CHAPTER 1: Manganese Transport, Trafficking and Function in Invertebrates

Manganese is a biologically important trace metal and is required for the growth and survival of most, if not all, living organisms. It is perhaps best known for its prominent role as a redox-active cofactor in free radical detoxifying enzymes.^1–8  However, the utilization of manganese in biological systems is substantially more diverse. The uptake and distribution of manganese is critical for proper function of manganese-requiring enzymes; however, this same metal can have deleterious effects in biological systems if homeostasis is disrupted.^9–12  In order to prevent toxicity, cells maintain manganese under tight homeostatic control. Adding complexity to the cellular control of manganese homeostasis is the presence of multiple types of manganese transporter that participate in the specific transport of manganese or in general divalent metal ion transport.

Cells appear to transport manganese solely as the divalent cation and several classes of manganese transporters have been characterized. These include Nramp H⁺-manganese transporters,^13–16  ATP-binding cassette (ABC) manganese permeases,^17–21  manganese transporting P-type ATPases,^22,23  cation diffusion facilitators (CDFs),^24–26  and inorganic phosphate transporters with high affinity for Mn–HPO₄ complexes.^27–29  Bacteria typically contain one or more of these types of transporter, and these classes of transporter are also present in eukaryotic cells.^30,31  These transporters comprise both high and low affinity manganese uptake systems and the transporter utilized depends on the concentration of manganese in the environment. The homeostatic range for manganese is quite wide, with cellular levels of manganese between 0.04 and 2.0 mM under optimal growth conditions.^{10,16,29,32,33}  Cells rarely experience optimal environmental levels of manganese and often face extreme conditions of either manganese deficiency or excess.³⁴  Cells activate stress response mechanisms in an attempt to return manganese levels to the homeostatic range. The response typically results in the upregulation or downregulation of cell surface and intracellular transport systems. The regulation of manganese uptake, distribution, and efflux can occur at both the transcriptional and post-translational levels, although the specific route of regulation varies in different organisms.

Manganese metalloenzymes are involved in a wide range of cellular functions, including detoxification of reactive oxygen species, protein glycosylation, polyamine biosynthesis, DNA biosynthesis, nucleic acid degradation, phospholipid biosynthesis and processing, polysaccharide biosynthesis, protein catabolism, the urea cycle, photosynthesis, and sugar catabolism.^2,35–45  Manganese-dependent enzymes that participate in these processes typically utilize manganese in Lewis acid–base reactions or as a reduction/oxidation center to facilitate catalysis. These types of reaction are exemplified by arginase (Lewis acid) and Mn superoxide dismutase (reduction/oxidation),^2,3,46,47  and the role of manganese in these reactions is shown in Figure 1.1.

The importance of manganese in biological systems is not limited to enzyme-mediated catalysis. Non-enzymatic manganese is involved in the formation of bacterial products, including secreted antibiotics,⁴⁸  and contributes to the stabilization of bacterial cell walls.⁴⁹  In addition, the accumulation of non-protein complexes of manganese can function in the removal of reactive oxygen species (ROS), especially superoxide.^50–53  These Mn-antioxidants are divalent manganese complexes of small metabolites, and while the nature of the intracellular Mn-complexes has not been clearly defined, phosphate and lactate Mn-complexes have been shown to display the capacity to react efficiently with superoxide in vitro.^52,54,55  Complexes of both iron and copper exhibit superoxide scavenging activity, however these metal ions also exhibit pro-oxidant activity.^56–59  In contrast, manganese ions react poorly with hydrogen peroxide and do not generate the highly toxic hydroxyl radical, providing a beneficial antioxidant activity without the pro-oxidant side effects of other redox active metals.^50,60 

It appears that Mn-antioxidants can serve to enhance oxidative stress protection when enzymatic antioxidants are insufficient in various organisms.^53,61–63  A critical role for Mn-antioxidants has been demonstrated in Deinococcus radiodurans, a bacterium that is extremely resistant to radiation and desiccation. In this organism, survival under extreme exposure to radiation and other oxidative stress conditions is not dependent on antioxidant enzymes but instead relies on the accumulation of millimolar concentrations of manganese and the subsequent formation of Mn-antioxidants.^50,51,60,63  Interestingly, Lactobacillus plantarum, while resistant to oxidative stress, does not express the antioxidant enzyme superoxide dismutase.^64,65  Indeed, L. plantarum appears to rely exclusively on Mn-antioxidants for protection against oxidative stress,^54,66,67  highlighting the power of this alternative ROS detoxification pathway.

The majority of the information on manganese antioxidants has come from investigation of bacterial and yeast systems; however, it is also likely that these complexes are present in multicellular organisms. Elevated manganese accumulation in the nematode Caenorhabditis elegans enhances thermotolerance and oxidative stress resistance, and extends life span.^68,69  The mechanism of the enhanced stress resistance due to manganese supplementation in C. elegans has not been fully elucidated but is suspected to involve elevated antioxidant activity.

Manganese is either known or proposed to be important for virulence in bacterial species such as Salmonella enterica, Mycobacterium tuberculosis, Staphylococcus aureus, Yersinia pestis, and Streptococcus pneumoniae.^19,30,31  Invasion and initial survival within host cells is not dependent on manganese; however, extended survival appears to require the element.^70,71  The expression of manganese transporters is required to enhance bacterial survival when challenged by host defenses.^{15,18,70,72–74}  Whether different classes of manganese transporter are redundant or involved at different stages of infection is not known. Models have been proposed in which manganese transporters, as well as iron transporters, are essential for virulence because of competition between the infecting bacterium and host cells for metal ions.^31,71  The need for manganese in bacterial virulence appears to go beyond its role as a cofactor in ROS detoxifying enzymes such as Mn-superoxide dismutase and catalase. Enterobacteria are capable of rapidly increasing uptake of manganese in response to stress, and can accumulate millimolar levels of manganese.¹⁷  This concentration of manganese far exceeds the level needed to supply Mn-superoxide dismutase with its cofactor. It appears that the formation of non-protein Mn-antioxidant complexes may also be an important virulence factor in some bacterial species. The additional protection against reactive oxygen species generated by the host cells may allow invading bacteria to survive the initial stages of infection, and thus promote colonization.

In prokaryotic cells, which lack internal compartmentalization, metal ion homeostasis is maintained primarily by tight regulation of metal cation flux across the cytoplasmic membrane. Manganese uptake in bacteria predominantly involves members of two transporter families, Nramp (MntH) and cation-transporting ABC permeases (MntABCD and related), with many species containing both types of transport system.^{17,20,70,73,75,76}  In addition, utilization of other transport systems for manganese, such as a P-type adenosine triphosphatase (ATPase) by Lactobacillus species (MntP) and Mycobacterium tuberculosis (CtpC), has also been observed.^23,77  Exposure to excess manganese leads to repression of these dedicated manganese transport systems.^20,74  However, the tight control of manganese influx can be bypassed via other transporters that are capable of facilitating the uptake of manganese but escape regulation by this metal. An example of this is PitA, an inorganic phosphate transporter with high affinity for Mn–HPO₄ complexes that appears to be a major source of manganese uptake during conditions of excess.²⁸ 

Members of the Nramp (natural resistance-associated macrophage protein) transporter family were first identified in yeast and mammalian cells and subsequently found to play a major role in metal ion homeostasis.^78–80  Nramp proteins function in general metal ion transport, and members of this transporter family have been shown to facilitate the movement of divalent metal ions including manganese, zinc, copper, iron, cadmium, nickel, cobalt, and lead.^14,81–85  Transport of metal ions through Nramp is energized by the symport of protons (Figure 1.2).^15,86 

The majority of bacterial Nramp1 homologues, typically designated as MntH,^{15,20,23,80,87}  appear to function in manganese homeostasis.^15,73,74  The MntH transporters are commonly found in bacterial species, although examples of bacteria lacking Nramp transporters have been described.³⁰  Metal accumulation studies revealed that overexpression of Staphylococcus aureus MntH resulted in increased cell-associated manganese but not calcium, copper, iron, magnesium or zinc, indicating that this Nramp1 transporter was selective for the uptake of manganese.²⁰  Consistent with these observations, mutants of mntH in Bacillus subtilis exhibited impaired growth in metal-depleted media that could be rescued by the addition of manganese.⁷⁴  Direct transport assays also indicated a preference for manganese in MntH from Salmonella enterica serovar Typhimurium and Escherichia coli. The affinity for manganese far exceeds that for iron in these MntH proteins, demonstrating the role of Nramp transporters in bacterial manganese uptake.³⁰ 

Species differences in MntH metal ion specificity have been observed, with some MntH homologues appearing to function in the transport of other metals in addition to manganese. While S. enterica, E. coli, and B. subtilis MntH exhibit a strong preference for manganese,^30,74  the M. tuberculosis MntH homologue, Mramp, appears to transport not only manganese but also significant amounts of iron and zinc.⁸⁸  Roles for Nramp transporters in the uptake of other metal ions, especially iron, have been documented in both prokaryotic and eukaryotic organisms.^{16,73,80,89–91}  Multiple Nramp isoforms can be present in a single species, and these Nramp transporters, although highly similar, may have divergent metal ion preferences. Pseudomonas aeruginosa expresses two distinct Nramp transporters capable of transporting manganese, and multiple Nramp isoforms are present in Burkholderia species, although the metal ion preferences of these transporters have not been determined.³⁰  While the most physiologically relevant substrate for the majority of bacterial MntH transporters appear to be manganese, it is clear that these transporters have the capacity to facilitate the uptake of other metals when they are present in excess. This broad metal ion selectivity in Nramp transporters also appears to enhance the uptake of toxic metal ions, such as cadmium and lead.^74,85,92 

The ATP-binding cassette (ABC) transporter superfamily is one of the largest classes of transporter, and this transporter family utilizes hydrolysis of ATP to facilitate the import or export of diverse substrates, ranging from ions to macromolecules.^93–95  These transporters are present in the plasma membrane or inner membrane of Gram-negative bacteria,^93,95,96  and are well known for their involvement in multi-drug resistance in both prokaryotic and eukaryotic cells by enhancing the export of toxins and drugs.^97,98  However, ABC transporters functioning as importers have only been described in prokaryotic systems.^93,95,96  Metal ion transporting ABC permeases have been identified with important roles in manganese acquisition.^{19,70,76,99,100}  The cation selectivity of manganese ABC-type permeases extends to other divalent metal ions including iron, zinc, cobalt, nickel, molybdenum, and cadmium; however, the typical affinities for these metal ions are 10- to 100-fold lower than for manganese.^100–103 

Examples of bacterial ABC transporters involved in manganese import include, but are not limited to, MntABCD (Bacillus subtilis, Staphylococcus aureus), SitABCD (Shigella flexneri), PsaABCD (Streptococcus pneumoniae), and YfeABCD (Yersinia pestis),^{17,18,21,31,70,74,104,105}  and these transporters exhibit similar subunit organization and function. The manganese transporter complex MntABCD (see Figure 1.2) consists of three subunits: MntC and MntD are integral membrane proteins that form the permease subunit and mediate cation import; MntB is the ATPase subunit; and MntA functions as a cation binding protein that delivers manganese to the permease complex.^{17,19,93,95,106}  MntA is present as a soluble periplasmic protein in Gram-negative bacteria.⁹⁴  In Gram-positive bacteria MntA is a lipoprotein anchored to the extracellular side of the plasma membrane,^31,74,107  because these bacteria do not possess an outer membrane. Similar organization is also present in the operons of other manganese ABC transporters such as sitABCD, yfeABCD, and psaABCD.^{17,19,21,31,70} 

P-type ATPases form a large superfamily of cation and lipid pumps and are distinct from the ABC class of ATPases in that ATP hydrolysis is coupled to transport within a single protein chain.¹⁰⁸  A manganese/cadmium transporting P-type ATPase, MntP (also known as MntA, although distinct from MntABCD) from Lactobacillus plantarum, was identified and proposed to be the major source of manganese for this organism.²³  Subsequent analysis of the L. plantarum genome revealed the presence of three Nramp transporters as well as a manganese ABC transporter.¹⁰⁹  Mutations of L. plantarum mntP or the Nramp and ABC transporters did not alter intracellular manganese concentrations under either manganese deficiency or excess.¹⁰⁹  A primary role for MntP in manganese acquisition in L. plantarum is not certain; however, Nramp and manganese ABC transporters were also not essential for manganese uptake. It appears that L. plantarum is highly adaptive in maintaining manganese uptake even in the absence of known transporters and additional, yet uncharacterized, transporters may participate in manganese accumulation. Three additional putative P-type calcium/manganese ATPases are present in L. plantarum and have been proposed as possible sources of manganese uptake in this bacterium.¹⁰⁹ 

In Salmonella lacking both the Nramp and manganese ABC transporters, manganese uptake activity has been observed, although at low levels.³⁰  The proposed source of this residual manganese uptake is PitA, a low affinity phosphate transporter.^27,110  The substrate for PitA is a neutral metal phosphate (metal–HPO₄) complex, and this transporter has a preference for phosphate complexes of magnesium, calcium, cobalt, and manganese.²⁷  In environments rich in metals and phosphate, PitA and related transporters have been proposed to be major suppliers of divalent metal cations¹¹¹  and may contribute to metal ion toxicity. Experimental evidence for manganese uptake in intact cells through PitA or other phosphate transporters is limited. However, stimulation of manganese uptake was produced in L. plantarum and B. subtilis by the addition of phosphate.^112,113 

A manganese efflux transporter, MntE, showing homology with the cation diffusion facilitator family (CDF) has been identified in several bacterial species.^26,114  Members of the CDF family are found in most prokaryotic and eukaryotic cells and typically function in metal tolerance by exporting cations from the cytoplasm to the cell exterior.^25,114,115  The most likely transport mechanism for MntE is an antiport cycle consisting of the efflux of manganese with the uptake of hydrogen and potassium ions.²⁵ 

Cells lacking functional MntE exhibit sensitivity to manganese but not other metal ions (cadmium, cobalt, copper, iron, nickel, and zinc) and accumulate three times the intracellular manganese seen in the wild-type strain.²⁶  The high levels of intracellular manganese in mntE mutants increased resistance to oxidative stress but did not lead to enhanced virulence. Bacteria lacking MntE were actually less pathogenic than wild-type cells,²⁶  indicating that control of manganese homeostasis is critical for both survival and virulence.

In addition to MntE, the P-type ATPase CtpC, from M. tuberculosis and M. smegmatis, also appears to facilitate manganese efflux. Deletion of ctpC leads to sensitivity to oxidative stress and elevated accumulation of cytosolic manganese.⁷⁷  Mutations that increase cytosolic manganese commonly enhance resistance to oxidative stress; however, this is not the case for cells lacking CtpC. This discrepancy appears to be explained by the function of CtpC in providing manganese for incorporation into secreted proteins, including Mn-superoxide dismutase enzymes. Thus the primary function of CtpC may not be simply to remove excess manganese from cells, but also to provide the manganese cofactor for secreted enzymes.⁷⁷ 

The only other example of a bacterial manganese efflux protein is YebN from Xanthomonas oryzae pv. oryzae. YebN does not belong to any known transporter family, although cells lacking YebN are manganese sensitive and accumulate high concentrations of intracellular manganese.^116,117  YebN may represent an uncharacterized class of manganese transporter, or alternatively the effect of this protein on manganese efflux may be indirect.

The uptake of essential transition metals, such as manganese, must be regulated in order to respond to changes in environmental conditions. Cells facilitate uptake when faced with deficiency and prevent import under conditions of metal excess. In bacteria this regulation is mediated primarily at the level of transcription.^30,31  The principal bacterial transcription factor involved in manganese-dependent gene expression is MntR, a manganese-specific member of the diphtheria toxin repressor family (DtxR).^74,106,118  MntR largely controls intracellular manganese levels in bacteria through regulated expression of mntH and mntABCD. Regulation of mntH and mntABCD is different under conditions of manganese deficiency and manganese excess, and variation among bacterial species has also been observed. In B. subtilis cells experiencing manganese deficiency, MntR activates expression of mntABCD although mntH is expressed independently of MntR.⁷⁴  However, under conditions of manganese sufficiency or excess, B. subtilis MntR functions as a typical repressor and inhibits the expression of both mntH and mntABCD,⁷⁴  thereby limiting manganese uptake. MntR dependent inhibition of both mntH and mntABCD expression has also been observed in E. coli and S. enterica.^119–121  In S. aureus, MntR acts as a negative regulator of mntABCD in the presence of excess manganese, similar to what is observed in B. subtilis. However, mntH levels in S. aureus are not decreased by excess manganese and positive expression of mntH appears to require MntR.²⁰  The manganese efflux systems mntE, ctpC, and yebN all exhibit positive regulation by MntR,^26,77,117  as would be expected to facilitate removal during condition of manganese excess. MntR homologues from different bacterial species often exhibit a low level of sequence identity,³¹  which may account for the contrasting modes of regulation observed.

In addition to MntR, the transcription factors Fur, PerR, and OxyR have been found to have roles in regulating the expression of mntH and mntABCD. Fur is well characterized for its role in the regulation of iron uptake genes in response to cellular iron status.¹²²  Expression of mntH and mntABCD is also regulated by Fur in some bacterial species including E. coli, S. meliloti, and S. enterica, but not in B. subtilis.^107,120,121  The ability of manganese transporters to facilitate the uptake of iron when present at high concentrations^15,31,101  may explain the dual regulation of mntH and mntABCD by manganese and iron in some bacterial species, although the physiological relevance of this regulation has not been established.

The principle roles of PerR and OxyR are in the regulation of genes in response to oxidative stress.^122,123  However, these factors are also involved in manganese homeostasis in several bacterial species including B. subtilis, S. aureus, S. pneumoniae, and S. enterica.^31,120,124  The regulation of mntH and mntABCD by PerR and OxyR is likely to be related to the involvement of manganese in defense against oxidative stress. Enhanced manganese uptake facilitates incorporation of manganese into antioxidant enzymes, such as Mn-superoxide dismutase, as well as production of non-protein antioxidant complexes.^{33,61–63,125,126}  However, when sufficient cellular antioxidant capacity is obtained, PerR and OxyR may sense the reduction in oxidative stress and limit manganese uptake to prevent toxicity from excessive manganese accumulation.

Limited information is available regarding the regulation of P-type manganese transporting ATPases (MntP) and cation-phosphate transporters (PitA). Expression of mntP from L. plantarum is increased under manganese deficiency,²³  consistent with regulation by MntR; however, regulators of L. plantarum mntP in response to manganese limitation or excess have not been determined. In the case of pitA, there is no report of manganese-mediated transcriptional control but regulation by zinc ions has been observed. The expression of pitA was increased approximately two-fold in response to elevated zinc concentrations and is also increased by phosphate limitation,¹²⁷  although the transcription factors involved in pitA regulation have not been identified. It has been proposed that the addition of excess zinc may compete with magnesium for binding to phosphate, reducing phosphate influx, because ZnHPO₄ complexes are less favored for transport by PitA than MgHPO₄.¹²⁷  This model suggests that pitA is regulated by alterations in intracellular phosphate levels and not directly by zinc ions. Excess manganese is not expected to limit phosphate uptake because MnHPO₄ complexes are preferred substrates for PitA,²⁸  thus this type of pitA regulation appears unlikely under conditions of manganese excess.

The yeast NRAMP transporters Smf1p and Smf2p form the primary manganese uptake system utilized by yeast experiencing manganese deficiency.^16,128–130  The Smf1p and Smf2p transporters have specific roles in the acquisition of manganese and are localized either at the cell surface or in intracellular vesicles (Figure 1.3). Regardless of their localiztion, the Smf1p and Smf2p transporters move manganese across membranes toward the cytosol.^{13,34,78,131,132}  A third NRAMP homologue, Smf3p, is also present in yeast but this transporter appears to function in the movement of iron from vacuolar stores.^133,134  Similar to other members of the Nramp family, yeast SMFs are capable of facilitating the translocation of divalent metals in addition to manganese, including iron, cobalt, copper, zinc, and cadmium, and may contribute to the toxicity of these metals when present in excess.^{13,34,78,79,81–83,89,132,133} 

Saccharomyces cerevisiae Smf1p and Smf2p were originally identified on the basis of their ability when overexpressed to bypass a mitochondrial protein-processing defect (Suppressor of Mitochondria import Function).¹³⁵  Subsequently Smf1p was characterized as a high affinity manganese transporter,⁸¹  revealing that the role of Smf1p in mitochondrial import was due to the requirement for manganese in mitochondrial protein processing.¹³⁵  Despite the high degree of similarity between Smf1p and Smf2p (50% identity and 67% similarity), these transporters are not functionally redundant and exhibit distinct roles in manganese uptake and trafficking.^{16,33,34,125,128,134,136}  Smf1p is localized at the cell surface under conditions of manganese deficiency; however, the majority of Smf1p is present in intracellular vesicles, thought to be endosomes, when manganese levels are sufficient.^{34,81,128–130,137}  Based on changes in Smf1p localization in response to manganese status it appears that Smf1p is functioning in manganese transport only under deficiency conditions and has no role in manganese uptake and distribution during manganese sufficiency. Consistent with this proposal, cellular manganese levels in yeast lacking SMF1 grown under manganese-sufficient conditions were unchanged when compared with the wild-type strain.⁸¹  In addition, no change in the activity of manganese-dependent enzymes, such as mannosyl-transferases and mitochondrial superoxide dismutase, was observed in cells lacking Smf1p.¹²⁵ 

In contrast to Smf1p, the second yeast Nramp transporter, Smf2p, exhibits many characteristics of a cell-surface manganese transporter and has a significant role in manganese homeostasis during manganese sufficient conditions. Deletion of SMF2 results in a dramatic reduction in cellular manganese accumulation, resulting in lowered activity of manganese-dependent enzymes in the cytosol, Golgi apparatus, and mitochondria.^125,138  Despite the cell-wide manganese deficiency in yeast lacking Smf2p, this transporter is observed in intracellular Golgi-like endosomal structures and cell surface localization is not detected.^16,34,125  Even in the absence of experimental evidence for plasma membrane associated Smf2p, it has been proposed that a small number of cell-surface Smf2p molecules, which are sufficient for manganese acquisition, may be present that have escaped detection by conventional microscopy or cell-fractionation techniques.¹³⁰ 

Smf1p is much more abundant than Smf2p under both manganese deficient and sufficient conditions,^82,128  suggesting that Smf1p is performing a needed function in cellular manganese homeostasis. While the total manganese content of cells lacking the Smf1p transporter is not altered, it appears that Smf1p is important for the transport of manganese that is utilized as a non-proteinaceous manganese antioxidant, similar to those observed in bacterial cells.³³  Addition of manganese salts to yeast cells lacking the cytosolic Cu/Zn superoxide dismutase is able to reverse all symptoms of oxidative stress, and this rescue is dependent on the presence of Smf1p but not Smf2p.³³  The presence of two manganese Nramp transporters in yeast appears to be necessary to provide separate pools of manganese, at least under manganese-sufficient conditions. Smf2p provides manganese for manganese-requiring enzymes while Smf1p plays a critical role in oxidative stress resistance by supplying manganese for non-proteinaceous manganese antioxidant molecules.

The Smf1p and Smf2p transporters are involved in the uptake of manganese that is needed for the activity of manganese enzymes and oxidative stress protection. However, other transporters participate in the uptake of manganese under conditions of manganese surplus. The primary low affinity manganese transporter in yeast cells is Pho84p, a member of the major facilitator superfamily (MFS). Pho84p is a high affinity phosphate/proton symporter²⁹  and appears to be analogous to the bacterial PitA.²⁷  Phosphate transport in yeast is mediated by at least six transporters; however, only Pho84p has a major role in manganese uptake.²⁹  The substrate for Pho84p is a divalent metal phosphate complex and in vitro reconstitution studies have shown a high preference for the transport of manganese and cobalt.²⁸  Manganese transport by Pho84p accounts for the majority of excess manganese accumulated when cells are exposed to manganese surplus, and yeast lacking Pho84p are resistant to manganese toxicity through reduced manganese accumulation.^29,33  In the absence of an excess of manganese, Pho84p appears to transport a magnesium phosphate complex. Magnesium can effectively inhibit manganese uptake through Pho84p,²⁹  and the higher concentrations of magnesium in yeast growth media compared to manganese probably facilitate the uptake of magnesium phosphate complexes. However, when yeast experience conditions in which a manganese surplus is present, Pho84p-mediated transport of MnHPO₄ complexes is favored.^28,29  The uptake of manganese through Pho84p appears to be unintended, and is a consequence of the presence, under manganese surplus conditions, of MnHPO₄ complexes that mimic the natural MgHPO₄ substrate. Manganese transported by Pho84p is capable of causing toxicity but does not appear to have a role as an essential nutrient. Cells lacking Pho84p do not exhibit defects in manganese-requiring enzymes²⁹  or in the formation of non-protein manganese antioxidants.³³ 

While much is known about the cellular uptake of manganese in both prokaryotic and eukaryotic systems, it is not entirely clear how cells target manganese to the correct recipient proteins.^34,131,139  The presence of multiple organelles in eukaryotic cells that contain manganese-requiring proteins increases the complexity of manganese trafficking. Proteins involved in the vectoral delivery of manganese inside cells have not been identified; however, manganese is correctly targeted under manganese-sufficient conditions, suggesting that a concerted process for manganese delivery exists. Movement of manganese to specific organelles within yeast cells is mediated by the action of intracellular transporters. Currently, manganese transporters for the Golgi complex^22,140,141  and vacuole^142,143  have been characterized. However, the identity of the manganese transporter for mitochondria is not known, although it is possible that manganese is taken up through the action of several transporters or transporters with dual specificity for other divalent metal ions.

One of the major functions of the Golgi apparatus is protein glycosylation, and manganese serves as an essential cofactor for several mannosyl-transferase enzymes present in this organelle.^22,144  Manganese transport from the cytosolic compartment into the Golgi is mediated by Pmr1p, a P-type calcium and manganese-transporting ATPase.^{22,34,141,144,145}  In the absence of Pmr1p, the sorting of proteins and glycosylation in the Golgi apparatus is impaired.^125,140,144  In addition, loss of function mutations in PMR1 lead to hyperaccumulation of manganese in the cytosol and severe manganese sensitivity.^145,146  The excess accumulation of manganese in cells lacking Pmr1p reveals that Pmr1p is not only important for supplying the manganese needed for proper activity of mannosyl-transferase enzymes, but it also plays an important role in manganese detoxification. Surplus manganese that has the potential to be toxic is transported into the secretory pathway by Pmr1p and is ultimately released from the cell through exocytic transport (see Figure 1.3).^{34,141,145–147}  Pmr1p-dependent manganese efflux appears to be the primary mechanism for removal of unwanted manganese from yeast cells, although a dedicated manganese efflux transporter has not been identified. The only example of a manganese efflux transporter in yeast cells is Hip1p, a high affinity histidine permease that is also involved in the transport of manganese;¹⁴⁸  nevertheless, the transport cycle that facilitates manganese efflux is not known. However, yeast lacking functional Hip1p do not display the severe manganese sensitivity seen in cells with mutations in PMR1,¹⁴⁸  indicating that the primary manganese efflux pathway is Pmr1p-dependent exocytic transport from the Golgi.

Manganese transported into the Golgi by Pmrp1 can also be redirected back to the cytosol through the action of Atx2p,³²  a putative transporter with similarity to the ZIP (Zrt1, Irt1-like Protein) family. The precise function of Atx2p is not clear but this protein has a role in maintaining the cytosolic concentration of manganese. Overexpression of Atx2p results in increased cellular manganese content, whereas cells lacking Atx2p exhibit decreased cytosolic manganese levels.³²  It appears that Atx2p and Pmr1p work in opposite directions to control manganese levels in both the cytosol and Golgi compartments.

In addition to the Pmr1p-mediated cellular export of manganese through the secretory pathway, vacuolar sequestration is another mechanism used to limit the toxicity of manganese. The yeast vacuole, a lysosome-like compartment, is a major site for metal ion storage and detoxification, and yeast strains defective in vacuolar function exhibit sensitivity to several transition metals including manganese.^149,150  Manganese is sequestered in the vacuole through the action of Ccc1p,^34,142,143  a manganese and iron transporter with similarity to the vacuolar iron transporter (VIT) family (see Figure 1.3). While the transport mechanism for the VIT class of transporters is not known, they are predicted to function by an H⁺ antiport carrier-type mechanism.¹⁵¹  Consistent with a role in manganese sequestration, cells lacking Ccc1p display enhanced sensitivity to manganese, and overexpression of Ccc1p reduces manganese toxicity in cells lacking Pmr1p.¹⁴²  A transporter that facilitates vacuolar efflux of manganese has not been identified and there is currently no evidence that manganese is released from the vacuole. It is possible that Ccc1p functions to sequester manganese in the vacuole to limit its toxicity, but not for storage and later use by the cell. Alternatively, vacuolar transporters typically associated with other metals, such as iron or calcium, may facilitate vacuolar manganese efflux under specific conditions.

The mitochondrial manganese transporter has yet to be identified; however, the search for this transporter has revealed other pathways important for incorporation of manganese into the mitochondrial superoxide dismutase, Sod2p. Movement of manganese to its proper destination does not always occur unhindered, and alterations in both manganese and iron metabolism can affect the delivery of manganese to Sod2p. The mitochondrial carrier protein Mtm1p was identified as an important molecule for manganese insertion into Sod2p.¹³⁸  Characterization of Mtm1p revealed that it was not the mitochondrial manganese transporter; instead, Mtm1p participates in mitochondrial iron metabolism, and dysregulation of mitochondrial iron alters manganese binding to Sod2p.^125,152,153  Iron atoms outnumber manganese by nearly two orders of magnitude in mitochondria; however, iron does not bind to Sod2p under these conditions.^152,153  The misincorporation of iron into Sod2p in yeast mutants with defects in mitochondrial iron metabolism is not caused by a global change in the chemical environment of mitochondrial iron. Instead, in the case of mutations that result in mitochondrial iron overload, failure to deliver iron to the proper recipient proteins results in accumulation of iron sulfur precursors on assembly proteins. Iron derived from these stalled iron cluster assembly scaffolds is highly reactive with Sod2p and prevents the proper binding of manganese.¹⁵³  A small pool of this Sod2p reactive iron appears always to be present, but under normal conditions the abundance of manganese is sufficient to promote proper insertion of manganese into Sod2p. However, when manganese levels are reduced, this reactive iron can effectively compete with manganese for binding to Sod2p.¹⁵³  These observations suggest that, similar to bacterial MnSOD, mitochondria Sod2p captures manganese on the basis of the differential availability of manganese and iron, and they do not support a requirement for a specific carrier protein to deliver manganese to Sod2p.¹⁴⁷ 

The regulation of many transition metal transporters, including those for copper, iron, and zinc, occurs primarily at the transcriptional level.¹⁵⁴  However, regulation of the Nramp transporters, Smf1p and Smf2p, occurs principally post-translationally in response to changes in manganese concentrations (Figure 1.4).^16,128,155  Smf1p and Smf2p exhibit multiple levels of post-translational regulation in response to changes in manganese from deficient, through sufficient, to toxic concentrations. When cells are deficient in manganese, the amount of Smf1p and Smf2p increases, allowing enhanced manganese accumulation.^16,128–130  Conditions of sufficient manganese redirect the majority of Smf1p and Smf2p to the vacuole for degradation, limiting the uptake of manganese, although a substantial fraction of these transporters is not degraded.^16,128–130  Exposure to toxic concentrations of manganese leads to increased vacuolar degradation of Smf1p and Smf2p, virtually eliminating these transporters from cells.^16,128–130 

When manganese levels are within a range that is sufficient for cellular needs but below the toxic threshold, the majority of newly synthesized Smf1p and Smf2p is directly targeted to the vacuole for degradation.^82,128  The constitutive degradation maintains Smf1p and Smf2p at levels that are sufficient to supply the cell with manganese but are not in an excess that would promote manganese overload. Vacuolar targeting of Smf1p and Smf2p during constitutive degradation occurs in the exocytic pathway, in contrast to most cell surface transporters that are targeted to the vacuole through endocytosis. As Smf1p and Smf2p transit the Golgi, the bulk of these proteins are conjugated with ubiquitin, marking them for vacuolar targeting through the multivesicular body (MVB) pathway.^130,137,156  The ubiquitin ligase Rsp5p is responsible for ubiquitination of Smf1p and Smf2p but requires adaptor proteins in order to interact with these transporters. One of these adaptor proteins, Bsd2p, interacts with both Rsp5p and the Smf1p and Smf2p transporters and thereby links Smf1p and Smf2p to Rsp5p, facilitating ubiquitin conjugation.^128,130,157  In addition to Bsd2p, two other adaptor proteins, Tre1p and Tre2p (transferin receptor-like), are also required for Rsp5p-mediated ubiquitination of Smf1p and Smf2p.^137,158  Cells lacking the adaptor protein Bsd2p, or both Tre1p and Tre2p, or containing mutations in RSP5 accumulate upwards of 10 times more Smf1p and Smf2p, and these transporters display localizations similar to those observed in manganese deficient conditions.^128,130,137  Bsd2p and Rsp5p are thought to form a complex first that is then bound by the Tre1p and Tre2p proteins. The Tre1/2p–Bsd2p–Rsp5 complex then interacts with and mediates the ubiquitination of Smf1p or Smf2p.¹³⁰  Subsequently, ubiquitinated Smf1p and Smf2p are directed through the MVB pathway for their eventual degradation in the vacuole.^{129,130,137,157} 

Manganese deficiency enhances the stability of Smf1p and Smf2p, resulting in increased accumulation of these transporters.^128,134,155  Under manganese deficient conditions, the Smf1p and Smf2p proteins do not interact with the Tre1/2p–Bsd2p–Rsp5 complex and vacuolar degradation is blocked.^128,137,158  Instead, these transporters are delivered either to the cell surface in the case of Smf1p or to intracellular vesicles for Smf2p.^16,128–130  The interaction of the Tre1/2p–Bsd2p–Rsp5 complex with Smf1p and Smf2p appears to be linked to the concentration of manganese in the Golgi.¹⁵⁵  Limiting manganese transport into the Golgi by deletion of PMR1 promotes the stabilization of Smf1p,¹⁵⁵  even though these conditions result in elevated cytosolic manganese.^141,145  How Smf1p and Smf2p detect manganese deficiency is not known but it has been proposed that these transporters are directly sensing the manganese status of the Golgi lumen.¹⁵⁵ 

In contrast to manganese sufficient conditions, the vacuolar degradation of Smf1p is enhanced to eliminate the residual levels of this transporter when intracellular manganese concentrations exceed the toxic threshold.¹⁵⁵  Degradation of Smf1p in response to toxic concentrations of manganese utilizes trafficking through the MVB pathway, similar to the case of sufficient manganese. However Smf1p vacuolar targeting in response to toxic manganese is independent of Bsd2p, Tre1p, and Tre2p.¹⁵⁵  Smf1p proteins localized to both the plasma membrane and intracellular vesicles are targeted for degradation following exposure to toxic concentrations of manganese. In contrast to conditions of manganese sufficiency, Smf1p appears to sense cytosolic manganese during chronic manganese toxicity. Plasma membrane associated Smf1p is rapidly internalized and delivered to the vacuole for degradation upon exposure to toxic concentrations of manganese. The endocytosis of Smf1p, while independent of Bsd2p, Tre1p, and Tre2p, does require the Rsp5p ubiquitin ligase.¹⁵⁵  However, Rsp5p adaptors that participate in the endocytosis of Smf1p in response to toxic manganese have not been defined. Smf1p localized to intracellular vesicles is slowly trafficked toward the vacuole with prolonged exposure to toxic concentrations of manganese. The slow degradation of Smf1p located in the intracellular vesicles during chronic exposure to toxic manganese does not appear to require Rsp5p or other known ubiquitin ligases. Smf2p is similarly slowly trafficked to the vacuole for degradation in response to chronic manganese toxicity. The slow degradation of the Smf1p and Smf2p proteins present in intracellular vesicles in response to manganese toxicity is unusual. It appears that cells do not immediately degrade these transporters during manganese exposure and Smf1p and Smf2p may be retained in an intracellular pool that can be rapidly recycled in the event of changes in the manganese status of the environment.¹⁵⁵ 

In contrast to the robust regulation of the Smf1p and Smf2p transporters by manganese status, other manganese transporters show limited or no manganese-dependent regulation. The phosphate content of the cell tightly regulates Pho84p, the major low affinity manganese transporter; however, transcription, localization, and stability of Pho84p do not appear to be regulated by manganese.²⁹  There is also no indication that expression or stability of the intracellular manganese transporters Pmr1p, Atx2p, and Ccc1p are modulated by manganese concentrations. Ccc1p is regulated by the iron status of the cell,¹⁵⁹  and it is possible that Pmr1p and Atx2p are similarly regulated by other environmental cues instead of the level of manganese in the environment.

In eukaryotic organisms, the uptake and trafficking of manganese appear to be mediated largely through the action of Nramp transporters. Among multicellular microorganisms, manganese homeostasis has been best characterized in the nematode Caenorhabditis elegans, which expresses three distinct Nramp proteins that function as cation transporters: SMF-1, SMF-2, and SMF-3.^160–162  Each of the C. elegans SMF proteins can function in the uptake of manganese, although it appears that they have overlapping but not identical substrate specificity and also participate in iron transport.¹⁶¹  In addition, each of the SMF proteins has specific functions and localization in intact worms.¹⁶⁰  Intestinal epithelial cells are the major site of manganese accumulation and contain the highest concentrations of manganese in the worm,¹⁶³  suggesting that ingestion is a likely route of manganese absorption. SMF-1 and SMF-3 are expressed in partially overlapping regions of intestinal epithelium and primarily localize to the apical plasma membrane; however, localization to intracellular vesicles has also been observed.^160,162,164  The overlapping yet distinct localization pattern for SMF-1 and SMF-3 suggests that these transporters have non-redundant roles in the uptake of intestinal manganese.¹⁶⁰  SMF-3 appears to be the primary manganese uptake transporter, with a minor role in iron uptake. Conversely, SMF-1 appears to have a limited role in manganese uptake and plays a major role in the import of iron.¹⁶⁰ 

In contrast to SMF-1 and SMF-3, SMF-2 is restricted to specialized pharyngeal cells and it exhibits a cytoplasmic localization.¹⁶⁰  In addition to functioning as a metal ion transporter, SMF-2 has been proposed to be involved in sensing environmental levels of metal ions.¹⁶⁰  This role for SMF-2 was inferred in part from the phenotypes of smf mutants and the localization of the SMF-2 protein. Worms with mutations in smf-1 or smf-3 display increased tolerance of manganese and decreased cellular manganese content following exposure to excess manganese, consistent with the role of the gene products in manganese uptake. Surprisingly, mutation of smf-2 resulted in enhanced sensitivity to manganese and increased manganese accumulation, indicating that SMF-2 may be involved in protecting against toxic manganese exposure.¹⁶⁰  SMF-2 may protect against excess manganese by enhancing excretion or sequestration of this metal; alternatively, SMF-2 may modulate manganese uptake by SMF-1 and SMF-2.¹⁶⁰  Expression of SMF-2 in the pharynx places this transporter at the entry point of manganese into the intestine, a reasonable site for sensing manganese concentrations in food. In this model, SMF-2 facilitates the uptake of manganese into pharyngeal cells, resulting in attenuation of a downstream signaling pathway that inhibits manganese uptake from SMF-1 and SMF-3. Possible mechanisms for SMF-2 mediated changes in manganese uptake by SMF-1 and SMF-2 include altering SMF-1/SMF-3 transporter activity, enhancing excretion through other transporters, or reducing pharyngeal pumping, resulting in reduced nutrient and manganese intake.¹⁶⁰ 

Transcriptional and post-translational regulation of Nramp transporters occurs in various organisms, and both modes of regulation appear to be present in C. elegans. In wild-type worms, the transcriptional response to manganese appears to be a minor component of SMF regulation. Exposure to non-toxic, micromolar levels of manganese results in a modest increase in transcription of the smf-1 and smf-2 genes. However, exposure to concentrations of manganese that result in toxicity produces a small decrease in smf-1 and smf-3 transcription. Expression of smf-2 appears to be insensitive to manganese at both low and high concentrations.¹⁶⁰ 

Among the C. elegans SMF proteins, only SMF-3 exhibits post-translational regulation in response to manganese. Both protein levels and intracellular localization of SMF-1 and SMF-2 are unchanged following exposure of worms to excess manganese. In contrast, manganese exposure results in the rapid translocation of SMF-3 to apical vesicular compartments and the eventual degradation of this protein.¹⁶⁰  SMF-3 appears to be internalized to endosomal compartments in response to manganese excess,¹⁶⁰  through a process that may be mechanistically similar to the vacuolar targeting of Smf1p and Smf2p in yeast under conditions of sufficient or excess manganese.^16,128,155  Consistent with its role as the primary manganese transporter, the post-translational regulation of SMF-3 in response to excess manganese appears to be the principle means utilized by C. elegans to limit the unwanted accumulation of this metal.

Currently, information regarding intracellular manganese transport in C. elegans is limited. A functional homologue of the yeast Pmr1p has been identified in C. elegans and was found localized to the Golgi. The C. elegans PMR-1 appears to perform a similar role to that observed for yeast Pmr1p in facilitating transport of manganese from the cytosol to the Golgi, as well as in protecting against manganese toxicity by enhancing exocytosis of manganese.^{68,140,145,165}  Intracellular manganese transporters identified in S. cerevisiae such as the Golgi efflux pump Atx2p³²  and vacuolar importer Ccc1p^142,143  have not been described in C. elegans. It is noteworthy that analysis of the C. elegans genome does not indicate the presence of an orthologue for yeast Ccc1p; however, a potential orthologue for yeast Atx2p is present.

Manganese is utilized as a cofactor for metalloenzymes and as part of non-protein antioxidant complexes in both prokaryotic and eukaryotic cells.^33,69,126  The uptake and efflux of manganese in prokaryotes and eukaryotes exhibit some overlap but are substantially distinct. Two major classes of manganese uptake transporter, the Nramp MntH and ABC-type manganese permeases MntABCD/SitABCD, have been characterized in prokaryotes.^15,17,73  Manganese efflux is mediated in most bacterial species by the cation diffusion facilitator (CDF) MntE.²⁶  Eukaryotes do not contain the ABC-type manganese permeases and rely on Nramp transporters (SMFs) for manganese acquisition under physiological conditions.^{81,134,160,162}  Intracellular distribution of manganese in eukaryotes utilizes several classes of transporter some of which, such as the Golgi transporter Pmr1p, are conserved across species.^68,140,165 

In prokaryotic cells, manganese transporters are regulated primarily at the level of transcription through the MntR transcription factor.^74,106,119  In contrast, the regulation of proteins involved in manganese uptake in eukaryotes appears to be principally post-translational, through targeted degradation in response to sufficient or excess manganese.^128,155,160  It is likely that the processes of manganese uptake, distribution, and elimination identified in bacteria, yeasts, and nematodes are conserved, at least in part, in more complicated invertebrate organisms.