intracellular zinc transporters are not significant in dietary zinc absorption
our aforementioned experiments, along with previously published work  <cit>  helped us to identify dzip <dig> and dzip <dig> as being required for transport of dietary zinc into the cytoplasm of the gut cells, and dznt <dig> as being required to pump zinc out of the enterocytes into the hemolymph  <cit> .
however, these zinc transporters are all plasma membrane zinc transporters, and whether the set of intracellular zinc transporters along the secretory pathway is involved in the zinc egress process is unknown.
in dietary copper absorption, for example, the golgi-resident menkes gene atp7a is critical and patients with menkes disease exhibit severe bodily copper shortage  <cit> .
if zinc absorption resembles that of copper, we would expect certain intracellular znt proteins, which mediate zinc efflux into the secretory pathway to be involved.
the drosophila genome encodes seven putative znt proteins, including five possible intracellular znts, as indicated by homology comparison with mammalian znt proteins   <cit> .
to analyze the functions of these znt proteins in gut zinc absorption, we collected all available rnai lines from the vienna drosophila rnai center , and custom-made another set at the tsinghua fly center.
znt35c was not further examined because it is not expressed in the midgut and was previously functionally characterized in malpighian tubules   <cit> .
of the remaining znts, cg <dig> is not expressed in the midgut ; cg <dig>  and cg <dig> are analyzed in detail as shown in figures  <dig> and 5; and the rnai effect of the other two znts, cg <dig> and cg <dig> based on rt-pcr analysis, are presented in additional file  <dig> .
most of these rnai lines against intracellular zinc transporter genes caused lethality when ubiquitously activated , suggesting that these rnai lines are working efficiently, and that the targeted znts are indispensable for fly development.
however, when these intracellular zinc transporters were knocked down specifically in the gut by the gut gal <dig> np <dig>  none resulted in any defects in viability, morphology, or fertility, either under normal or zinc-deficient conditions , nor even at 29°c, a temperature at which the gal <dig> protein is thought to be more potent and produces a stronger rnai effect.
to further scrutinize their involvement in dietary zinc absorption, rnai of these intracellular transporters was carried out in a sensitized background with dznt <dig> knockdown.
when dznt <dig> is knocked down, flies are more sensitive to zinc shortage   <cit> .
however, even under these conditions, the additional knock down  of each of these intracellular znts did not in any case noticeably increase the sensitivity of the dznt1-rnai flies to zinc deficiency .
as an independent and more sensitive test for bodily zinc deficiency, we quantified the alp activity in the whole body minus the gut when these intracellular znts were gut-specifically knocked down.
we reasoned that a slight alteration in the zinc level might be reflected by a change in alp, but might not result in overall survival or developmental phenotypes.
we found that the alp activity of all the gut-specific rnai lines of intracellular znts did not change , indicating that intracellular zinc exocytosis is not significantly involved in dietary zinc absorption in the gut.
the golgi apparatus is the harbor of the secretory pathway.
znt <dig> has been shown to be important for the control of zinc levels in the golgi apparatus  <cit> .
a previous mouse znt <dig> knockout study presented a perplexing scenario regarding the role of znt <dig> in dietary zinc absorption: while the znt7-null mouse has a low overall zinc level, its tissues are not deficient in zinc.
furthermore, zinc supplementation cannot rescue the phenotype at all  <cit> .
if indeed exocytosis is involved in dietary zinc absorption, we would expect zinc loading by the drosophila znt <dig> counterpart to play a significant role.
drosophila has only one likely znt <dig> homolog, cg <dig>  to confirm that this is indeed the drosophila znt <dig>  first we tried to determine the subcellular location of the cg <dig> protein.
to facilitate visualization, we fused egfp in frame to the c terminal of cg <dig>  the golgi marker was produced by fusing red fluorescent protein  behind the golgi-targeted peptide of human β- <dig> -galactosyltransferase  <cit> .
we found that when cg <dig> was co-transfected into caco- <dig> cells, it apparently colocalized with the golgi marker , as described in a previous report  <cit> .
furthermore, the larval lethality resulting from ubiquitous silencing of cg <dig>   <cit>  could be partially rescued to late pupal or adult stage by expression of hznt <dig>  but not of dznt <dig> , corroborating that cg <dig>  hereafter named dznt <dig>  is the drosophila znt <dig>
alp activity is dependent on zinc loading in the golgi.
as expected, ubiquitous knockdown of dznt <dig> significantly reduced zinc-dependent alp activity, but had little effect on iron-dependent aconitase activity .
the affected flies exhibited a severe but hznt7-rescuable phenotype: they died as late third-instar larvae.
this experiment also showed the potent rnai effect of the lines used.
consistent with the mouse knockout study, ubiquitous dznt <dig> rnai led to an overall reduction of zinc in the whole body .
to address precisely the function of dznt <dig> in dietary zinc absorption, we tissue-specifically knocked down dznt <dig> in the gut , and then examined the effects of this knockdown on the rest of the body.
np3084-driven dznt <dig> rnai did not appreciably affect the alp activity of the whole body .
the above results suggest that dznt <dig> in the gut does not contribute to systemic zinc levels, but rather that dznt <dig> functions locally to regulate the activity of zinc-dependent enzymes.
therefore, zinc efflux to the golgi does not seem to be significantly involved in dietary zinc absorption.
cg <dig> , a close homolog of dznt <dig>  also participates in the exit of zinc from enterocytes for systemic use
in the process of our screen for znt transporters involved in zinc absorption in the gut, the knockdown of cg <dig>  localized at 77c in the genomic region, was found to cause sensitivity in flies on edta-supplemented food when ubiquitously or gut-specifically knocked down .
in the phylogenetic tree of drosophila znts, cg <dig> lies closest to dznt <dig> , sharing with it 26% identity and 48% similarity.
consistent with the plasma-membrane residence of dznt <dig>  cg <dig> is also localized to the plasma membrane as shown by the fluorescence signal emitted by cg5130-egfp in caco- <dig> cells .
this is consistent with the previous finding showing the basolateral membrane localization of cg <dig> in salivary glands  <cit> .
cg <dig> expression in cho cells led to zinc reduction as indicated by zinpyr- <dig> staining .
to further address the zinc-absorption role of cg <dig> in the gut, we examined the zinc-dependent alp activity of the rest of the body when cg <dig> was gut-specifically knocked down.
consistent with the observed sensitivity to zinc deficiency, gut-specific rnai of cg <dig> led to decreased alp activity of the whole body minus gut, but did not significantly change aconitase activity .
considering the sequence similarity between dznt <dig> and cg <dig>  we investigated further, and found that this rnai effect is not mediated by reduction of dznt <dig>  as cg <dig> knockdown did not affect the expression of dznt <dig> .
we next investigated whether dznt <dig> and cg <dig> function cooperatively in the exit of zinc out of the gut into the circulation.
we found that when the dznt1-rnai line was recombined with the cg5130-rnai line, the double rnai line displayed greater sensitivity to zinc deficiency compared with either of the individual single rnai lines .
these data indicate that zinc efflux from the enterocytes is mediated by a collaborative function of dznt <dig> and cg <dig>
expressions of the midgut zinc uptake genes are mainly influenced by changes in dietary zinc
one important question in the regulation of zinc absorption is how zinc transporters are regulated by available zinc.
from the physiological point of view, the expression of zinc uptake proteins dzip <dig> and dzip <dig> should ideally be reduced when the diet is rich in zinc to avoid excessive zinc toxicity, and their expression should be increased when the diet is low in zinc, in order to facilitate dietary zinc uptake from zinc-limited food.
using semi-quantitative rt-pcr analysis , we found that this is indeed the case.
of all the drosophila zip genes, only dzip <dig> and dzip <dig> appear to be transcriptionally regulated by dietary zinc levels .
when the food was replete with zinc the rna levels of these two zinc uptake proteins, dzip <dig> and dzip <dig>  were repressed, and conversely, when the food was depleted in zinc, the rna levels of dzip <dig> and dzip <dig> were increased.
the induced dzip <dig> expression was further confirmed by immunostaining at the midgut constriction when dietary zinc was limited .
these data suggest that dzip <dig> and dzip <dig> respond to dietary zinc availability, and function cooperatively to ensure appropriate zinc absorption under zinc-limited and zinc-supplemented conditions.
assessing the transcription of all the znt genes in response to excessive dietary zinc, we detected a slight increase in expression only in the case of dznt <dig> .
the immunohistochemical staining results suggest that most of this increase can be explained by de novo or ectopic induction of znt <dig> expression in regions other than the midgut constriction region  <cit> .
this lack of obvious transcriptional control in the zinc absorption area  prompted us to investigate whether dznt <dig> is subject to post-transcriptional control.
quantitative analysis of dznt <dig> in the midgut by western blotting demonstrated a strong post-transcriptional regulation of dznt1; when zinc was high, the dznt <dig> protein was dramatically reduced .
we also assayed the expression of cg <dig> by fusing it to an ha tag at the c terminal.
however, there was no obvious change in the cg <dig> protein level in response to zinc fluctuations .
the midgut zinc uptake genes are unresponsive to bodily zinc status
limiting zinc efflux when zinc is replete will benefit the rest of the body at the expense of the gut.
this led us to investigate another important zinc regulation question: does zinc absorption reflect the bodily zinc requirement?
to address this question, we genetically manipulated drosophila to create these scenarios: high zinc in the rest of the body but low in the gut; high zinc in the gut but low in the body.
we then determined how the influx and efflux zinc transporters responded.
by introducing dznt <dig> rnai, we could make the fly zinc replete in the gut but zinc deficient in the rest of the body  <cit> .
under this scenario, dzip <dig> and dzip <dig> expression was much reduced , despite the unsatisfied need for zinc in the body.
by overexpressing dznt <dig>  we created the opposite scenario: higher zinc level in the body but lower in the gut  <cit> .
in this case, dzip <dig> and dzip <dig> were both up-regulated .
using dzip <dig> antibody, we can clearly see a much stronger signal of dzip <dig> when dznt <dig> is over-expressed .
another way to control body zinc level is through manipulating znt35c expression  <cit> .
when znt35c is repressed or over-expressed, the body zinc level is correspondingly increased or decreased   <cit> .
again, we did not see any alteration of dzip <dig> and dzip <dig> expression .
further, we did not see obvious dznt <dig> expression change either .
taking these results together, we conclude that dietary zinc uptake is not responsive to the zinc status or need of the body.
the regulation is, strictly speaking, controlled by the zinc status of the enterocytes, and not even directly by the diet itself.
dietary zinc influenced the expression of these uptake genes by affecting the zinc levels of the enterocytes.
discussion
in this work, we systematically dissected the repertoire of zinc transporter candidates for their functions in dietary zinc absorption.
through this systematic work in a drosophila model, the mechanism of zinc absorption has started to take shape, and the important players have been clarified.
zinc absorption is mediated by several transporters localized to the plasma membrane, whereas transporters in the exocytosis pathway appear to be insignificant in this process.
the regulation of dietary zinc absorption is mediated through changes in the expression of some of these plasma membrane-localized transporters.
a model summarizing this process is shown in figure  <dig>
in order to analyze the tissue-specific functions of these zinc transporters, we utilized tissue-specific rnai.
one caveat of this approach is that when a phenotype is seen, it is not clear whether this is due to an off-target effect, and when no effect is observed, it is not clear whether this is due to low rnai efficiency.
to address the first concern, we used multiple rnai lines that were generated by targeting different gene regions, and we also tested their zinc-specific effects.
for example, if an rnai line has a zinc-responsive phenotype, this suggests that the resultant phenotype is probably real, and unlikely to be the result of off-targeting because the probability of a random off-targeting event happening to a zinc metabolism gene is low.
the second concern is more of an issue because rnai is never a genetic null, so to argue that a gene is not involved in a process simply because no phenotype is associated with rnai needs extra caution.
for the purpose of this study, we wanted to test whether intracellular znts are involved in dietary zinc absorption.
gut-specific rnai of the plasma membrane-resident znt genes dznt <dig> and cg <dig> resulted in a change in the zinc phenotype of the body, while gut-specific rnai of the intracellular znts had no discernible effect on dietary zinc absorption.
when we performed a ubiquitous knockdown we saw specific effects for many of these intracellular znt genes, suggesting effective rnai and their functionality in other aspects of zinc metabolism.
ubiquitous rnai of two of these genes in particular, the golgi-resident dznt <dig> and the vesicle-resident cg <dig> , induced larval or embryonic lethality in treated flies   <cit> , however, specifically targeting each of the intracellular transporters by rnai in the gut did not appear to have any effect on dietary zinc absorption.
worth mentioning is that cg <dig> is the closest drosophila homolog of cdf- <dig>  which has been shown to function in zinc storage in the gut granules of caenorhabditis elegans.
further, in drosophila, suppression of cg <dig> greatly decreases mtnb-eyfp expression  <cit> .
it appears therefore that cg <dig> is an important regulator of intracellular zinc homeostasis, although it plays an insignificant role in the process of dietary zinc absorption.
the exocytosis pathway involves many organelles.
in contrast to the dramatic effect seen for the plasma membrane-resident znts, none of the multiple rnai lines for the whole set of znts located in different parts of this pathway displayed any zinc absorption defect, and this was the case even in an sensitized background  and using the sensitive alp activity assay.
this strongly suggests that the intracellular pathway plays no role or at least no significant role in dietary zinc absorption.
a central part of the secretion pathway is the golgi apparatus.
there is only one golgi-resident znt member, dznt <dig>  in the fly.
znt <dig> has been previously implicated in dietary zinc absorption in a mouse knock-out study  <cit> , and znt7-null mice have a reduced body zinc level.
nevertheless their phenotype is not ameliorated by intravascular zinc injection.
we consider the complex phenotype of znt7-null mice as arising from a universal golgi zinc transport defect, resulting lack of zinc in the golgi apparatus, instead of a systemic zinc deficiency.
in our study, we found that universal knockdown of dznt <dig> also caused a severe phenotype, including a reduction in bodily zinc level.
however, gut-specific dznt <dig> knockdown produced little effect on bodily zinc metabolism, except for a local change of the golgi zinc level, as reflected by alp activity reduction in the gut only.
among the several zinc transporters involved in dietary zinc absorption, the two zips  and at least one dznt  involved in zinc uptake are strongly influenced by the dietary zinc level.
when there is an excess of zinc available, uptake and efflux of zinc are reduced.
when zinc is scarce, the uptake transporters dzip <dig> and dzip <dig> are upregulated, whereas the efflux of zinc is not greatly affected.
the ectopic induction of dznt <dig> in otherwise non-expressing gut tissues may be a self-protecting mechanism for the gut when zinc overdosing happens, because zinc accumulation is generally toxic to cells.
some general characterization of the zip and znt transporters was previously performed  <cit> , although the roles of different zinc transporters in the process of dietary zinc absorption were not previously explored except that of dznt <dig>  in this study, the zinc transporters that were newly found to be involved in dietary zinc absorption were characterized in more detail at the molecular and cellular levels.
our results, when applicable, are largely consistent with previous work, but some inconsistencies do arise.
it was previously reported that ubiquitous dzip <dig> knockdown resulted in lethality  <cit> we did not see this lethality under normal culturing conditions.
however, we did see lethality when the zinc supply was lowered.
the difference in results is likely attributable to different culturing conditions in different laboratories such as variations in food mineral content.
likewise, midgut-specific overexpression of dzip <dig> was reported to be lethal  <cit> , but in our hands this happened only when extra zinc was added.
further, we found that ubiquitous knockdown of dzip <dig> produced a reduced alp phenotype without much of a decrease in viability, whereas lye et al.
<cit>  found that ubiquitous knockdown of dzip <dig> with a stronger gal <dig> driver, tubulin-gal <dig>  resulted in larval lethality.
this discrepancy may be a consequence of either the difference in the drivers used, or in the food nutrient supply, as mentioned above.
therefore, in the characterization of the general properties of these zinc transporters, our results are largely consistent with previous studies, except in a few cases in which the extent of the phenotype is different, which could be attributable to differences in the experimental conditions used.
partial redundancy of the influx and efflux transporters were seen in dietary zinc absorption in drosophila.
dzip <dig> and dzip <dig> are responsible for zinc uptake, whereas dznt <dig> and cg <dig> are responsible for the efflux.
knocking any of these transporters down caused zinc deficiency under zinc-limited conditions, suggesting that all of them are important in the absorption of dietary zinc.
nevertheless, slight differences do exist.
although downregulation of either dzip <dig> or dzip <dig>  had equivalent effects on alp levels when flies were cultured on  <dig>  mmol/l edta food, on  <dig>  mm edta food dzip <dig> knockdown flies developed well while dzip <dig> flies were arrested at the early larval stage .
it is possible that dzip <dig> is slightly more important than dzip <dig> or is a more potent zinc transporter when zinc is severely depleted.
this notion is consistent with the observation that overexpression of dzip <dig> causes a more severe phenotype than does overexpression of dzip <dig>
in the case of the zinc efflux transporters, dznt <dig> and cg <dig>  knockdown of either presents a similar phenotype, namely, death at around the third-instar larval stage when zinc is limited.
interestingly, dznt <dig> is regulated by zinc, whereas cg <dig> is not, suggesting that dznt <dig> is a regulatory transporter whereas cg <dig> may play a more constitutive role in zinc absorption in the gut.
further, overexpression of dznt <dig> produced a zinc-sensitive phenotype whereas cg <dig> overexpression did not.
we speculate that cg <dig> might have a lower zinc-transporting capacity so that under conditions of zinc excess, the amount of zinc it can transport is limited, so that zinc toxicity is avoided.
alternatively, cg <dig> might work together with dznt <dig>  to export zinc.
either scenario can explain why it is not important for cg <dig> to have zinc-responsive expression.
together our results offer a glimpse into the overall process of dietary zinc absorption in a drosophila model.
zinc absorption, regulated by uptake, occurs through a relatively direct route from the gut lumen to the basolateral sides of the gut epithelium and hence into the circulation.
although most intracellular znts are expressed in the gastrointestinal tract, these intracellular zinc transporters are not significant in dietary zinc absorption.
dzip1/ <dig> and dznt1/cg <dig> are respectively responsible for zinc intake and efflux in the enterocytes.
in human studies, the dzip1/ <dig> counterpart hzip <dig> was shown to be important for zinc absorption.
hzip <dig> is haploinsufficient as mutation in one allele may cause zinc deficiency  <cit> .
whether any other zips are involved in this process in mammals is not well-established  <cit> .
the lack of an ortholog of zip <dig> in drosophila makes it possible that dzip1/ <dig> may substitute for zip <dig> in mediating zinc absorption.
therefore, the specific transporters for a particular role may vary between different organisms.
for zinc efflux, participation of mammalian znt <dig>  the counterpart of dznt <dig>  is almost a certainty, but whether znt <dig> is the sole player in zinc efflux in the mammalian gut remains to be seen.
overall, our results are notably consistent with some findings regarding mammalian zinc absorption: expression of zip <dig> and znt <dig> is reduced when the diet is replete in zinc  <cit> , and the zinc status of the body appears to have little effect on the efficiency of dietary zinc absorption  <cit> .
CONCLUSIONS
the genetic amenability of drosophila enabled us to show which zinc transporters are involved in the process of dietary zinc absorption, and which are not.
we found that dietary zinc absorption is mediated by a set of plasma membrane-resident zinc transporters with partially overlapping functions, including the importers dzip1/dzip <dig> and the exporters dznt1/cg <dig> .
the array of intracellular zinc transporters, such as the golgi-resident znt <dig>  is not involved in dietary zinc absorption.
zinc absorption is subject at the influx side to the rna-level control of zinc importers, and at the efflux side to the translational or post-translational control of the exporters.
bodily zinc needs do not exert a feedback control on zinc absorption; instead, zinc absorption is controlled solely by the zinc levels in the enterocytes.
dietary zinc influences zinc absorption through its effects on enterocyte zinc.
this work assessed for the first time all potential zinc transporters for their roles in dietary zinc absorption, and we have outlined a relatively intact picture of the dietary zinc absorption and its control in a model organism .
the deciphering of dietary zinc absorption in drosophila should be helpful for zinc absorption research in general, and offers a reference point for future studies in other organisms including comparative evolutionary analysis of this important biological process.

 zinc, an essential micronutrient, serves as a structural, catalytic, or regulatory component of many zinc-containing proteins, and is important in almost all aspects of biology. zinc deficiency is a worldwide health problem, and it is estimated that at least 25% of the population is at risk. low zinc can cause pleiotropic problems, such as abnormal morphogenesis, growth retardation, and dysfunction of the reproductive and immune systems. too much zinc accumulation is also harmful to cells and organisms. maintenance of zinc homeostasis is therefore critical, and multiple mechanisms participate in ensuring the proper uptake, storage, and efflux of zinc. zinc dyshomeostasis has been shown to cause or influence many common diseases including diabetes, neurodegeneration, and cancer. given the multi-faceted roles of zinc in biology and the alarmingly high deficiency rate observed in human populations, the importance of research into zinc is starting to be appreciated. recent research indicates that zinc transport across membranes is mediated by two subfamilies of mammalian zinc transporters, znt and zip. members of the zip protein family function in zinc influx from the extracellular medium or vesicular organelles into the cytoplasm, and those of the znt family mediate zinc efflux or compartmentalization. systemically, dietary zinc is absorbed through the enterocyte and transported into the circulation and then to the tissues where it is needed. adjustments in zinc absorption and excretion are the primary means of maintaining zinc homeostasis. human zip has been identified as the gene responsible for acrodermatitis enteropathica, a disease caused by impaired absorption of dietary zinc in the intestine. the zip protein has been proposed to absorb zinc from the lumen, a role which is supported by its localization in the apical membrane of the enterocyte and its functionality in the mouse. expression of zip was found to be strongly responsive to dietary zinc concentrations, exhibiting upregulation with zinc limitation and downregulation with zinc excess, and thus indicating a mechanism by which the absorptive rate of dietary zinc can be beneficially regulated. mutations in zip zip or zip also confer on mice a decreased ability to survive under dietary zinc limitation, particularly during pregnancy, when zinc absorption is normally increased. in vitro studies additionally showed that human zip can regulate zinc homeostasis in intestinal epithelial caco- cells. however, direct supporting evidence for the involvement of zip zip and zip in mammalian dietary zinc absorption is still lacking. it has been suggested that znt on the basal membrane is involved in pumping of zinc from the cytosol of enterocytes into the circulation, and this was functionally confirmed in drosophila. despite this progress, our knowledge of zinc absorption is still fragmented and limited, and mostly derived from studies in mammalian organisms, some of which were not functional studies. targeted mutagenesis of many zinc transporters in mice have not yet been performed and for those that have been targeted, mutations were usually generated ubiquitously instead of in a tissue-specific manner, making analysis of their specific role in dietary zinc absorption difficult. for example, znt knockout mice die at the embryonic stage, precluding further functional analysis of znt in dietary zinc absorption. znt mutant mice have an overall lower bodily zinc level, suggesting that znt might be a player in dietary zinc absorption, but it can be argued that this is a secondary effect due to the intracellular zinc dyshomeostasis in znt7-mutant cells. indeed, znt7-mutant mice do not display typical zinc deficiency symptoms, and their phenotypes cannot be rescued by zinc supplementation. as a result of these limits, after years of studies we still do not have a complete picture of dietary zinc utilization in a single platform. it remains unknown exactly how many zinc transporters are involved in dietary zinc utilization, how they are regulated, and while western and immunohistochemical evidence has shown that some intracellular zinc transporters are expressed in the mouse gastrointestinal tract, it is not known whether those transporters found on membranes of the intracellular exocytosis pathway are important for dietary zinc absorption. to gain a more comprehensive picture of zinc absorption, we took advantage of the powerful genetics of drosophila and systematically dissected the specific involvement of all potential dzips and dznts in gut zinc absorption. prior to this study, our understanding of dietary zinc absorption in drosophila was extremely limited. although some analyses of dzips and dznts have been undertaken, none of these transporters, except for dznt, have been studied for their involvement or regulation in the process of dietary zinc absorption.  identification of two close homologs, dzip and dzip as specific zinc transporters involved in dietary zinc absorption we previously demonstrated that dznt is involved in the efflux of zinc from the midgut enterocytes for systemic use. however, it was not known which zip is responsible for zinc uptake into the enterocytes. according to blastp searches for drosophila homologs of mammalian zip family members, the drosophila genome encodes putative zip proteins. notably, the d. melanogaster genome lacks a close homolog of zip a key player in mammalian absorption of dietary zinc. this was further confirmed when hzip and its closest d. melanogaster homolog cg or foi were used as queries to blast across all genomes of various drosophila species, suggesting that the role of zip is executed by some other zip homologs in the fly. to identify the zip protein that mediates zinc uptake, we knocked down individually each of these putative zinc transporters, both ubiquitously and gut-specifically, and tested the sensitivity of the larvae to zinc depletion. a dramatic effect was observed with cg when cg expression was knocked down, either ubiquitously or gut-specifically, only around to 15% of the larvae survived to adulthood on a zinc-limited diet whereas the eclosion of the control flies was only slightly affected under the same conditions, suggesting that cg is indispensable for proper zinc uptake. the protein encoded by cg shows high homology to human zip and zip. interestingly, an immediately adjacent gene in this genomic region, cg is very closely related to cg. compared with the cg protein, the cg protein displays slightly better homology to hzip and is in fact the closest homolog of hzip in the fly genome. we have therefore named cg and cg as drosophila zip and dzip respectively, hereafter. both dzip and dzip are predicted to have the typical features of zip family members, including eight transmembrane domains, and extracellular amino and carboxyl termini. the high similarity between dzip and dzip and their adjacent locations on the genome prompted us to further investigate whether dzip also participates in gut zinc uptake. consistent with this notion, these two genes are the most highly expressed zip genes in the gut, according to the fly atlas. because ubiquitous knockdown of dzip did not cause significant aberrance in viability, morphology, or fertility in flies fed on either normal or edta food, we decided to use a more sensitive assay to measure the effect on zinc absorption. activity of the secretory enzyme alkaline phosphatase is very sensitive to zinc deficiency. indeed, ubiquitous or gut-specific rna interference of either dzip or dzip significantly reduced the activity of alp, but had no effect on the activity of the iron-dependent enzyme aconitase. when we performed rnai of both dzip and dzip and examined the additive effect when both genes were suppressed, virtually no larvae survived to adulthood, whereas a few larvae survived to adulthood when dzip alone was knocked down. adding zinc back into the edta-containing food was able to restore the survival rates of dzip2-rnai and dzip dzip2-rnai flies to nearly normal level and to about 50% of normal level respectively, but other metals, including copper, manganese, and iron, did not have any ameliorating effect. these results suggest that dzip and dzip are zinc-specific transporters, and while both are required for dietary zinc uptake, they are functionally partially redundant as well. dzip and dzip are plasma membrane-resident zinc transporters responsible for zinc uptake into midgut enterocytes during larval development, dzip is mainly expressed in the midgut, and is also present in trachea and testis, according to flyatlas expression data. to examine the endogenous expression pattern of dzip at the protein level, we raised a polyclonal antibody against dzip and performed immunofluorescence staining on dissected larval gut. intensive expression of dzip can be detected in the midgut constriction. under higher magnification, the endogenous dzip was found to be localized to the plasma membrane of the enterocytes, and interestingly, it was mainly restricted to the apical membrane of the enterocytes, which lines the lumen of the midgut. this apical localization is consistent with the role of dzip in dietary zinc uptake. it has been reported that dzip when fused to enhanced green fluorescent protein, presents a somewhat basolateral expression in the salivary glands. we therefore investigated its location, particularly in the midgut. because there is no dzip antibody currently available, we fused egfp in frame to the c terminal of dzip and expressed the fusion protein in human caco- cells. as predicted, dzip2-egfp was found to be located on the plasma membrane of the caco- cells. to examine whether dzip localizes to the apical side of midgut cells, we generated a dzip2-ha transgenic fly by fusing an hemagglutinin tag to the c terminal of dzip and expressed it in the midgut. a clear signal was observed on the apical membrane of the midgut. these results thus suggest that both dzip and dzip mediate the absorption of dietary zinc from lumen into the cytosol of the enterocyte. to confirm that dzip and dzip function as zinc importers, we monitored cytoplasmic zinc levels when they were overexpressed. we used the zinc-activating reporter mtnb-eyfp for this purpose. mtnb-eyfp comprises the regulatory sequence of the zinc-responsive gene metallothionein b, an intracellular zinc binding protein, fused to an enhanced yellow fluorescent protein. the mtnb-eyfp fluorescence signal was enhanced in dzip1-overexpressing larvae at the midgut constriction, indicating excessive zinc accumulation in the cytosol of these cells. consistent with the mtnb-eyfp fluorescence result, semi-quantitative reverse transcriptase -pcr also showed that both mtnb and mtnc were induced in flies with ubiquitous dzip overexpression, and these flies displayed specific sensitivity to dietary overload of zinc, but not to dietary overload of copper or iron. zinc accumulation, as detected by the zinc indicator zinpyr- was also evident when dzip was expressed in chinese hamster ovary cells. these observations confirmed that overexpression of dzip leads to zinc accumulation in the cytosol of cells. ubiquitous dzip overexpression leads to embryonic or first-instar larval lethality. this happened even when we used the gut-specific gal driver. however, we identified a comparably weaker dzip line, in which ubiquitous expression still resulted in early larval lethality but gut-specific activation did not. this line, when gut-activated, exhibited zinc-specific sensitivity. we then used caco- and cho cells to study the zinc influx function of dzip consistently, in dzip2-expressing cells, the mt2a transcriptional level, a reflection of cytoplasmic zinc level, was much higher than that of the control. this zinc elevation was also evident when zinpyr- was used as the zinc indicator. these results indicate that dzip also transports zinc into the cytoplasm. intracellular zinc transporters are not significant in dietary zinc absorption our aforementioned experiments, along with previously published work helped us to identify dzip and dzip as being required for transport of dietary zinc into the cytoplasm of the gut cells, and dznt as being required to pump zinc out of the enterocytes into the hemolymph. however, these zinc transporters are all plasma membrane zinc transporters, and whether the set of intracellular zinc transporters along the secretory pathway is involved in the zinc egress process is unknown. in dietary copper absorption, for example, the golgi-resident menkes gene atp7a is critical and patients with menkes disease exhibit severe bodily copper shortage. if zinc absorption resembles that of copper, we would expect certain intracellular znt proteins, which mediate zinc efflux into the secretory pathway to be involved. the drosophila genome encodes seven putative znt proteins, including five possible intracellular znts, as indicated by homology comparison with mammalian znt proteins. to analyze the functions of these znt proteins in gut zinc absorption, we collected all available rnai lines from the vienna drosophila rnai center, and custom-made another set at the tsinghua fly center. znt35c was not further examined because it is not expressed in the midgut and was previously functionally characterized in malpighian tubules. of the remaining znts, cg is not expressed in the midgut; cg and cg are analyzed in detail as shown in figures  and 5; and the rnai effect of the other two znts, cg and cg based on rt-pcr analysis, are presented in additional file. most of these rnai lines against intracellular zinc transporter genes caused lethality when ubiquitously activated, suggesting that these rnai lines are working efficiently, and that the targeted znts are indispensable for fly development. however, when these intracellular zinc transporters were knocked down specifically in the gut by the gut gal np none resulted in any defects in viability, morphology, or fertility, either under normal or zinc-deficient conditions, nor even at 29°c, a temperature at which the gal protein is thought to be more potent and produces a stronger rnai effect. to further scrutinize their involvement in dietary zinc absorption, rnai of these intracellular transporters was carried out in a sensitized background with dznt knockdown. when dznt is knocked down, flies are more sensitive to zinc shortage. however, even under these conditions, the additional knock down of each of these intracellular znts did not in any case noticeably increase the sensitivity of the dznt1-rnai flies to zinc deficiency. as an independent and more sensitive test for bodily zinc deficiency, we quantified the alp activity in the whole body minus the gut when these intracellular znts were gut-specifically knocked down. we reasoned that a slight alteration in the zinc level might be reflected by a change in alp, but might not result in overall survival or developmental phenotypes. we found that the alp activity of all the gut-specific rnai lines of intracellular znts did not change, indicating that intracellular zinc exocytosis is not significantly involved in dietary zinc absorption in the gut. the golgi apparatus is the harbor of the secretory pathway. znt has been shown to be important for the control of zinc levels in the golgi apparatus. a previous mouse znt knockout study presented a perplexing scenario regarding the role of znt in dietary zinc absorption: while the znt7-null mouse has a low overall zinc level, its tissues are not deficient in zinc. furthermore, zinc supplementation cannot rescue the phenotype at all. if indeed exocytosis is involved in dietary zinc absorption, we would expect zinc loading by the drosophila znt counterpart to play a significant role. drosophila has only one likely znt homolog, cg to confirm that this is indeed the drosophila znt first we tried to determine the subcellular location of the cg protein. to facilitate visualization, we fused egfp in frame to the c terminal of cg the golgi marker was produced by fusing red fluorescent protein behind the golgi-targeted peptide of human β- -galactosyltransferase. we found that when cg was co-transfected into caco- cells, it apparently colocalized with the golgi marker, as described in a previous report. furthermore, the larval lethality resulting from ubiquitous silencing of cg could be partially rescued to late pupal or adult stage by expression of hznt but not of dznt, corroborating that cg hereafter named dznt is the drosophila znt alp activity is dependent on zinc loading in the golgi. as expected, ubiquitous knockdown of dznt significantly reduced zinc-dependent alp activity, but had little effect on iron-dependent aconitase activity. the affected flies exhibited a severe but hznt7-rescuable phenotype: they died as late third-instar larvae. this experiment also showed the potent rnai effect of the lines used. consistent with the mouse knockout study, ubiquitous dznt rnai led to an overall reduction of zinc in the whole body. to address precisely the function of dznt in dietary zinc absorption, we tissue-specifically knocked down dznt in the gut, and then examined the effects of this knockdown on the rest of the body. np3084-driven dznt rnai did not appreciably affect the alp activity of the whole body. the above results suggest that dznt in the gut does not contribute to systemic zinc levels, but rather that dznt functions locally to regulate the activity of zinc-dependent enzymes. therefore, zinc efflux to the golgi does not seem to be significantly involved in dietary zinc absorption. cg, a close homolog of dznt also participates in the exit of zinc from enterocytes for systemic use in the process of our screen for znt transporters involved in zinc absorption in the gut, the knockdown of cg localized at 77c in the genomic region, was found to cause sensitivity in flies on edta-supplemented food when ubiquitously or gut-specifically knocked down. in the phylogenetic tree of drosophila znts, cg lies closest to dznt, sharing with it 26% identity and 48% similarity. consistent with the plasma-membrane residence of dznt cg is also localized to the plasma membrane as shown by the fluorescence signal emitted by cg5130-egfp in caco- cells. this is consistent with the previous finding showing the basolateral membrane localization of cg in salivary glands. cg expression in cho cells led to zinc reduction as indicated by zinpyr- staining. to further address the zinc-absorption role of cg in the gut, we examined the zinc-dependent alp activity of the rest of the body when cg was gut-specifically knocked down. consistent with the observed sensitivity to zinc deficiency, gut-specific rnai of cg led to decreased alp activity of the whole body minus gut, but did not significantly change aconitase activity. considering the sequence similarity between dznt and cg we investigated further, and found that this rnai effect is not mediated by reduction of dznt as cg knockdown did not affect the expression of dznt. we next investigated whether dznt and cg function cooperatively in the exit of zinc out of the gut into the circulation. we found that when the dznt1-rnai line was recombined with the cg5130-rnai line, the double rnai line displayed greater sensitivity to zinc deficiency compared with either of the individual single rnai lines. these data indicate that zinc efflux from the enterocytes is mediated by a collaborative function of dznt and cg expressions of the midgut zinc uptake genes are mainly influenced by changes in dietary zinc one important question in the regulation of zinc absorption is how zinc transporters are regulated by available zinc. from the physiological point of view, the expression of zinc uptake proteins dzip and dzip should ideally be reduced when the diet is rich in zinc to avoid excessive zinc toxicity, and their expression should be increased when the diet is low in zinc, in order to facilitate dietary zinc uptake from zinc-limited food. using semi-quantitative rt-pcr analysis, we found that this is indeed the case. of all the drosophila zip genes, only dzip and dzip appear to be transcriptionally regulated by dietary zinc levels. when the food was replete with zinc the rna levels of these two zinc uptake proteins, dzip and dzip were repressed, and conversely, when the food was depleted in zinc, the rna levels of dzip and dzip were increased. the induced dzip expression was further confirmed by immunostaining at the midgut constriction when dietary zinc was limited. these data suggest that dzip and dzip respond to dietary zinc availability, and function cooperatively to ensure appropriate zinc absorption under zinc-limited and zinc-supplemented conditions. assessing the transcription of all the znt genes in response to excessive dietary zinc, we detected a slight increase in expression only in the case of dznt. the immunohistochemical staining results suggest that most of this increase can be explained by de novo or ectopic induction of znt expression in regions other than the midgut constriction region. this lack of obvious transcriptional control in the zinc absorption area prompted us to investigate whether dznt is subject to post-transcriptional control. quantitative analysis of dznt in the midgut by western blotting demonstrated a strong post-transcriptional regulation of dznt1; when zinc was high, the dznt protein was dramatically reduced. we also assayed the expression of cg by fusing it to an ha tag at the c terminal. however, there was no obvious change in the cg protein level in response to zinc fluctuations. the midgut zinc uptake genes are unresponsive to bodily zinc status limiting zinc efflux when zinc is replete will benefit the rest of the body at the expense of the gut. this led us to investigate another important zinc regulation question: does zinc absorption reflect the bodily zinc requirement? to address this question, we genetically manipulated drosophila to create these scenarios: high zinc in the rest of the body but low in the gut; high zinc in the gut but low in the body. we then determined how the influx and efflux zinc transporters responded. by introducing dznt rnai, we could make the fly zinc replete in the gut but zinc deficient in the rest of the body. under this scenario, dzip and dzip expression was much reduced, despite the unsatisfied need for zinc in the body. by overexpressing dznt we created the opposite scenario: higher zinc level in the body but lower in the gut. in this case, dzip and dzip were both up-regulated. using dzip antibody, we can clearly see a much stronger signal of dzip when dznt is over-expressed. another way to control body zinc level is through manipulating znt35c expression. when znt35c is repressed or over-expressed, the body zinc level is correspondingly increased or decreased. again, we did not see any alteration of dzip and dzip expression. further, we did not see obvious dznt expression change either. taking these results together, we conclude that dietary zinc uptake is not responsive to the zinc status or need of the body. the regulation is, strictly speaking, controlled by the zinc status of the enterocytes, and not even directly by the diet itself. dietary zinc influenced the expression of these uptake genes by affecting the zinc levels of the enterocytes. discussion in this work, we systematically dissected the repertoire of zinc transporter candidates for their functions in dietary zinc absorption. through this systematic work in a drosophila model, the mechanism of zinc absorption has started to take shape, and the important players have been clarified. zinc absorption is mediated by several transporters localized to the plasma membrane, whereas transporters in the exocytosis pathway appear to be insignificant in this process. the regulation of dietary zinc absorption is mediated through changes in the expression of some of these plasma membrane-localized transporters. a model summarizing this process is shown in figure  in order to analyze the tissue-specific functions of these zinc transporters, we utilized tissue-specific rnai. one caveat of this approach is that when a phenotype is seen, it is not clear whether this is due to an off-target effect, and when no effect is observed, it is not clear whether this is due to low rnai efficiency. to address the first concern, we used multiple rnai lines that were generated by targeting different gene regions, and we also tested their zinc-specific effects. for example, if an rnai line has a zinc-responsive phenotype, this suggests that the resultant phenotype is probably real, and unlikely to be the result of off-targeting because the probability of a random off-targeting event happening to a zinc metabolism gene is low. the second concern is more of an issue because rnai is never a genetic null, so to argue that a gene is not involved in a process simply because no phenotype is associated with rnai needs extra caution. for the purpose of this study, we wanted to test whether intracellular znts are involved in dietary zinc absorption. gut-specific rnai of the plasma membrane-resident znt genes dznt and cg resulted in a change in the zinc phenotype of the body, while gut-specific rnai of the intracellular znts had no discernible effect on dietary zinc absorption. when we performed a ubiquitous knockdown we saw specific effects for many of these intracellular znt genes, suggesting effective rnai and their functionality in other aspects of zinc metabolism. ubiquitous rnai of two of these genes in particular, the golgi-resident dznt and the vesicle-resident cg, induced larval or embryonic lethality in treated flies, however, specifically targeting each of the intracellular transporters by rnai in the gut did not appear to have any effect on dietary zinc absorption. worth mentioning is that cg is the closest drosophila homolog of cdf- which has been shown to function in zinc storage in the gut granules of caenorhabditis elegans. further, in drosophila, suppression of cg greatly decreases mtnb-eyfp expression. it appears therefore that cg is an important regulator of intracellular zinc homeostasis, although it plays an insignificant role in the process of dietary zinc absorption. the exocytosis pathway involves many organelles. in contrast to the dramatic effect seen for the plasma membrane-resident znts, none of the multiple rnai lines for the whole set of znts located in different parts of this pathway displayed any zinc absorption defect, and this was the case even in an sensitized background and using the sensitive alp activity assay. this strongly suggests that the intracellular pathway plays no role or at least no significant role in dietary zinc absorption. a central part of the secretion pathway is the golgi apparatus. there is only one golgi-resident znt member, dznt in the fly. znt has been previously implicated in dietary zinc absorption in a mouse knock-out study, and znt7-null mice have a reduced body zinc level. nevertheless their phenotype is not ameliorated by intravascular zinc injection. we consider the complex phenotype of znt7-null mice as arising from a universal golgi zinc transport defect, resulting lack of zinc in the golgi apparatus, instead of a systemic zinc deficiency. in our study, we found that universal knockdown of dznt also caused a severe phenotype, including a reduction in bodily zinc level. however, gut-specific dznt knockdown produced little effect on bodily zinc metabolism, except for a local change of the golgi zinc level, as reflected by alp activity reduction in the gut only. among the several zinc transporters involved in dietary zinc absorption, the two zips and at least one dznt involved in zinc uptake are strongly influenced by the dietary zinc level. when there is an excess of zinc available, uptake and efflux of zinc are reduced. when zinc is scarce, the uptake transporters dzip and dzip are upregulated, whereas the efflux of zinc is not greatly affected. the ectopic induction of dznt in otherwise non-expressing gut tissues may be a self-protecting mechanism for the gut when zinc overdosing happens, because zinc accumulation is generally toxic to cells. some general characterization of the zip and znt transporters was previously performed, although the roles of different zinc transporters in the process of dietary zinc absorption were not previously explored except that of dznt in this study, the zinc transporters that were newly found to be involved in dietary zinc absorption were characterized in more detail at the molecular and cellular levels. our results, when applicable, are largely consistent with previous work, but some inconsistencies do arise. it was previously reported that ubiquitous dzip knockdown resulted in lethality we did not see this lethality under normal culturing conditions. however, we did see lethality when the zinc supply was lowered. the difference in results is likely attributable to different culturing conditions in different laboratories such as variations in food mineral content. likewise, midgut-specific overexpression of dzip was reported to be lethal, but in our hands this happened only when extra zinc was added. further, we found that ubiquitous knockdown of dzip produced a reduced alp phenotype without much of a decrease in viability, whereas lye et al. found that ubiquitous knockdown of dzip with a stronger gal driver, tubulin-gal resulted in larval lethality. this discrepancy may be a consequence of either the difference in the drivers used, or in the food nutrient supply, as mentioned above. therefore, in the characterization of the general properties of these zinc transporters, our results are largely consistent with previous studies, except in a few cases in which the extent of the phenotype is different, which could be attributable to differences in the experimental conditions used. partial redundancy of the influx and efflux transporters were seen in dietary zinc absorption in drosophila. dzip and dzip are responsible for zinc uptake, whereas dznt and cg are responsible for the efflux. knocking any of these transporters down caused zinc deficiency under zinc-limited conditions, suggesting that all of them are important in the absorption of dietary zinc. nevertheless, slight differences do exist. although downregulation of either dzip or dzip had equivalent effects on alp levels when flies were cultured on  mmol/l edta food, on  mm edta food dzip knockdown flies developed well while dzip flies were arrested at the early larval stage. it is possible that dzip is slightly more important than dzip or is a more potent zinc transporter when zinc is severely depleted. this notion is consistent with the observation that overexpression of dzip causes a more severe phenotype than does overexpression of dzip in the case of the zinc efflux transporters, dznt and cg knockdown of either presents a similar phenotype, namely, death at around the third-instar larval stage when zinc is limited. interestingly, dznt is regulated by zinc, whereas cg is not, suggesting that dznt is a regulatory transporter whereas cg may play a more constitutive role in zinc absorption in the gut. further, overexpression of dznt produced a zinc-sensitive phenotype whereas cg overexpression did not. we speculate that cg might have a lower zinc-transporting capacity so that under conditions of zinc excess, the amount of zinc it can transport is limited, so that zinc toxicity is avoided. alternatively, cg might work together with dznt to export zinc. either scenario can explain why it is not important for cg to have zinc-responsive expression. together our results offer a glimpse into the overall process of dietary zinc absorption in a drosophila model. zinc absorption, regulated by uptake, occurs through a relatively direct route from the gut lumen to the basolateral sides of the gut epithelium and hence into the circulation. although most intracellular znts are expressed in the gastrointestinal tract, these intracellular zinc transporters are not significant in dietary zinc absorption. dzip1/ and dznt1/cg are respectively responsible for zinc intake and efflux in the enterocytes. in human studies, the dzip1/ counterpart hzip was shown to be important for zinc absorption. hzip is haploinsufficient as mutation in one allele may cause zinc deficiency. whether any other zips are involved in this process in mammals is not well-established. the lack of an ortholog of zip in drosophila makes it possible that dzip1/ may substitute for zip in mediating zinc absorption. therefore, the specific transporters for a particular role may vary between different organisms. for zinc efflux, participation of mammalian znt the counterpart of dznt is almost a certainty, but whether znt is the sole player in zinc efflux in the mammalian gut remains to be seen. overall, our results are notably consistent with some findings regarding mammalian zinc absorption: expression of zip and znt is reduced when the diet is replete in zinc, and the zinc status of the body appears to have little effect on the efficiency of dietary zinc absorption.  the genetic amenability of drosophila enabled us to show which zinc transporters are involved in the process of dietary zinc absorption, and which are not. we found that dietary zinc absorption is mediated by a set of plasma membrane-resident zinc transporters with partially overlapping functions, including the importers dzip1/dzip and the exporters dznt1/cg. the array of intracellular zinc transporters, such as the golgi-resident znt is not involved in dietary zinc absorption. zinc absorption is subject at the influx side to the rna-level control of zinc importers, and at the efflux side to the translational or post-translational control of the exporters. bodily zinc needs do not exert a feedback control on zinc absorption; instead, zinc absorption is controlled solely by the zinc levels in the enterocytes. dietary zinc influences zinc absorption through its effects on enterocyte zinc. this work assessed for the first time all potential zinc transporters for their roles in dietary zinc absorption, and we have outlined a relatively intact picture of the dietary zinc absorption and its control in a model organism. the deciphering of dietary zinc absorption in drosophila should be helpful for zinc absorption research in general, and offers a reference point for future studies in other organisms including comparative evolutionary analysis of this important biological process. 
zinc is key to the function of many proteins, but the process of dietary zinc absorption is not well clarified. current knowledge about dietary zinc absorption is fragmented, and mostly derives from incomplete mammalian studies. to gain a comprehensive picture of this process, we systematically characterized all zinc transporters for their possible roles in dietary zinc absorption in a genetically amenable model organism, drosophila melanogaster. somewhat surprisingly, it appears that the array of intracellular znt proteins, including the golgi-resident dznt is not directly involved in dietary zinc absorption. by modulating zinc status in different parts of the body, we found that regulation of dietary zinc absorption, in contrast to that of iron, is unresponsive to bodily needs or zinc status outside the gut. the zinc transporters that are involved in dietary zinc absorption, including the importers dzip and dzip and the exporter dznt are respectively regulated at the rna and protein levels by zinc in the enterocyte. our study using the model organism drosophila thus starts to reveal a comprehensive sketch of dietary zinc absorption and its regulatory control, a process that is still incompletely understood in mammalian organisms. a set of plasma membrane-resident zinc transporters was identified to be responsible for absorbing zinc from the lumen into the enterocyte and the subsequent exit of zinc to the circulation. dzip and dzip two functionally overlapping zinc importers, are responsible for absorbing zinc from the lumen into the enterocyte.
 zinc is key to the function of many proteins, but the process of dietary zinc absorption is not well clarified. current knowledge about dietary zinc absorption is fragmented, and mostly derives from incomplete mammalian studies. to gain a comprehensive picture of this process, we systematically characterized all zinc transporters for their possible roles in dietary zinc absorption in a genetically amenable model organism, drosophila melanogaster.  a set of plasma membrane-resident zinc transporters was identified to be responsible for absorbing zinc from the lumen into the enterocyte and the subsequent exit of zinc to the circulation. dzip and dzip two functionally overlapping zinc importers, are responsible for absorbing zinc from the lumen into the enterocyte. exit of zinc to the circulation is mediated through another two functionally overlapping zinc exporters, dznt and its homolog cg. somewhat surprisingly, it appears that the array of intracellular znt proteins, including the golgi-resident dznt is not directly involved in dietary zinc absorption. by modulating zinc status in different parts of the body, we found that regulation of dietary zinc absorption, in contrast to that of iron, is unresponsive to bodily needs or zinc status outside the gut. the zinc transporters that are involved in dietary zinc absorption, including the importers dzip and dzip and the exporter dznt are respectively regulated at the rna and protein levels by zinc in the enterocyte.  our study using the model organism drosophila thus starts to reveal a comprehensive sketch of dietary zinc absorption and its regulatory control, a process that is still incompletely understood in mammalian organisms. the knowledge gained will act as a reference for future mammalian studies, and also enable an appreciation of this important process from an evolutionary perspective. dietary zincdrosophilaintracellular zinc traffickingregulationzinc absorption 