A mutagenic study identifying critical residues for the structure and function of rice manganese transporter osmtp8. 1

ABSTRACT Rice (_Oryza sativa_) MTP8.1 (_Os_MTP8.1) is a tonoplast-localized manganese transporter of the cation diffusion facilitator family. Here we present a structure-function analysis of

_Os_MTP8.1 based on the site-directed and random mutagenesis and complementation assays in manganese hypersensitive yeast, in combination with three-dimensional (3D) structure modeling

based on the crystal structure of the _Escherichia coli_ CDF family member, _Ec_YiiP. Two metal-binding sites are conserved in _Os_MTP8.1 with _Ec_YiiP, one is between transmembrane helices

TM2 and TM5, the other is the cytoplasmic C-terminus. In addition to these two metal-binding sites, there may exist other Mn-binding sites such as that at the very end of the CTD. Two

residues (R167 and L296) may play an important role for the hinge-like movement of CTDs. Several mutations such as E357A and V374D may affect dimer formation and S132A may induce a

conformational change, resulting in a loss of transport function or modification in metal selectivity. The N-terminus of _Os_MTP8.1 was not functional for Mn transport activity and the real

function of NTD remains to be investigated in the future. The findings of the present study illustrate the structure-function relationship of _Os_MTP8.1 in Mn transport activity, which may

also be applied to other plant Mn-CDF proteins. SIMILAR CONTENT BEING VIEWED BY OTHERS STRUCTURAL INSIGHTS INTO ION SELECTIVITY AND TRANSPORT MECHANISMS OF _ORYZA SATIVA_ HKT2;1 AND HKT2;2/1

TRANSPORTERS Article 03 April 2024 SOS1 TONOPLAST NEO-LOCALIZATION AND THE RGG PROTEIN SALTY ARE IMPORTANT IN THE EXTREME SALINITY TOLERANCE OF _SALICORNIA BIGELOVII_ Article Open access 20

May 2024 ARCHITECTURE AND AUTOINHIBITORY MECHANISM OF THE PLASMA MEMBRANE NA+/H+ ANTIPORTER SOS1 IN _ARABIDOPSIS_ Article Open access 26 July 2023 INTRODUCTION Manganese (Mn) is an

essential micronutrient for plant growth and development. In addition to being a cofactor in many enzymatic processes involved in glucose metabolism and energy production, it also acts as a

cofactor of MnSOD in the mitochondrion and as part of the water-splitting complex in PSII in the chloroplast1,2. Despite its importance, Mn toxicity is common in acidic soils because the

amount of exchangeable Mn increases in the soil solution3. Recently, some of the genes responsible for Mn uptake and homeostasis in rice (_Oryza sativa_ L.) have been identified. Members of

the transporter gene family include natural resistance-associated macrophage protein (NRAMP)4,5,6,7, yellowstripe-like (YSL)8,9, metal tolerance protein (MTP)10,11 and vacuolar iron

transporter (VIT)12. Mn uptake in rice roots is mediated by two polarized plasma membrane transporters, _Os_Nramp5 and _Os_MTP913. _Os_Nramp5 is an influx transporter that has been localized

to the distal side of the exodermis and endodermis6. In contrast, _Os_MTP9 is an efflux transporter that has been localized to the proximal side of both cell layers11. _Os_YSL2 and _Os_YSL6

have been implicated in Mn homeostasis. _Os_YSL2 is a metal-nicotianamine transporter that is required for long-distance transport of iron and Mn8. _Os_YSL6 translocates Mn from the

apoplast to the symplast when plants are exposed to high levels of Mn9. _Os_MTP8.1 that was isolated from a rice shoot cDNA library conferred Mn tolerance in _Saccharomyces cerevisiae_ and

its expression was found to enhance tolerance and accumulation of Mn, but not other heavy metals. _Os_MTP8.1 has been localized to the tonoplast and transports cytosolic Mn2+ into the

vacuole. Disruption of _Os_MTP8.1 results in decreased chlorophyll content in the youngest leaf blades. Rice mtp8.1 Tos-17 insertion mutants and transgenic _Os_MTP8.1 RNAi knockdown lines

are hypersensitive to Mn and accumulate less Mn in shoots and roots than wild-type plants10. However, its structure-function relationship remains elusive to date. MTPs belong to the cation

diffusion facilitator (CDF) transporter family and are important for the maintenance of cation homeostasis in bacteria, yeast, plants and mammals14,15,16. MTPs play a critical role in

removing transition metals from the cytosol either by intracellular sequestration or by cellular export17. Phylogenetic analysis has indicated that the CDF family members can be divided into

three major groups based on metal ion specificity: Zn-CDF, Fe/Zn-CDF and Mn-CDF18. To date, _Ec_YiiP is the only full-length CDF member that has been crystallized. It was initially

characterized at a 3.8 Å resolution19 and subsequently improved to 2.9 Å20. _Ec_YiiP is a Y-shaped homodimer and each protomer contains three zinc-binding sites. Site A is located at the

center of TM2 and TM5 and acts as an active site for zinc transport. This active site is tetrahedrally coordinated by D45 and D49 of TM2 and H153 and D157 of TM5. Site B is located in the

intracellular loop that connects TM2 and TM3. Site C hosts four Zn2+ ions that mediate an interaction between CTDs and stabilize dimers. The six helices of the transmembrane domain (TMD) are

grouped into two bundles with four (TM1–TM2–TM4–TM5) and two (TM3–TM6) helices. When Zn2+ binds to site C, the C-terminus moves the TM3-TM6 pair, causing reorientation of TM5, which

allosterically changes the geometry of the active Zn2+-binding site (site A) and thereby accomplishes transmembrane transport of Zn2+. By using site-directed mutagenesis, Kawachi _et al_.

studied the structural basis of Zn2+ selectivity and transport activity in _At_MTP121. They found that two Zn-binding sites (sites A and C) are conserved in _At_MTP1 with _Ec_YiiP. They also

found that the N-terminus of _At_MTP1 is very important for metal selectivity. They suggested that the N-terminal domain, the His-rich loop and the leucine zipper motif in TM6 may

contribute to the high Zn2+ selectivity in _At_MTP1. The His-rich loop of _At_MTP1 acts as a sensor of cytosolic Zn to maintain an essential level of Zn in the cytosol22. The sequence

characteristics of _Os_MTP8.1 differs from that of _At_MTP1; for example, _Os_MTP8.1 does not have the His-rich loop between TM4 and TM5 and no leucine zipper motif exists within TM6. The

sequence of plant Mn-CDF members are highly similar and differ from _Ec_YiiP and _At_MTP1 only in terms of structure-function relationships. In the present study, we are used site-directed

and random mutagenesis to investigate the structural basis of Mn transport activity in a plant Mn-CDF member, _Os_MTP8.1. RESULTS SUBCELLULAR LOCALIZATION AND METAL SELECTIVITY OF _OS_MTP8.1

The full-length cDNA fragment of _Os_MTP8.1 (Os03g0226400) was RT-PCR amplified using sequence information derived from the Rice Annotation Project of NCBI (http://www.ncbi.nlm.nih.gov).

_Os_MTP8.1 encodes a putative protein of 396 amino acids in length. Phylogenetic analysis of _Os_MTP8.1 indicated that it belongs to the Mn clade of the CDF family10. Transport activity of

_Os_MTP8.1 was assessed by complementation assay using a number of yeast mutants that were deficient in various metal transporters. _Os_MTP8.1 was expressed in _S. cerevisiae_ yeast mutants

lacking the endogenous metal transporters for Zn (_zrc1_), Co (_cot1_), Cu (_cup2_), Cd (_ycf1_), Ni (_smf1_) and Mn (_pmr1_). The results showed that a pmr1 strain but not other mutants

were restored after transformation with _Os_MTP8.1 (Fig. 1A), thereby suggesting that _Os_MTP8.1 may be a Mn-specific transporter. _Os_MTP8.1 was localized to the vacuolar membrane when it

is expressed heterologously in yeast (Fig. 1B). To further confirm the localization of _Os_MTP8.1 in rice, the cDNA of _Os_MTP8.1 was fused in frame to GFP in the expression vector pXZP008.

Transient expression of _Os_MTP8.1:GFP in rice mesophyll protoplasts showed that green fluorescence was localized to the tonoplast (Fig. 1C). The tonoplast localization of _Os_MTP8.1 was

consistent with the results of a previous study involving onion epidermal cells10. SITE-DIRECTED AND RANDOM MUTANT _OS_MTP8.1 EXPRESSION IN YEAST FROM TM1 TO TM6 We aligned _Os_MTP8.1 and

some other reported Mn-CDF, which included _At_MTP8, _At_MTP11, _Cs_MTP8, _Hv_MTP8.1, _Pt_MTP11.1, _Pt_MTP11.2 and _Os_MTP9 with _Ec_YiiP (Fig. 2). Amino acids that were conserved within

Mn-CDF were selected for replacement with aliphatic amino acid Ala. Random mutagenesis was performed on pFL61-OsMTP8.1 by using GeneMorph II random mutagenesis kits (Stratagene) according to

the manufacturer’s instructions. The mutation products were transformed into _E. coli_, more than 200 plasmids were extracted. Each plasmid was transformed into the Mn-sensitive mutant

_pmr1_, cells were grown for three days on SD-Ura plates containing 6 mM MnCl2. The corresponding plasmids from transformed yeast cells which unable to grow on high manganese plates were

selected for further study (Fig. S1). Primers for the mutations from random mutagenesis are shown in Table S1. Yeast strain _pmr1_ transformed with an empty vector pFL61 was unable to grow

on medium supplemented with 3 mM or higher Mn concentrations, but yeast cells expressing pFL61-_Os_MTP8.1 could grow on medium supplemented with up to 8 mM Mn. Plate growth assays indicated

that the mutants have different sensitivities to Mn (Fig. 3). A total of 63 single-substitution mutations conferred nearly the same level of Mn tolerance as that of wild-type _Os_MTP8.1

(Fig. S2). A reduced level of Mn tolerance was conferred by 23 mutations, which included N107A, N110A, L113A, S126A, I127A, L140A, I145A, S155A, I156A, V158A, K160A, I163A, K165A, V171A,

G184A, V189A, L226A, W227A, N235A, V238A, L277A, A293T and S295P. These mutants were able to grow in the presence of 3 mM Mn, but not as well as that of the wild type in the presence of 8 mM

Mn. These findings suggest that these amino acids contribute to the activity of _Os_MTP8.1. The other 20 residues possibly play a critical role in the activity of _Os_MTP8.1, as

substitution of these amino acids resulted in yeast cells growing only in the presence of 3 mM Mn but not with 8 mM Mn. These residues included S106A, I122A, S136A, G143A, F148A, G164A,

G172A, I173A, F185A, I237A, Y241A, H245A, N252A, G255A, D270A, G273A, A278T, Y280A, N284K and G287A. Finally, 18 mutations that included K117A, T133I, D135A, D139A, L146A, Y161A, P162A,

R167A, A177R, M180A, M216A, K223A, D244A, F247A, D248A, V260A, W285A and L296A resulted in a complete loss of ability to confer Mn tolerance, which indicated that these amino acids are

critical for Mn transport activity of OsMTP8.1. THE FUNCTION OF N- AND C-TERMINAL DOMAINS OF OSMTP8.1 The _Os_MTP8.1 protein has six transmembrane (TM) domains, an N-terminal domain and a

C-terminal domain. To study the function of the N- and C-terminal domains of _Os_MTP8.1, we construct several deletion mutants. For the N-terminal domain, Mn tolerance conferred by these

deletion mutants was equivalent to that conferred by wild-type _Os_MTP8.1 (Fig. 4A). These results indicate that the N-terminal domain does not contribute to Mn transport activity in

_Os_MTP8.1. For the C-terminal domain, we constructed 7 deletion mutants and only the △392–396 mutant conferred Mn tolerance in the presence of 3 mM and 8 mM Mn, whereas other mutants did

not confer any Mn tolerance even at a concentration of 3 mM Mn (Fig. 4B). To further determine which residues are required for Mn transport of _Os_MTP8.1, several substitute mutants were

constructed. Mutations E304A, T325A, R327A, F336S, L359A and F373L conferred reduced levels of Mn tolerance. The ability to confer Mn tolerance was significantly reduced in the H316A, D324A,

L349A, E357A, Q360A and E379A mutants. Approximately 15 mutants showed a complete loss in their ability to confer Mn tolerance; these mutants included T310A, G332A, V337D, E338A, D340N,

H353A, G356R, E370A, R371A, V374D, H375Q, D377A, H382Y, E385A and H386A (Fig. 4B). Mutations conferred nearly the same level of Mn tolerance as wild-type were shown in Fig. S2. ZINC

TOLERANCE ASSAYS The _Os_MTP8.1 mutants were also tested for Zn tolerance in the Zn-hypersensitive yeast strain _zrc1_. The _ZRC1_ gene encodes a multicopy suppressor of Zn toxicity that has

been localized to the vacuolar membrane of _S. cerevisiae_23. Two mutants, F75A and S132A, conferred tolerance to high levels of Zn (18 mM) but not other metals including Cd, Ni, Cu, Co or

Fe (Figs 5A and S3). F75 was localized to the NTD and S132 to EL1. Furthermore, to investigate the metal transport activity of these two mutants, the accumulation of Zn and other metal

cations was compared in _zrc1_ that expressed OsMTP8.1 or the mutants. The results showed that two mutants accumulated 1.6-fold the amount of Zn of the wild type OsMTP8.1 (Fig. 5B).

Significant differences were not detected in the concentrations of other metals (data not shown). Cellular localization of the mutants within yeast cells showed a vacuolar localization

similar to the wild-type proteins (Fig. S4), confirming that the mutant proteins are expressed and functional. A summary of effects of all mutations in _Os_MTP8.1 as determined by yeast

complementation assays are presented in Fig. 6. 3D STRUCTURAL MODELING OF _OS_MTP8.1 To determine the function of mutated residues involved in the determination of metal transport activity,

we constructed a 3D model of _Os_MTP8.1 by using the SWISSMODEL software (Fig. S5). The final models turned out to be strongly biased toward _E. coli_ YiiP (3H90). The N-terminal domain of

_Os_MTP8.1 was much longer than _Ec_YiiP (Fig. 2) and was not reflected in the 3D model. Biochemical studies and X-ray structural analysis of _Ec_YiiP indicated that it has three

Zn2+-binding sites: site A is a tetrahedral Zn2+-binding site situated in the transmembrane domain and is composed of two residues from TM2 (Asp45 and Asp49) and two residues from TM5

(His153 and Asp157), two cytoplasmic binding sites, sites B and C. Site B is localized to the intracellular loop that connects TM2 and TM3 (IL1), in which Zn2+ is coordinated with one water

molecule and three residues Asp68, His71 and His75 and site C in the C-terminal domain that hosts two Zn2+ ions bound in a binuclear cluster by Asp285, His232, His248, His283 and His261 from

the neighboring subunit of _Ec_YiiP. This site hosts four Zn2+ ions that mediate a tight interaction between CTDs and play an important role in stabilizing the dimer19,20. According to the

3D model, the active Mn-binding site (site A) of _Os_MTP8.1 consists of D135 and D139 from TM2 and D244 and D248 from TM5 (Fig. 7A). The sequence DxxxD (x = any amino acid) in TM2 and TM5

are conserved in Mn-CDF. Mutations in any of these four residues appeared to abolish Mn transport activity of _Os_MTP8.1. These results suggested functional conservation of site A between

_Ec_YiiP and _Os_MTP8.1. Sequence alignment indicated no conserved regions between _Ec_YiiP and _Os_MTP8.1 in site B (Figs 2 and 7B). However, mutations Y161A and P162A in the intracellular

loop between TM2 and TM3 appeared to abolish Mn transport function and mutation G164A apparently strongly reduced the transport function of OsMTP8.1. These three residues are conserved among

Mn-CDFs. These results indicated that IL1 is essential for _Os_MTP8.1 function, but may not likely form a metal-binding site as _Ec_YiiP. A number of highly conserved residues were detected

at site C of _Os_MTP8.1, including D324, D340, H353, H375 and D377, thereby implicating its role in metal binding (Fig. 7C). Mutations D340A, H353A, H375A and D377A showed a complete loss

in the ability to confer Mn tolerance and D324A presented a significant reduction in function. In addition to the conserved residues, 9 mutations also abolished Mn tolerance, namely, T310A,

G332A, V337A, E338A, G356A, E370A, H382A, E385A and H386A. Sequence alignment indicated that these residues are highly conserved in Mn-CDF but less conserved with _Ec_YiiP, indicating that

the structure of CTD for Mn binding in Mn-CDF may be different from that of _Ec_YiiP. Previous biochemical and X-ray analyses have indicated that the _Ec_YiiP homodimer structure is

stabilized by an interlocked salt bridge near the membrane surface that is formed by K77 and D20720. The corresponding residues for these two residues in _Os_MTP8.1 were R167 and G298, these

two residues apparently did not form a salt bridge and G298A conferred nearly the same level of Mn tolerance as that of wild-type _Os_MTP8.1. Interestingly, we found that R167 and L296 were

critical for Mn transport activity of _Os_MTP8.1. These two residues were located near the membrane surface (Fig. 8B); we speculate that they play an important role in hinge movement of

_Os_MTP8.1. DISCUSSION According to the phylogenetic study conducted by Montanini _et al_., Mn-CDF proteins can be differentiated from other CDF members by the consensus sequence DXXXD in

TM2 and TM518. The active Mn-binding site (site A) of _Os_MTP8.1 is formed by four Asp residues, of which D135 and D139 are located in TM2 and D244 and D248 in TM5 (Fig. 7A). These key

residues of the Mn-binding site are highly conserved in other Mn-CDF family proteins (Fig. 2). In contrast to _Ec_YiiP, _Os_MTP8.1 does not contain the Mn-binding site B in the cytosolic

loop (IL1) between TM2 and TM3. This site is also not conserved within other CDF family members such as _Mm_CDF3 from _Maricaulis maris_ MCS1024 and the plant zinc transporter _At_MTP121.

IL1 is the interface for dimerization of _Ec_YiiP protomers19. In _Os_MTP8.1, IL1 contains critical residues (Y161, P162 and G164) for transport function (Fig. 7B); these residues may be

involved in dimerization of _Os_MTP8.1. These results also suggest that there is an alternative access mechanism for Mn to reach the active transport site from CTD of _Os_MTP8.1 via the

intracellular cavity. The cytoplasmic binding site (site C) of _Os_MTP8.1 has been localized to the CTD-CTD interface and is composed of several highly conserved residues (D324, D340, H353,

H375 and D377) (Fig. 7C). _Ec_YiiP showed extensive outer-shell constraints around the binuclear zinc coordination in site C, which were composed of D233, E250, D265, I282 and Q284. These

residues apparently establish an extensive network of outer-shell interactions at the CTD interface, thereby stabilizing the CTD-CTD association20. In _Os_MTP8.1, these residues correspond

to T325, E342, E357, V374 and L376 (Fig. 7C). Yeast complementation assays support the essential role of T325, E357 and V374 and E357 and V374 may contribute to the function of stabilizing

the CTD-CTD association in _Os_MTP8.1 (Fig. 7C). The sequence HKPEH from residue 382 to 386 in _Os_MTP8.1 is highly conserved among Mn-CDF and distinguishable from _Ec_YiiP (Fig. 2) and

mutations H382A, E385A and H386A abolished Mn transport activity. We therefore propose that there may be another Mn-binding site at the end of the CTD of _Os_MTP8.1. The 3D model of

_Os_MTP8.1 ended at 380 C and lost the structural information of this site and the binding information remains to be elucidated in future research investigations. Recently, the 3D structures

of the CTDs of several CDF proteins were resolved, e.g., the CzrB of _T. thermophiles_25, the apo form of a CDF protein of _T. maritime_26 and MamM of _M. gryphiswaldense_27. These findings

demonstrate that CTDs adopt a metallochaperone-like fold and the metallochaperones can carry metal ions to various protein targets. It has been suggested that the CTD plays a role in

sensing the Mn ions and delivering these to the TMD region of _Os_MTP8.1. The six transmembrane helices of the TMD in _Ec_YiiP consist of two independent bundles; TM1, TM2, TM4 and TM5 form

a four-helix bundle, whereas TM3 and TM6 compose another bundle. The orientation of the TM3–TM6 bundle is stabilized by four interlocking salt bridges formed by K77 of TM3 and D207, which

are located in IL3. The charge interlock also plays an important role in stabilizing dimer associations and is essential for Zn transport20. When Zn binds to site C, it triggers a hinge-like

movement of two CTDs, inter-CTD movements alter the TM3-TM6 bundle orientation and causes reorientation of TM5. The orientation of TM5 with respect to TM2 affects the coordination geometry

of the active Zn-binding site, thereby facilitating Zn transport20. In _Os_MTP8.1, two residues (R167 from TM3 and L296 from TM6) near the membrane surface play critical roles in Mn

transport (Fig. 8B). Our results suggest these two residues may play an important role in the hinge-like movement of CTDs when these bind Mn. Mutation M180A in TM3 and W285A in TM6 abolished

Mn transport activity of _Os_MTP8.1 (Fig. 8B). These two hydrophobic residues are localized face-to-face between TM3 and TM6. These mutations may weaken the hydrophobic interactions between

TM3 and TM6, thereby resulting in a different orientation for the TM3- TM6 bundle. In _At_MTP1, Ala-substituted S101 or N258 conferred increased Zn2+ tolerance21. From the cytosolic side

view of the 3D model, these two residues obstruct the entrance to the pore of the active Zn2+-binding site between TM2 and TM5. Asn258 is directly adjacent to the His-rich loop and may

function as a gate that is controlled by the His-rich loop to control Zn flux. In _Os_MTP8.1, two residues, L146 and I237, were conserved to S101 and N258 of _At_MTP1 (Fig. 8A) and mutant

L146A completely lost its ability to transport Mn and I237A conferred reduced levels of Mn tolerance (Fig. 3). There is a long His-rich loop between TM4 and TM5 of _At_MTP1, but the loop

between TM4 and TM5 of _Os_MTP8.1 was very short (Fig. 2). Residues L146 and I237 of _Os_MTP8.1 may have different mechanisms for metal transport due to S101 and N258 of _At_MTP1. We propose

that mutations L146A and I237A affect the coordination geometry between TM2 and TM5 and abolish Mn transport activity in _Os_MTP8.1. The N-terminal deletions showed no effect of Mn

transport activity compared to that in wild-type _Os_MTP8.1 (Fig. 4A). In _At_MTP1 and _Ptd_MTP1, two Cys residues are required for Zn transport activity; these two residues are well

conserved among plant Zn-CDF proteins21,28. The N-terminal deletion mutant, which lacks the Cys residues, also did not confer Zn tolerance. Compared to the Zn-CDF proteins, the function of

the N-terminal domain of _Os_MTP8.1 requires further investigation. Previous studies have shown that several sites influence metal specificity in CDF family proteins. In _At_MTP1, the

His-rich loop of IL2 and sequences within NTD are responsible for metal selectivity and several residues within transmembrane domain were also found to influence metal selectivity21. In

_Os_MTP1, residue L82, which is located in EL1, may also play a role in substrate specificity29. This also found in other CDF family proteins such as _Sc_ZRC130. Two mutants, F75A located in

the NTD and S132A located in EL1, also conferred tolerance to Zn besides Mn. In conclusion, by using site-directed and random mutagenesis and complementation assays in yeast and in

combination with 3D structure modeling based on the _E. coli_ Zn transporter YiiP, we have identified residues that are essential for Mn transport in the plant Mn-CDF member, _Os_MTP8.1. We

also presented a structure-function relationship involving _Os_MTP8.1 for Mn transport activity that may also be applied to other plant Mn-CDF proteins. Two metal binding sites are conserved

in _Os_MTP8.1 with _Ec_YiiP, one is the active Mn-binding site between TM2 and TM5 and the other is the CTD-binding site. In addition, there may also be other Mn binding sites such as the

site at the very end of CTD. Several mutations may affect dimer formation or cause a conformational change in the protein, thereby resulting in a loss of transport function or modification

in metal selectivity. The N-terminus of _Os_MTP8.1 is not functional for Mn transport activity and the real function of NTD remains to be investigated in future studies. METHODS

SITE-DIRECTED AND RANDOM MUTAGENESIS OF _OS_MTP8.1 _Os_MTP8.1 was inserted into the _Not_I sites of pFL6131 to obtain the expression plasmid. Site-directed mutagenesis and random mutagenesis

were performed on the pFL61-_Os_MTP8.1 vector using QuikChange II XL site-directed mutagenesis and GeneMorph II random mutagenesis kits (Stratagene) according to the manufacturer’s

instructions32. Sequences encoding N- or C-terminally truncated mutants of OsMTP8.1 were amplified by PCR and the DNA fragments obtained were inserted into the _Not_I site of the pFL61

vector. The primers used and the mutations induced are listed in Table S2. All constructs generated in the present study were verified by DNA sequencing. HETEROLOGOUS EXPRESSION OF MUTATED

OSMTP8.1 IN YEAST The mutants and the wild-type yeast strains used in this research were obtained from the Euroscarf collection33. Plasmids were transformed into the Mn-sensitive mutant

Y04534 (_pmr1_) or Zn-sensitive mutant Y00829 (_zrc1_) and its parental strain BY4741 for Mn and Zn complementation analyses. Yeast transformation was performed by using the lithium

acetate/PEG transformation method34. Positive colonies were selected on synthetic dropout (SD) plates containing the appropriate selective markers. Yeast strains expressing empty vector,

wild-type _Os_MTP8.1, or mutated OsMTP8.1 variants were pre-cultured in SD-Ura liquid medium at 30 °C for 16 h. Pre-cultured cells were diluted to an OD600 nm of 1.0 and 10-μL aliquots were

spotted onto SD-Ura plates containing various concentrations of metals as indicated elsewhere35. Plates were incubated at 30 °C for 3 days. SUBCELLULAR LOCALIZATION IN RICE PROTOPLAST AND

YEAST For the subcellular localization in rice protoplasts, the _Os_MTP8.1 cDNA fragment without a stop codon was amplified and was cloned into the _BamH_I and _Kpn_I sites of pXZP00836. The

construct was transformed into rice mesophyll protoplasts and GFP fluorescence was observed using a confocal laser scanning microscopy (Confocal System-UitraView VOX, Perkin Elmer). For the

subcellular localization in yeast, the ORFs were amplified by using primers containing the _Mlu_I site and cloned into the yeast expression vector pFL61-GFP. The construct was transformed

into _pmr1_ and GFP signal was observed by confocal laser scanning microscopy. BIOINFORMATICS ANALYSIS OF OSMTP8.1 Multiple sequence alignments of TtCzrB (GenBank Acc. No. AJ307316.1) from

Thermus thermophilus, _Arabidopsis thaliana_ AtMTP1 (GenBank Acc. No. At2g46800), _At_MTP8 (GenBank Accession Number At3g58060) and _At_MTP11 (GenBank Acc. No. At2g39450), _Cucumis sativus

Cs_MTP8 (GenBank Acc. No. AFJ24703), _Hordeum vulgare Hv_MTP8.1 (GenBank Acc. No. AFP33387), _Pt_MTP11.1 (Phytozome: POPTR_0010s21810) and _Pt_MTP11.2 (Phytozome: POPTR_0008s04940) from

_Populus trichocarpa_, _Os_MTP9 (Phytozome: LOC_Os01g03914) and _Os_MTP8.1 (Phytozome: LOC_Os03g12530) from _Oryza sativa,_ and _E. coli_ K12 YiiP (P69380) were performed using multalin37.

The 3D model of OsMTP8.1 was generated by homology modeling using the SWISSMODEL software (http://swissmodel.expasy.org/)38,39,40,41 based on the structure of _Ec_YiiP (PDB ID 3H90). Images

were generated by using PyMOL 1.6.x. ADDITIONAL INFORMATION HOW TO CITE THIS ARTICLE: Chen, X. _et al_. A mutagenic study identifying critical residues for the structure and function of rice

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Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (31171326 to W.Z., 31301839 to X.C.), the

Natural Science Foundation of Jiangsu Province of China (BK20130672 to X.C.) and a project funded by the Fundamental Research Funds for the Central Universities-Nanjing Agricultural

University Youth Science and Technology Innovation Fund (KJ2013030 to X.C.). AUTHOR INFORMATION Author notes * Chen Xi and Li Jiyu contributed equally to this work. AUTHORS AND AFFILIATIONS

* Department of Biochemistry & Molecular Biology, College of Life Sciences, Nanjing Agricultural University, Nanjing, 210095, Jiangsu, China Xi Chen, Jiyu Li, Lihua Wang, Gang Ma & 

Wei Zhang Authors * Xi Chen View author publications You can also search for this author inPubMed Google Scholar * Jiyu Li View author publications You can also search for this author

inPubMed Google Scholar * Lihua Wang View author publications You can also search for this author inPubMed Google Scholar * Gang Ma View author publications You can also search for this

author inPubMed Google Scholar * Wei Zhang View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS X.C., J.L. and W.Z. planned the experiments,

X.C., J.L., L.W. and G.M. performed the experiments and X.C., J.L. and W.Z. analyzed the data and wrote the paper. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing

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http://creativecommons.org/licenses/by/4.0/ Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Chen, X., Li, J., Wang, L. _et al._ A mutagenic study identifying critical residues

for the structure and function of rice manganese transporter OsMTP8.1. _Sci Rep_ 6, 32073 (2016). https://doi.org/10.1038/srep32073 Download citation * Received: 13 May 2016 * Accepted: 02

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