Orphan lysosomal solute carrier MFSD1 facilitates highly selective dipeptide transport

Our previous studies demonstrating a crucial role for MFSD1 in regulating cell migration (7, 10), spurred us to try to identify its substrates. Since MFSD1 localizes to the lysosome (11) we examined the metabolites whose presence in this organelle changed upon MFSD1’s absence. For an initial untargeted metabolomics screen, the LysoIP protocol (12) was applied on murine colon carcinoma MC-38 WT and MFSD1^−/− cells, both expressing the tagged lysosomal protein Tmem192-3xHA for lysosome immunopurification. We observed a significant increase of dipeptides containing lysine and arginine in lysosomes of MFSD1^−/− cells when compared to control WT lysosomes (Fig. 1A and SI Appendix, Fig. S1 A and B), with a >80-fold increase for the dipeptide KP. For targeted metabolomics, we prepared WT and MFDS1^−/− human 293LX cells (SI Appendix, Fig. S1C) expressing the lysosomal bait protein Tmem192-3xHA (SI Appendix, Fig. S1D). We confirmed proper targeting by colocalization of Tmem192 with the lysosomal marker LAMP2 in both WT and MFSD1^−/− 293LX cells (SI Appendix, Fig. S1 E and F). The relative abundance of dipeptides in purified lysosomes from WT and MFSD1^−/− cells was compared to a generated library of 361 dipeptides. Strikingly, we observed a specific increase in the accumulation of dipeptides containing either arginine or lysine, and to a reduced degree also of histidine, while other dipeptides were not affected in MFSD1^−/− lysosomes (Fig. 1B). Interestingly, we also observed an accumulation of the single amino acids lysine and arginine in the purified MFSD1^−/− lysosomes from three different MFSD1^−/− single-cell clones, when compared to the corresponding WT single-cell clones (SI Appendix, Fig. S1G). From these initial targeted metabolomics experiments, we conclude that MFSD1 transports dipeptides containing at least one positively charged amino acid.

MFSD1 requires the presence of its accessory protein GLMP (13), and GLMP^−/− phenocopies MFSD1^−/− (7, 13). Nontargeted metabolomics of GLMP^−/− lysosomes showed the highest accumulation for positively charged dipeptides, with a 47-fold increase for the dipeptide RD (SI Appendix, Fig. S1H), thus, recapitulating the results obtained from the nontargeted analysis of MFSD1^−/− (SI Appendix, Fig. S1B). Additionally, we observed an increase in lipid metabolites in both, GLMP^−/− and MFSD1^−/−, lysosomes (SI Appendix, Fig. S1 B and H), suggesting a compensatory process common to both knock-outs. We continued to perform a targeted metabolomics analysis on GLMP^−/− cells (SI Appendix, Fig. S1C). We found that the accumulation of arginine, lysine, and histidine-containing dipeptides in GLMP^−/− and MFDS1^−/− lysosomes was similar (Fig. 1C). Inducible re-expression of MFSD1 in 293LX Tmem192-3xHA MFSD1^−/− cells (SI Appendix, Fig. S1I) rescued dipeptide levels in these lysosomes to WT amount (Fig. 1D). These results suggest that MFSD1 likely functions as a dipeptide exporter.

MFSD1 can be redirected to the plasma membrane by mutating its dileucine lysosomal targeting motif to alanine (13, 14) allowing patch clamp analysis. We confirmed the expected orientation of MFSD1^AA in the plasma membrane (i.e., lysosome luminal side exposed to the extracellular side, SI Appendix, Fig. S2D). Lysosomal pH conditions were achieved by setting extra- and intracellular pH to 5.5 and 7.2, respectively (Inset in Fig. 2A). The dipeptide KG was selected for further transport analysis on HEK293S GnT1^−/− cells expressing MFSD1^AATST. KG induced an inwardly directed current, which was absent in control cells (Fig. 2A). Its amplitude increased at higher concentrations of KG and started to level out at around 30 mM (Fig. 2A). A Michaelis–Menten fit to the normalized current amplitudes measured at −70 mV resulted in a K_m = 4.11 ± 0.41 mM (Fig. 2B). The rate of current deactivation upon removal of the substrate from the bath solution is a surrogate for the substrate turnover rate of a SLC (15, 16). A monoexponential fit to the current decay (see Inset in Fig. 2C, current deactivation indicated by the red line) yielded a decay rate of 34 ± 6 s⁻¹ (Fig. 2C). In the voltage range between −140 mV to +30 mV (Fig. 2D) amplitudes were larger at negative potentials and leveled out above −80 mV (Fig. 2E). Under physiological lysosomal membrane voltages between −40 mV to −20 mV (17) MFSD1 operates at about 90 % of its maximal capacity.

A variety of positively and negatively charged as well as electroneutral (AA) dipeptides were tested as potential substrates using initial substrate concentrations of 30 mM. Lysine, arginine, and histidine-containing dipeptides induced inwardly directed currents (Fig. 3A). Dipeptides with a net negative charge (DA, DD, and AD), as well as the neutral dipeptide AA, failed to stimulate a current. Compared to the amplitudes induced by 30 mM KG only AK and KK created higher currents (Fig. 3 A and B), with the highest transport affinity for AK and KA with K_m = 0.97 ± 0.05 mM and 1.82 ± 0.11 mM (Table 1 and SI Appendix, Fig. S3 A–F). The different K_ms partly explained the different current levels shown in Fig. 3B. At a concentration of 30 mM the current induced by KG, KA, AK, HG, RG, and KK, reaches about 88%, 94%, 97%, 92%, 91%, and 76% of the maximal amplitude, respectively.

At a saturating dipeptide concentration, the current amplitude is determined by the substrate turnover rate and the charge of the substrate. To separate the distinct influences of these two factors, we plotted the concentration-dependent current amplitudes of RG, KA, AK, and KK (Fig. 3C), which are all positively charged (SI Appendix, Fig. S3J). When normalizing the respective current amplitude against the saturating V_max of KG, we obtained relative V_max values of RG, KA, AK, and KK of 0.76, 0.84, 1.03, and 2.2, respectively. The different dipeptides are transported at different rates but these differences are not large enough to obscure the influence of the substrate charge on the current amplitudes. Our results thus unequivocally show that MFSD1 is a dipeptide transporter, which can mediate the transit of dipeptides that contain at least one positively charged amino acid.

We investigated whether MFSD1 could transport single amino acids or tripeptides. We applied three positively charged (H, K, and R), one negatively charged (D), and one neutral amino acid (A), at a concentration of 20 mM (Fig. 3 D and E). None of the tested amino acids was able to induce a current. This suggests that single amino acids are not substrates of MFSD1. The same holds true for tripeptides (GKG, RGD, and GHK) for which no currents were detected when applied at a concentration of 30 mM (Fig. 3 F and G). These data indicate a very high specificity of MFSD1 for dipeptides.

We sought to explore whether the transport function of MFSD1 differs in the presence or absence of GLMP. We first expressed MFSD1^AAeGFP in WT and GLMP^−/− cells, respectively, and found that MFSD1^AAeGFP was present at the plasma membrane of both cell lines (SI Appendix, Fig. S2A). Notably, the current amplitudes evoked by the dipeptide KG and the K_m values in WT and GLMP^−/− cells were indistinguishable (Fig. 3H, Table 2, and SI Appendix, Fig. S3 G and H). We also expressed the fusion protein GLMP^Y402AMFSD1^AAeGFP in WT cells and confirmed cell surface localization (SI Appendix, Fig. S2B) and proper orientation (i.e., lysosome luminal side exposed to the extracellular side) (SI Appendix, Fig. S2E). As seen, the fusion protein GLMP^Y402AMFSD1^AAeGFP and MFSD1^AAeGFP had essentially the same K_m value for the dipeptide KG (Fig. 3H, Table 2, and SI Appendix, Fig. S3I). These data, therefore, suggest that MFSD1 requires GLMP to resist lysosomal degradation (13, 18) but not for dipeptide transport.

Dipeptide transporters such as SLC15A1-A5 or their bacterial homologue DtpB are known to be proton-coupled (19, 20). The proton gradient across the lysosomal membrane would suggest that MFSD1 might as well be proton-driven, although recently lysosomal uniporters were identified (21). We varied extra- and intracellular proton concentrations to investigate the pH dependence of MFSD1-mediated currents induced by 3 mM KG at a membrane potential of −70 mV. We found that a successive increase of the pH from 5.5 to 8.5 on the cis-side (i.e., the side from which the substrate is applied) led to a reduction of the current amplitude. Conversely, changing the pH on the trans-side (i.e., the intracellular side) from 5.5 to 8.5 had no appreciable effect on the current amplitude (Fig. 4 A–C). However, if a proton was an obligatory cosubstrate of MFSD1, raising the proton concentration intracellularly ought to have reduced the current amplitude. This is because the proton can rebind to the transporter after it is released and thereby hamper the progress of the transport cycle. Rebinding, will more frequently occur if the proton concentration is high. The absence of current inhibition upon a raise in proton concentration by a factor of 1,000 (from pH 8.5. to 5.5), thus, refutes the idea that protons are obligatory cosubstrates of MFSD1 (Fig. 4C). Hence, we conclude that MFSD1 is a uniporter.

We then explored alternate hypotheses for the influence of pH on the current amplitude. We reasoned that increases in pH could be decreasing the turnover of substrates by deprotonating one of the transporter’s acidic amino acids. We examined the current deactivation rate as a surrogate for the rate of substrate turnover and determined whether it was inhibited at a higher pH. We extracted current deactivation rates from experiments conducted both at 25 °C (Fig. 4D) and 15 °C (Fig. 4E), to slow down the transport kinetics of MFSD1 and thus better resolve rate differences. While the deactivation rate was reduced at 15 °C, it was unaffected by changes in pH_out (Fig. 4F). These data indicate that increases in pH do not alter the transporter’s capacity to turnover substrate.

We then hypothesized that the deleterious effects of higher pH on current amplitude could be due to i) a loss in substrate affinity or ii) a change of the charge carried by the substrate. We first determined the K_m of KG at pH_out7.5. At this pH_out the K_m of KG was essentially the same as that at pH_out5.5 (Fig. 4G and SI Appendix, Fig. S4A and Table 1). Then we turned to potential alterations in the charge carried by the dipeptides. The pK_A value of a dipeptide depends on its amino acid composition. Accordingly, if the loss of the current amplitude at higher pH_out values is caused by a change in the protonation state of the transported substrate, the proton concentration dependence of this effect ought to vary between the different tested dipeptides. Indeed, we observed a varying decline in the currents evoked by the dipeptides upon increasing the pH_out (Fig. 4H and SI Appendix, Fig. S4 B–E), with a rank order by pKa value of DK < HG < KK < KG. The observation that the pH dependence of the current amplitude is a property of the chosen substrate supports the idea that the decline in current amplitude at higher pH_out values is due to the gradual deprotonation of the dipeptide. For KK we confirmed that the reduction in current amplitude at higher pH values was not caused by a loss of its affinity for MFSD1 (SI Appendix, Fig. S4 F and G and Table 1). This could not be tested for DK and HG because these dipeptides failed to produce a current at higher pH_out values.

In summary, the data presented here show that MFSD1 functions as a uniporter and that its transport rate is not affected by the ambient pH.

MC-38 and HEK293 Lenti-X cells (Takara, #632180, Kusatsu, Japan) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Thermo Fisher Scientific, #31966-021) supplemented with 10% Fetal Calf Serum (FCS) (Sigma, #9665) and incubated at 37 °C with 5% CO₂. HEK293S GnT1^−/− cells (ATCC, #CRL-3022, Manassas, USA) were cultivated in DMEM-Ham’s F12 (Thermo Fisher Scientific, #10565-018) supplemented with 10% FCS and incubated at 37 °C with 5% CO₂. The expression of ectopic MFSD1 variants in respective cells was induced with Doxycycline for 18 to 24 h.

The constructs used in this study are listed in SI Appendix, Table S1. All restriction enzymes and T4 ligase used for cloning were from New England Biolabs (NEB, Ipswitch, USA) and the Gateway BP and LR Clonase II were from Thermo Fisher Scientific. All Gateway cloning constructs were cloned via the donor plasmid pDONR221 (Addgene, #12536017, Watertown, USA) into the Doxycycline inducible expression plasmid pINDUCER20 (65). Constructs were heat-shock transformed into One Shot Stbl3 E. coli bacteria (Thermo Fisher Scientific, #C737303). Proper sequence of all constructs has been confirmed by sequencing (Microsynth, Balgach, Switzerland) (SI Appendix, Table S1).

Constructs generated for mammalian expression were separately packed into lentiviral particles using pdelta8.9 (Addgene, #2221) and pCMV-VSV-G (Addgene, #8454) cotransfected using Lipofectamine 3000 (Thermo Fisher Scientific) into Lenti-X 293T cells (TaKaRa, #632180). Crude lentiviral supernatant was used for infection of target mammalian cells. Cells were selected for stable integration of the constructs via respective antibiotic selection.

The generation of MC-38 WT and MFSD1^−/− is described elsewhere (7). HEK293 Lenti-X (Takara, #632180) were seeded on a 24-well plate and at ~80% confluency cells were transfected with Lipofectamine CRISPRMAX Cas9 Transfection reagent, as suggested by the manufacturer (Qiagen, #CMAX00001, Hilden, Germany) and shown in SI Appendix, Table S2. All RNA oligos were from Integrated DNA Technologies (IDT).

One day after transfection cells were expanded on a T25 Tissue culture flask (TPP, #90026). Additional 3 d later, cells were single-cell cloned into 96-well plates. Two to three weeks later single-cell clones were determined with an Olympus CKX41 microscope with a Olympus Plan C N 4×/0.10 objective (Olympus, Shinjuku, Japan) and expanded. MFSD1^−/− and GLMP^−/− was verified by qPCR as follows: RNA was isolated from single-cell clones using the RNeasy Mini Kit (Qiagen, #74104) according to manufacturers’ instructions. cDNA synthesis and qPCR was performed in a single tube using the Luna Universal One-Step RT-qPCR Kit (NEB, #E3005) with an input of 0.1 µg to 1.0 µg of total isolated RNA per reaction on a LightCycler 480 machine (Roche, Basel, Switzerland) using primers listed in SI Appendix, Table S3.

MC-38 and HEK293 Lenti-X WT, MFSD1^−/−, MFSD1^−/−::MFSD1-TST (rescue cell line), and GLMP^−/− cells were stably transfected to express the HA-tagged lysosomal marker Tmem192, suitable for immunopurification of intact lysosomes (12). Specifically, cells were expanded onto three p100 Tissue Culture Dishes (VWR, #734-2321), washed with cold PBS and scraped into 2 mL Potassium Phosphate Buffered Saline (KPBS) (136 mM KCl, 10 mM KH₂PO₄, pH 7.25) and centrifuge for 2 min, at 1,000 × g, 4 °C. The cell pellet was resuspended in 1 mL KPBS and cells were homogenized with a glass dounce homogenizer, applying 20 strokes. The cell lysate was cleared by centrifugation for 2 min, at 1,000 × g, 4 °C. The cell lysate containing the lysosomes was added to 100 µL pre-equilibrated Peirce Anti-HA Magnetic beads (Thermo Fisher Scientific, #88836) and incubated rotating at 4 °C for 3 min. The beads were washed three times with 1 mL KPBS, and the lysosomal metabolites were extracted using 50 µL of Extraction buffer (methanol/acetonitrile/ water = 40/40/20) for 10 min at room temperature. The beads were removed and the extract was snap-frozen and stored at −80 °C until further analysis.

Data-dependent LC–MS/MS (liquid chromatography-tandem mass spectrometry) was performed with an Ultimate 3000 High-Performance Liquid Chromatography (HPLC) system, coupled to a Q Exactive Focus mass spectrometer (both Thermo Fisher Scientific) via electrospray ionization. A flow rate of 100 μL/min was used and 1 μL of each sample was injected onto the respective column and guard column. For reversed phase chromatography, an ACQUITY UPLC HSS T3 (Waters; 150 mm × 2.1 mm; 1.8 μm) was used. Employing a 20 min linear gradient of 99% A (0.1% formic acid in water) to 60% B (acetonitrile with 0.1% formic acid). In HILIC (hydrophilic interaction chromatography), a SeQuant ZiC-pHILIC, (Merck; 5 µm, 100 × 2.1 mm) was used with a 20 min gradient from 90% A (acetonitrile) to 80% B (20 mM ammonia bicarbonate in water). Mass spectra were acquired with a resolution of 70,000 in polarity switching mode with full MS acquisition from m/z 70 to m/z 1,045. MS/MS data were collected in data-dependent acquisition mode in a pooled quality control sample, containing aliquots of all samples and using a normalized collision energy of 25. Data analysis and statistical evaluation was performed with Compound Discoverer (Thermo Fisher Scientific).

Detection and quantification of amino acids and dipeptides was done by LC–MS/MS, using a Vanquish HPLC system coupled to a TSQ Altis mass spectrometer (both Thermo Fisher Scientific), employing the Selected Reaction Monitoring (SRM) mode and positive polarity. In brief, dried samples were resolved in 0.1% formic acid in water and 1 µL was injected onto a Kinetex (Phenomenex) C18 column (100 Å, 150 × 2.1 mm), employing a 9-min-long linear gradient from 100% A (1% acetonitrile, 0.1% formic acid in water) to 90% B (0.1% formic acid in acetonitrile) at a flow rate of 100 µL/min. Retention times, SRM transitions, and optimal collisional energies were determined by authentic standards and degenerate dipeptide libraries. All data interpretation was performed using Xcalibur (Thermo Fisher Scientific).

Cell lysates or purified lysosomes were separated on 4 to 15% Sodium Dodecyl Sulfate - PolyAcrylamide Gel Electrophoresis (SDS-PAGE) gradient gels (Bio-Rad, Hercules, USA) and transferred on Protran 0.45 nitrocellulose membranes (GE Healthcare, Chicago, USA). The nitrocellulose membrane was blocked with 1× Pierce Clear Milk Blocking Buffer (Thermo Fisher Scientific, #37587) for 1 h at room temperature. Primary antibodies were incubated in blocking buffer overnight at 4 °C, while secondary antibodies were incubated in blocking buffer for 1 h at room temperature. Following washing with Tris Buffered Saline-Tween 20 (TBS-T), membranes were incubated with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, #34096) and the chemiluminescent signal was detected with the ChemiDoc MP Gel Imaging System (Bio-Rad). Antibodies used are listed in SI Appendix, Table S4.

Cells were grown on Nunc Lab-Tek Chamber Slides (Thermo Fisher Scientific, #154534) and recombinant gene expression was induced by 100 ng/µL doxycycline for 24 h. Cells were fixed with 4% formaldehyde in PBS (Thermo Fisher Scientific, #28906) for 10 min at room temperature (RT) followed by blocking/permeabilization with 1% BSA/0.1% Triton X-100 in PBS for 1 h at RT. Cells were stained with primary antibodies in blocking/permeabilization buffer for 2 h at RT, followed by secondary antibody staining for 1 h at RT. Cells were counterstained with DAPI for 10 min at RT and mounted in ProLong Diamond Antifade Mountant (Thermo Fisher Scientific, #P36970). Between incubation steps cells were washed with PBS for 3 × 5 min. Immunofluorescent pictures were taken on a Zeiss LSM880 inv. Fast Airyscan confocal microscope with a Plan-Apochromat ×40/NA 1.3 OIL objective. Colocalization (Pearson’s R value with the Coloc 2 tool) was determined by Image J. Antibodies used are listed in SI Appendix, Table S4.

Cells were grown on poly-D-lysine coated glass-bottom dishes and expression of MFSD1^AAeGFP or GLMP^Y402AMFSD1^AAeGFP was induced for 24 h with 5 µg/mL Doxycycline. Prior confocal imaging, cells were rinsed in Live cell imaging solution (Gibco, #A59688DJ), and stained with 0.4% Trypan Blue solution (Sigma, T8154) for 3 min at room temperature. Cells were washed three times with Live cell imaging solution and confocal pictures were taken on a Nikon Eclipse Ti (Nikon, Minato, Japan) confocal microscope with a Plan-Apochromat VC ×60/NA 1.4 Oil DIC N2 objective. Images were processed by Image J.

Cells were washed off the cell culture plate and washed once in cold Fluorescence Activated Cell Sorting (FACS) buffer (1% BSA, 10 mM Ethylenediaminetetraacetic acid in PBS). Cells were incubated for 10 min on ice with Fc block (BioLegend, #422301) and stained with anti-myc 4A6 (Sigma-Aldrich, #05-724) or anti-GFP 5G4 (E. Ogris lab, MPL Vienna) for 30 min. Cells were washed once with FACS buffer and stained with secondary anti-mouse AF633 antibody (Thermo Fisher Scientific, #A21050) for additional 30 min. After the final wash, cells were resuspended in 250 µL FACS buffer and analyzed on a Beckton Dickinson CytoFLEX S (Beckton Dickinson, Franklin Lakes, USA) flow cytometer. Data were processed by FlowJo software (Tree Star Inc., Ashland, USA).

HEK293S GnT1^−/− cells expressing hsMFSD1^AATST, HEK293 Lenti-X wild-type or GLMP^−/− cells expressing hsMFSD1^AAeGFP, and HEK293 Lenti-X wild-type cells expressing the fusion protein GLMP^Y402AMFSD1^AAeGFP upon Doxycycline induction for 18 to 24 h, or control, were grown on poly-D-lysine coated cell culture dishes. During experiments, cells were maintained in a buffer consisting of 150 mM NaCl, 3 mM KCl, 2.5 mM CaCl₂, 2 mM MgCl₂, 20 mM glucose, and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), with the pH adjusted to 7.4 using NaOH.

The patch pipette solution was composed of 150 mM NMDG (N-methyl-D-glucamine), 1 mM CaCl₂, 0.7 mM MgCl₂, 10 mM HEPES, and 10 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid. The pH was titrated to 5.5, 7.2, or 8.5 with MsOH (methanesulfonic acid) or NMDG.

For dipeptide application, the buffer comprised 150 mM NMDG, 2.5 mM CaCl₂, 2 mM MgCl₂, 20 mM glucose, and, depending on the desired pH, 10 mM MES (2-(N-morpholino)ethanesulfonic acid) for pH 5.5 and for pH 6.5, 10 mM HEPES for pH 7.5, and 10 mM tris(hydroxymethyl)aminomethane for pH 8.5. Peptides and amino acids were purchased at Bachem Holding AG (Bubendorf, Switzerland), Carl Roth GmbH + Co (Karlsruhe, Germany), or Sigma-Aldrich (St. Louis, MO, USA), respectively (SI Appendix, Table S5). All buffers were prepared freshly before each experiment. All chemicals utilized were of analytical grade and were employed without further purification, sourced from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified.

Dipeptides were applied via a perfusion system (Octaflow II, ALA Scientific Instruments, Inc.), which allowed for rapid and complete solution exchange around the cells within a 20-ms timeframe.

Experiments were conducted at 15 °C or 25 °C, as indicated, with precise temperature control provided by a temperature control unit (cell microcontrol inline preheater, Green Leaf Scientific), which was linked to a temperature control system (PTC-20, npi electronic GmbH, Tamm, Germany). Both, the patch-clamp pipette, and the perfusion system were manipulated using the PatchStar micromanipulator (Scientifica Ltd., Uckfield, East Sussex, United Kingdom).

Patch-clamp recordings were conducted in the whole-cell configuration using an Axon 200B amplifier equipped with an Axon 1550 digitizer. Recordings were sampled at 100 µs intervals, and current amplitudes and associated kinetics were analyzed using Clampfit software (Molecular Devices, LLC, San José, CA, USA). Passive holding currents were subtracted, and the traces were filtered using an 80 Hz digital 8-pole Bessel low-pass filter.

All analyses were performed using Graph Pad Prism version 9 (GraphPad Software, Boston, MA, USA). Data shown are as mean ± SD.

D.B., T.K., W.S., and M.R. designed research; D.B., N.B., T.K., and M.R. performed research; G.M.M., G.H., T.K., H.H.S., D.S., C.Z., W.S., and M.R. contributed new reagents/analytic tools; D.B., N.B., T.K., D.S., C.Z., W.S., and M.R. analyzed data; and D.S., C.Z., W.S., and M.R. wrote the paper.

The authors declare no competing interest.