Key Residues and Phosphate Release Routes in the Saccharomyces cerevisiae Pho84 Transceptor

Pho84, a major facilitator superfamily (MFS) protein, is the main high-affinity Pi transceptor in Saccharomyces cerevisiae. Although transport mechanisms have been suggested for other MFS members, the key residues and molecular events driving transport by Pi:H+ symporters are unclear. The current Pho84 transport model is based on the inward-facing occluded crystal structure of the Pho84 homologue PiPT in the fungus Piriformospora indica. However, this model is limited by the lack of experimental data on the regulatory residues for each stage of the transport cycle. In this study, an open, inward-facing conformation of Pho84 was used to study the release of Pi. A comparison of this conformation with the model for Pi release in PiPT revealed that Tyr179 in Pho84 (Tyr150 in PiPT) is not part of the Pi binding site. This difference may be due to a lack of detailed information on the Pi release step in PiPT. Molecular dynamics simulations of Pho84 in which a residue adjacent to Tyr179, Asp178, is protonated revealed a conformational change in Pho84 from an open, inward-facing state to an occluded state. Tyr179 then became part of the binding site as was observed in the PiPT crystal structure. The importance of Tyr179 in regulating Pi release was supported by site-directed mutagenesis and transport assays. Using trehalase activity measurements, we demonstrated that the release of Pi is a critical step for transceptor signaling. Our results add to previous studies on PiPT, creating a more complete picture of the proton-coupled Pi transport cycle of a transceptor.

The major facilitator superfamily (MFS) 4 is one of the largest families of secondary active transporters (1). Currently, 64 crys-tal structures from 10 different families are available, and several of these structures were resolved in substrate-free and substrate-bound conformational states, therefore providing significant insights into the molecular mechanisms involved in nutrient transport (2). The key feature of MFS transporters is that transport across the membrane occurs via an alternatingaccess mechanism, the "rocker-switch" model (3), which involves a variety of distinct conformations including outwardopen, substrate-bound occluded, and inward-open states. A limitation of the rocker-switch model, however, is that because it is based solely on rigid body rotation, it does not explain the presence of the occluded state. To account for this limitation, the "clamp-and-switch" model has recently been proposed (2). This model suggests that the conformational transition process is more dynamic than previously assumed and that the switch between conformations is driven by pore-lining helices that bend to form the occluded state and that allow for outwardfacing to inward-facing rotations and vice versa.
Inorganic phosphate (P i ) is required for numerous cellular functions, such as synthesis of DNA and membrane lipids, intracellular signaling, and the generation of high-energy phosphate esters in ATP. Because of the central role of P i , cells have adopted strategies to ensure rapid cellular responses to internal and external fluctuations in P i levels that may disturb cellular phosphate homeostasis. Moreover, several P i transporters have been shown to transport P i by H ϩ -coupled symport (4).
Currently, the only available crystal structure of a P i :H ϩ transporter is the eukaryotic Piriformospora indica transporter PiPT, which was resolved in the substrate-bound inward-facing occluded conformation (5). The current model for P i :H ϩ -transport is based on this crystal structure and describes three major conformational variations (i.e. substrate-docking, binding, and release) (5). A weakness of this model is that it lacks experimental data on the regulatory residues in each stage of the transport cycle.
The Saccharomyces cerevisiae Pho84 high-affinity phosphate transporter is a member of the phosphate:H ϩ symporter family (2.A. 1.9) and is the main transporter during P i -limiting condi-tions. Furthermore, Pho84 activates the protein kinase A (PKA) pathway, which senses and signals the uptake of external P i in phosphate-starved cells. This dual functionality of Pho84 has led to its classification as a transceptor (6). A three-dimensional model of Pho84 in the open inward-facing conformation has been created by homology modeling using the glycerol 3-phosphate transporter GlpT as a template (7). This model has been used to verify the roles of key residues in P i binding, transport, and signaling (8). A comparison of the Pho84 and PiPT structures revealed a series of conserved residues in the P i binding site (5). Interestingly, in contrast to Tyr 150 in PiPT, Tyr 179 in Pho84 is not in the binding site of the open inward-facing conformation. This difference may result from protonation/deprotonation of the nearby Asp 178 residue (Asp 149 in PiPT), which may affect the active conformation of the transporter. In addition, Asp 178 has been shown to participate in H ϩ -transfer (8). Nevertheless, the protonation/deprotonation mechanism of regulating conformational changes in P i :H ϩ transporters remains putative.
In this study, we examined the role of Tyr 179 on the P i release step in the open inward-facing conformation of Pho84. To address the finer details of the release mechanism and to build on the transport model previously suggested for PiPT, we performed a series of unrestrained molecular dynamics (MD) and steered molecular dynamics (SMD) simulations using different protonation states of P i and Asp 178 .
These simulations revealed that the protonation state of Asp 178 alters the conformational state of Pho84. Upon protonation of Asp 178 , Tyr 179 underwent a rotameric change to become part of the binding site. This agrees with the contact found between P i and Tyr 150 in the PiPT occluded inward-facing conformation. Based on SMD simulations, different P i release routes were suggested. The lowest-energy release route was found with H 2 PO 4 Ϫ and deprotonated Asp 178 . We also confirmed the importance of Tyr 179 in regulating P i transport by a series of site-directed mutagenesis studies and biochemical assays. Finally, we measured trehalase activity to determine whether Tyr 179 regulated PKA signaling, and we demonstrated that the release of P i is critical for signaling. Altogether, our data contributes to a more complete picture of the dual functions of phosphate transceptors.

Results and Discussion
Tyr 179 Is Crucial for the Substrate Release Step of P i Transport-The two-dimensional topology of Pho84 consists of a C-domain and an N-domain, each domain is made up of six transmembrane segments (Table 1), and both the N and C termini are oriented toward the cytoplasm. The three-dimensional in silico model displays a Mayan temple shape (3) (Fig.  1A) with a distinct N-and C-terminal domain organization and a clearly visible transport channel in the center of the protein (Fig. 1B). The transport channel is a structural feature common in MFS proteins.
A multiple sequence alignment (MSA) analysis of Pho84 with other P i :H ϩ transporters revealed that Tyr 179 is highly conserved (Fig. 2). Furthermore, the corresponding Tyr 150 is proposed to be in the P i binding site of the PiPT crystal structure. In the inward-open conformation of the Pho84 model, Tyr 179 orients toward the cytoplasm and points away from the binding cavity. A growth spot test with strains expressing mutant alleles

loops (L), and the N and C termini according to Lagerstedt and co-workers (7)
Amino acid sequence (the position is given in superscript)
Our data for Tyr 179 clearly shows that this residue plays a crucial role in P i transport. By introducing a side chain that is relatively inert, such as a residue that is non-charged, weakly polar, or lacking an aromatic residue, transport activity was largely abolished. In contrast, by introducing side chains with   increased polarity (Tyr 179 -Ser) or hydrophobicity (Tyr 179 -Phe), transport activity was restored to the wild-type level.
Protonation/Deprotonation of Asp 178 and Its Impact on P i Release-A protonation/deprotonation cycle has been shown to be crucial for the functionality of many transporters. The Escherichia coli lactose permease LacY serves as a paradigm for understanding the importance of protonation/deprotonation in the transport cycle that begins in the outward-open conformation and requires functional protonation to facilitate substrate binding (10). MD simulations of the protonation/deprotonation of Glu 325 showed significant changes in the struc-ture leading to a transition from the inward-facing to the occluded conformation (11). MD simulations of the E. coli sugar fucose:H ϩ symporter FucP also showed that protonation of Glu 135 is required to trigger conversion from the outwardopen to the inward-open conformation (12). These examples show that protonation/deprotonation events are crucial to the alternating-access dynamics of MFS transporters.
In a previous site-directed mutagenesis study, the Pho84 residue Asp 178 was mutated to Glu and Asn, and Asn was found to mimic a protonated aspartate (8). These results led to the hypothesis that protonation of Asp 178 plays a role in a late phosphate release step of the transport cycle. To investigate this hypothesis, a series of unrestrained MD simulations were performed on Pho84 models with protonated or deprotonated Asp 178 . P i was inserted in the Pho84 binding site at approximately equal distances from Lys 492 and Asp 178 . This allowed us to investigate the roles of these residues on P i binding and on proton transfer. In addition, simulations were conducted using different protonation states of P i (H 3 PO 4 , H 2 PO 4 Ϫ , and HPO 4 2Ϫ ) to resolve the role of charge balance in the binding site and its influence on the energetics of cytosolic release of P i (see supplemental Table S1 for information on the number of molecules in each system and the denotations that are used throughout this paper). All of the systems were subsequently subjected to 30 ns of equilibration to reach stable energy, temperature, and density values (supplemental Figs. S1-S16). Equilibration of the transmembrane helices ( Fig. 1) in the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer were verified by recording root mean square deviation values over time for FIGURE 4. A, total transport activity was measured using radioactive phosphate in a short-term uptake assay with a saturating phosphate concentration (110 M KH 2 PO 4 ). Cells were grown in LP i conditions. Experiments were performed in triplicate. Data are expressed as percentages of WT activity (14.658 nmol min Ϫ1 mg Ϫ1 cells, dry weight set as 100%), mean Ϯ S.D. **, p Ͻ 0.05, significantly different (Student's t test). B, immunoblot with anti-c-Myc antibody to detect membrane enrichment. CEN.PK 113 7D strain was used as the (ϩ) control, the CEN.PK 5D PHO84⌬ was used as the (Ϫ) control. The band for the recombinant Pho84 -6xHis-2Myc (apparent molecular weight of 66 kDa) is indicated with a square bracket. C, loading controls using complete yeast extract (10 g) and anti-␤-actin. The band for ␤-actin is indicated with a square bracket. Extracted snapshots of the Pho84 binding site after 30 ns of equilibration revealed the position of P i in the binding site, the location of residues important for P i binding, and the location of proton transfer (Fig. 6). Analysis of the position of H 3 PO 4 and its variations in distance over time (Fig. 6, A1,  Ϫ or HPO 4 2Ϫ revealed weakened interactions with Asp 178 due to repulsion between monoand divalent species of P i and the deprotonated state of Asp 178 (Fig. 6, B1 and C1, and supplemental Fig. S18). Moreover, systems with either H 2 PO 4 Ϫ or HPO 4 2Ϫ in the binding site and a protonated state of Asp 178 (Fig. 6, B2 and C2, supplemental Figs. S18 and S19) revealed the formation of a hydrogen bond between Asp 178 and the adjacent Asp 76 residue resulting in less distance between helix I that harbors Asp 76 and helix IV that harbors Asp 178 . Similar interactions were seen in the absence of P i indicating that the proton is not donated by P i (supplemental Fig. S19). These data confirm the role of Asp 178 in proton-coupling (8). In addition, it has been suggested that the symport of protons through Pho84 occurs at a P i :H ϩ stoichiometry of 1:3 (13).
The largest shift in the distance between Asp 178 and Asp 76 upon protonation of Asp 178 was observed for H 2 PO 4 Ϫ rather than for HPO 4 2Ϫ suggesting that H 2 PO 4 Ϫ has the largest impact on the conformational flexibility of Pho84. Simulations with H 3 PO 4 in the binding site and protonated Asp 178 resulted in a helical rotation that affected helices I and IV such that Asp 76 , rather than Asp 178 , bound to P i (Fig. 6, A2, and supplemental  Fig. S18). These data demonstrate that the charge distribution in the Pho84 binding site regulates the active conformational state of Pho84.
In addition to the observed roles of Asp 178 in regulating P i binding and proton-coupling, the change in the conformational state of helix IV in Pho84 that occurred upon protonation of Asp 178 led to Tyr 179 becoming more accessible for P i binding (Fig. 6, B2 and C2, and supplemental Fig. S20). X-ray crystallographic analysis of PiPT in the substrate-bound inward-facing occluded conformation shows that the corresponding tyrosine (Tyr 150 ) is involved in P i binding (5). As shown in a comparison of Fig. 6, panels A1/A2 versus panels B1/B2/C1/C2, the shift in the position of Tyr 179 as correlated to the protonation state of Asp 178 was not as prominent for models that incorporated H 3 PO 4 .
We investigated the stability of salt bridges involving Asp 76 and Asp 79 and their link to Pho84 conformational changes (Fig.  6). We found stable contacts between Asp 76 -Arg 267 and Asp 79 -Arg 168 in all of the simulations with the exception of the model incorporating H 3 PO 4 and protonated Asp 178 in which Asp 178 and Asp 76 exchanged positions (Fig. 6, A2). Moreover, a Pho84 simulation without P i in the binding site showed a disruption of the Asp 79 -Arg 168 salt bridge after 15 ns of equilibration. Because protonation of Asp 178 led to a stable Asp 79 -Arg 168 salt bridge, we suggest that the presence of P i in the binding site may increase the conformational stability of Pho84 (data not shown).
Characterizing Alternative Release Routes Using SMD Simulations-A series of SMD simulations were performed to investigate alternative P i release routes in Pho84. For each model investigated, five separate SMD simulations were performed to sample the configurational space provided by the flexible Lys 492 residue and to identify possible Pho84 exit routes using various protonation states of both Asp 178 and P i . Although, according to the Jarzynski equality method that requires a large number of samplings to be performed (14 -16), five simulations for each system is not enough to estimate the free energies of the possible release pathways, we used the simulations to qualitatively, rather than quantitatively, describe the alternative release routes and their corresponding forces and energies.
Calculation of force profiles (supplemental Figs. S21-S22), work (supplemental Figs. S23-S24), as well as hydrogen bond contacts between residues and P i (supplemental Tables S2-S5 and Figs. S25-S28) and water molecules and P i (supplemental Fig. S29 -S30) along the series of routes populated allowed for a deeper understanding of the molecular basis of the regulation of P i release in Pho84. The steered MD simulations suggested that the lowest-energy release route involved H 2 PO 4 Ϫ and deprotonated Asp 178 (supplemental Figs. S23-S34). SMD (Fig.  7) and unrestrained simulations demonstrated that protonation of Asp 178 resulted in a rotation of helix IV, which increased the work required to release P i via this route. The increased work required could be a result of the increase in hydrogen bond contacts and the number of water molecules encountered by H 2 PO 4 Ϫ when Asp 178 is protonated. A 30-ns unrestrained simulation showed that rotation of Tyr 179 into the binding site made it more accessible for P i binding. This increased binding between P i and Tyr 179 upon protonation of Asp 178 indicated that Pho84 regulates its conformational state preceding P i release. The lowest-energy route for the release of H 2 PO 4 Ϫ from Pho84 involves helices IV and XI and a minimum number of contacts with residues in L-VI. Upon protonation of Asp 178 , however, the transport channel of Pho84 narrowed as a result of increased contacts with Gln 119 in helix II and increased contacts with L-VI residues. In this protonated Pho84, H 2 PO 4 Ϫ must be released via a higher energy route (Fig. 7). A detailed analysis of the number of hydrogen bond contacts formed between H 2 PO 4 Ϫ and water along the release route (Fig. 7) dem-onstrated a reduction in hydrogen bonding between P i and water. This reduction resulted from increased hydrogen bonding to Tyr 179 in the binding site upon protonation of Asp 178 ; these data support the observation that the release route narrowed. In addition, H 3 PO 4 was found to contact Gln 119 in helix II and to make contacts with L-VI residues suggesting that this alternate high-energy release route is used for H 3 PO 4 as well as for H 2 PO 4 Ϫ . SMD simulations of the cytosolic release of HPO 4 2Ϫ showed that this protonation state of P i binds more strongly to Pho84 than any of the other investigated protonation states, thus charge balance in the transport channel appears to regulate P i release routes (supplemental Figs. S22, S28, S30, and S34 and Table S5). The Proposed Mechanism for P i Release in P i :H ϩ Transporters Using Pho84 as a Model-Results from unrestrained and steered MD simulations showed that the protonation states of P i and Asp 178 and the resulting orientations of Tyr 179 impact regulation of the P i release pathway. Protonation of Asp 178 is a crucial step in triggering the conformational change of Pho84 from an open inward-facing conformation to a more occluded conformation. However, this switch in conformation appears to be independent of the protonation state of P i . As a result of Pho84 adopting the occluded conformation, Tyr 179 becomes part of the binding site as seen in the PiPT crystal structure.
Based on our observations, we propose a sequential mechanism for the release of P i (Fig. 8).
(i) The open inward-facing conformation of Pho84 containing deprotonated Asp 178 is the active form that releases P i . We found that H 2 PO 4 Ϫ is the most favorable protonation state of P i to be released due to the distribution of the charges of the residues along the release pathway. In this active conformation of Pho84, Tyr 179 points away from the binding site and may regulate the possible H 2 PO 4 Ϫ release routes and/or may act as a gatekeeper, which blocks more hydrophobic phosphate substrates.
(ii) Protonation of Asp 178 results in a conformational change from an open inward-facing conformation to an occluded con- FIGURE 7. Representative data from steered dynamics simulations for the release of P i from the Pho84 binding site after initially restraining the Lys 492 -P i distance for 0.1 ns. The calculated force profiles and the Pho84 residues (and their transmembrane helix) that form hydrogen bonds with P i along the release pathway or the average number of hydrogen bonds formed with water along the route for models involving deprotonated and protonated Asp 178 . Numbers in brackets represent hydrogen bonding between the residue and H 2 PO 4 Ϫ as a percentage of the total simulation time.  DECEMBER 16, 2016 • VOLUME 291 • NUMBER 51 formation in which Tyr 179 is reoriented into the binding site and Pho84 is locked into a closed and inactive conformation, which is unable to release H 2 PO 4

Mechanistic Studies on the Pho84 Transporter
Ϫ . Transceptor Function Is Coupled to the Transport Cycle-In addition to its well known transport function, the Pho84 transceptor has the unique property of PKA signaling (17,18). Previous point mutation studies have shown that these functions can be uncoupled resulting in a non-transporting protein that is still able to activate PKA upon phosphate detection (8). These studies focused on mutating residues that bind P i or that could bind phosphate analogues that trigger PKA activation. In this study, we addressed whether limited release of P i from the Pho84 binding site influenced the ability of Pho84 to activate PKA (Fig. 9). Because transport activity data clearly showed that Tyr 179 is crucial for Pho84 functionality, trehalase activity assays were performed with relevant mutants.
The ability of the Tyr 179 -Ala mutant strain to activate PKA was slightly reduced, whereas the ability of the Tyr 179 -Gly mutant strain to activate PKA was abolished. The Tyr 179 -Phe and Tyr 179 -Ser mutant strains were able to activate PKA equivalent to the wild-type strain. Modest to strong reductions in transport activity in Asp 178 and Asp 358 mutants, respectively, were shown to not affect signaling. Abolishing transport by introducing an Asp 358 -Glu mutation resulted in a 50% reduction in signaling capacity (8). These observations show that, although transport and signaling are tightly intertwined, there are subtle yet profound differences in the residues that regulate the dual functions of Pho84 (Table 2).
In this study, we showed that Tyr 179 , which is associated with the substrate release step during transport, has a stronger influence on signaling than Asp 358 (Table 2), which has been shown to be crucial for substrate binding (8). We hypothesize that this difference originates from the different phosphate binding and release mechanisms operating in transport and in signaling.
Strain Construction-Mutants were created by oligonucleotide-directed site-specific mutagenesis using a plasmid containing the PHO84 WT gene as described previously (8). The synthetic oligonucleotides used are listed in Table 3. The mutagenesis was performed using the Stratagene (USA) QuikChange TM II mutagenesis kit according to the manufacturer's protocol. All of the mutant constructs were confirmed by DNA sequencing the entire PHO84 WT gene. Using pU6H2MYC/PHO84 WT and pU6H2MYC/PHO84 MUT as templates for PCR, cassettes containing the last 1.3 kb of PHO84 WT or PHO84 MUT , c-myc, a His 6 epitope, and a selection (Kan r ) marker were transformed into CEN.PK 113-7D and were incorporated into the genome by homologous recombination.

Residues involved in substrate transport and sensing/signaling
The importance of residues in Pho84 for transport and signaling/sensing are as follows: ϩϩϩ, crucial; ϩϩ, contributing; ϩ, little contribution. This is based on data obtained by site-directed-mutagenesis and relevant biochemical assays performed in this study and in previous work (8). Yeast Growth Spot Tests-Strains were cultured overnight in YPD at 30°C under continuous agitation. Cells were collected, washed twice with sterile dH 2 O, and resuspended in sterile dH 2 O at an A 600 of 0.3. A ϫ10 dilution series was spotted onto HP i and LP i SC medium agar plates. The plates were incubated at 30°C, and growth was recorded every 24 h for a total of 3 days. To visualize the reduction in growth of the pho84⌬ and mutant strains under LP i conditions, images were acquired with a higher illumination setting resulting in a brighter background. The figures were cropped and no further image processing was applied.

Amino acid
Phosphate Transport Measurements-Phosphate uptake in intact S. cerevisiae cells expressing Pho84 WT -myc or Pho84 MUT -myc was measured as previously described (8). Briefly, cells were grown in LP i medium and assayed by addition of 2 l of [ 32 P]orthophosphate (carrier-free, 0.18 Ci mol Ϫ1 ; 1 mCi ϭ 37 MBq) (PerkinElmer, USA) and phosphate (10 mM KH 2 PO 4 ) to a final concentration of 220 M. To determine total transport activity, a final phosphate concentration of 110 M was used. Cells were resuspended to 1 mg ml Ϫ1 (wet weight) in buffer containing 25 mM Tris succinate, pH 4.5, and 3% glucose. Aliquots of 30 l were incubated for 10 min at room temperature. After 10 min, 3 ml of ice-cold 25 mM Tris succinate, pH 4.5, buffer was added to stop the reaction. The cells were rapidly filtered (Whatman GF/F, USA) and the radioactivity retained on the filters was measured by liquid scintillation spectrometry.
Immunoblot Analysis of Pho84 Expression-Immunoblot analysis was performed as previously described (8). Briefly, membrane fractions were collected and 10 g of protein samples were separated by SDS-PAGE using a 10% Laemmli system (21). Anti-myc HRP-conjugated antibody (anti-myc-HRP, 1:5000, Novex) was used to detect expression of the Pho84 WT/ MUT-myc constructs. The (Ϫ) control was cut from the blot to avoid visualization of irrelevant samples, but detection was performed simultaneously with the mutant sample blot. As a load-ing control, 10 g of total cell extract was loaded onto a separate gel according to the previously described protocol, and anti-␤actin HRP-conjugated antibody (1:5000) (Abcam, UK) was used for detection. After 1 min of incubation with chemiluminescent substrate (GE Healthcare, UK), the membrane-enriched sample blot was exposed to X-ray film for 1.5 min. The control blot was visualized using the Bio-Rad ChemiDoc TM MP imaging system with an exposure time of 30 s. The molecular masses of the separated proteins were determined by their mobility relative to the pre-stained protein markers (Fermentas, Germany). Figures were cropped and no further image processing was applied.
Acid Phosphatase Assays-rAPase activity was assayed in liquid using an adapted protocol for the colorimetric Abcam Acid Phosphatase Assay Kit. Briefly, whole cells were used as the source of the enzyme and p-nitrophenyl phosphate was used as the substrate. Yeast strains were grown overnight in 5 ml of YPD at 30°C, centrifuged, and washed twice with LP i SC medium. Washed cells were inoculated into 15 ml of LP i or HP i phosphate SC medium to an A 600 of 0.4. Cells were grown at 30°C under continuous agitation for 3 h after which 80 l of the cell suspension was harvested, washed once with acetate buffer (60 mM, pH 4.5), and resuspended in 80 l of acetate buffer containing 1 mM p-nitrophenyl phosphate (final concentration). The reaction was incubated at 25°C for 1 h and was then stopped with the addition of 20 l of saturated Na 2 CO 3 . The cells were removed from the reaction by centrifugation before measuring the A 405 . The relative rAPase activity was determined by the formula A 405 /A 600 ϫ t, in which t is the time of incubation (min).
Trehalase Activity Measurements-Cells were cultured at 30°C to exponential phase (A 600 ϭ 1.0 -1.5) in YP medium with 2% (w/v) glucose. Mid-exponential phase cells were harvested and transferred to phosphate starvation medium (5.7 g liter Ϫ1 YNB without phosphate, with ammonium sulfate) with 4% (w/v) glucose and appropriate auxotrophic supplements. Cells were starved of phosphate for 3 days at 30°C under continuous shaking, and starvation medium was refreshed daily. The phosphate-starved glucose-repressed cells were rapidly cooled on ice and harvested by centrifugation (5000 ϫ g for 5 min at 4°C). The pellet was washed twice with ice-cold 25 mM MES buffer, pH 6.0, resuspended in phosphate starvation medium with 4% (w/v) glucose, and incubated at 30°C with shaking. After 30 min of incubation, 1 mM KH 2 PO 4 was added to the culture. 75 mg ml Ϫ1 cell samples were taken at the indicated time points. Cells were rapidly cooled by the addition of ice-cold dH 2 O, centrifuged (5000 ϫ g for 5 min at 4°C), and re-suspended in 0.5 ml of ice-cold 25 mM MES buffer, pH 7.0. Crude cell extracts were prepared as described previously (22) and dialyzed (BRL microdialysis system) against 25 mM MES buffer, pH 7.0, with 50 M CaCl 2 at 4°C. Trehalase activity in the dialyzed cell extracts was determined using a coupled enzymatic reaction of glucose oxidase and peroxidase with glucose as described previously (22). The specific activity was expressed as nanomole of glucose liberated per min per mg of protein. The total amount of protein in the samples was determined using the standard Lowry method described previously (23). way out of the receptor site and to ensure an energetically stable trajectory. The constant velocity chosen in this study (0.01 Å ns Ϫ1 ) is among the slowest found in the literature and was selected based on the results of unbinding studies on similar systems (39 -42).
Author Contributions-D. S. and J. V. d. V. performed the site-directed mutagenesis, phosphate uptake assays, acid phosphatase activity studies, and the spot tests. G. V. Z. performed the trehalase activity measurements. B. C. G. K. performed all of the theoretical calculations and analyses. B. C. G. K., D. S., and B. P. conceived the idea and the experimental design of the project. B. C. G. K. and D. S. led the writing of this paper. All of the authors have taken part in the preparation of this manuscript, have reviewed the results, and have approved the final version of this manuscript.