Materials and Reagents

1. 50 ml polypropylene conical tubes (Falcon, catalog number: 352098 )
2. High-g-rated microfuge tubes (Beckman Coulter, catalog number: 357448 )
3. 15 ml 50k MWCO Centrifugal Filter (Millipore, catalog number: UFC905024 )
4. 4 ml 50k MWCO Centrifugal Filter (Millipore, catalog number: UFC805024 )
5. 0.4 μm extruder filter (Whatman, catalog number: 800282 )
6. 0.22 μm mixed cellulose ester membrane (Millipore, catalog number: GSTF02500 )
7. 7 ml glass scintillation vials (Fisher Scientific, catalog number: 03-340-4A )
8. SEC Column (GE Life Sciences Superdex 200 Increase 10/30 GL; catalog number: 28-9909-44 )
9. 1 cm diameter glass column (Kimble Flex-Column, catalog number: 420401 )
10. Bio-Beads SM-2 adsorbent media (Bio-Rad, catalog number: 152-8920 )
11. cDNA for the Slc35a1 gene; mouse CMP-sialic acid transporter; mCST (Biobasic, Uniprot: Q61420)
12. EasySelect Pichia Expression Kit (Thermo Fisher, catalog number: K174001 )
13. Pichia pastoris strain SMD1168H (Thermo Fisher, catalog number: C18400 )
14. PreScission protease
    Note: We express and purify this ourselves, but it can also be obtained commercially, e.g., GE Healthcare, catalog number: 27084301 .
15. Cytidine 5’-monophospho-N-acetylneuraminic acid; CMP-sialic acid; CMP-Sia (Carbosynth, catalog number: MC04391 )
16. [³H]CMP-sialic acid (American Radiolabeled Chemicals, catalog number: ART 0147-50 μCi )
17. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HEPES (Fisher Scientific, catalog number: BP310-1 )
18. Sodium chloride; NaCl (Fisher Scientific, catalog number: S271-3 )
19. Potassium chloride; KCl (Fisher Scientific, catalog number: P217-3 )
20. Magnesium chloride; MgCl₂ (Fisher Scientific, catalog number: M33-500 )
21. Zinc chloride; ZnCl₂ (Acros Organics, catalog number: 380130050 )
22. Benzamidine hydrochloride hydrate (Acros Organics, catalog number: 105240250 )
23. Imidazole (Fisher Scientific, catalog number: O3196-500 )
24. Deoxyribonuclease I; DNase I grade II, from bovine pancreas (Sigma-Aldrich, catalog number: 10104159001 )
25. Pepstatin A (Fisher Scientific, catalog number: BP2671-25 )
26. Leupeptin hemisulfate (Fisher Scientific, catalog number: BP2662-100 )
27. Aprotinin from bovine lung (Fisher Scientific, catalog number: BP2503-10 )
28. Phenylmethanesulfonyl fluoride (Acros Organics, catalog number: 215740010 )
29. N-dodecyl-β-D-maltopyranoside solgrade; DDM (Anatrace, catalog number: D310S )
30. N-dodecyl-β-D-maltopyranoside anagrade; DDM (Anatrace, catalog number: D310 )
31. 20% DDM (solgrade) solution made in water; stored at -20 °C
32. Methanol (Fisher Scientific, catalog number: A452-SK4 )
33. 1,3-Bis[tris(hydroxymethyl)amino]propane; Bis-tris-propane-HCl (Acros Organics, catalog number: 202640250 )
34. Sodium hydroxide; NaOH (Fisher Scientific, catalog number: S318-1 )
35. TALON metal affinity resin (Takara, catalog number: 635504 )
36. DL-1,4-Dithiothreitol; DTT (Acros Organics, catalog number: 16568-0250 )
37. Ethylenediamine tetraacetic acid, disodium salt; EDTA (Fisher Scientific, catalog number: S311-500 )
38. Yeast polar lipid extract; YPL (Avanti, catalog number: 190001C )
39. Pentane (Fisher Scientific, catalog number: P399-1 )
40. Antarctic phosphatase (New England Biolabs, catalog number: M0289S )
41. Ultima Gold scintillation cocktail (PerkinElmer, catalog number: 6013329 )
42. Argon gas, at least grade 4.8
43. Ultrapure water
44. Liquid nitrogen
45. Lysis Buffer (see Recipes)
46. Buffer A (see Recipes)
47. Buffer B (see Recipes)
48. Buffer C (see Recipes) 
49. AnP Buffer (see Recipes)
    

Equipment

1. Pipettes
2. -80 °C freezer
3. Miller (Retsch, model: MM400 )
4. Centrifuge
5. 50 ml Grinding Jars with 25 mm Grinding Balls (Retsch, catalog numbers: 01.462.0216 and 05.368.0105 , respectively)
6. Glass Hamilton syringe (Hamilton, catalog number: 81365 )
7. 12-well filter vacuum manifold (Millipore, model: 1225 , catalog number: XX2702550)
8. Sonicator (Avanti, model: G112SP1T_B )
9. Mini extruder (Avanti, catalog number: 610000 )
10. Liquid Scintillation Counter, e.g., Beckman LS 6000IC
    

Software

1. Prism 6 (GraphPad)

Procedure

1. CST Protein Expression and Purification
   1. Generate Pichia pastoris stable cell lines expressing CST according to manufacturer instructions (EasySelect Pichia Expression Kit, Thermo Fisher).
      Notes:
      
      1. Briefly, this entails first sub-cloning CST cDNA into the pPICZ vector. This vector is linearized and then introduced into Pichia pastoris (strain SMD1168H) cells by electroporation. Successful transformants are selected on zeocin-containing growth media.
      2. While the bulk of our structural and biochemical characterization of CST has been with the mouse protein, we have observed that the human protein has near-identical properties. Therefore, the methods described here should be applicable to the study of either type of CST.
   2. Express CST in P. pastoris according to manufacturer instructions. We typically induce expression in methanol-containing media for 20-24 h at 25 °C. We routinely grow 6 x 1 L cultures, yielding approximately 10 g cell paste per liter of culture.
   3. Harvest cells by centrifugation at 4,000 x g for 15 min. Discard supernatant and freeze cell pellets with liquid nitrogen.
   4. Lyse frozen cells in a mixer mill. We typically lyse 10 g of frozen cell paste per 50 ml milling chamber for 5 cycles of 3 min at 25 Hz. Chambers are cooled in liquid nitrogen between cycles.
   5. Resuspend milled cells in 2.5 ml of lysis buffer (Recipe 1) per gram of cells and incubate for 1.5 h at 4 °C.
   6. Centrifuge lysate at 35,000 x g for 35 min at 4 °C.
   7. Collect supernatants, adjust pH to 7.2 using 5 M NaOH, and then add imidazole to 5 mM.
   8. Equilibrate TALON resin in Buffer A (Recipe 2). Use 0.175 ml of packed resin per gram of cells.
   9. Add pH-adjusted supernatant to TALON resin and incubate at 4 °C for 2 h under gentle rotation.
   10. Wash resin in batch with 5 column volumes of Buffer A containing 5 mM imidazole. Pellet at 1,250 x g for 5 min and resuspend in 1 column volume of Buffer A.
   11. Load resin onto a 1 cm diameter glass column, and wash using a peristaltic pump at approximately 1 ml/min with 5 column volumes of Buffer A containing 20 mM imidazole, followed by 2 column volumes of Buffer A containing 40 mM imidazole.
   12. Elute protein with at least 5 column volumes of Buffer A containing 300 mM imidazole.
   13. Pool eluted protein and add 1 mM DTT and 1 mM EDTA.
   14. Add PreScission protease at a mass ratio of 1:20 protease:total protein, and dialyze overnight at 4 °C against Buffer A. Dialyze against a sufficient volume to reduce the imidazole concentration to 5 mM or lower. For example, dialyze against a volume that is at least 60 times the sample volume.
   15. Run cleaved protein over a 1 ml TALON column equilibrated in Buffer A. Collect the initial flow-through. Wash the column twice with one column volume buffer A. Pool the flow-through and washes.
       Note: This step is necessary to remove contaminants that are not resolved from CST during size exclusion chromatography (next step).
   16. Concentrate the pooled sample to at least 500 μl in a 50k MWCO concentrator and run on a 10/300 Superdex 200 Increase gel filtration column equilibrated in Buffer B (Recipe 3).
   17. Combine peak fractions for incorporating into proteoliposomes.
       
       
2. Proteoliposome Reconstitution
   1. To incorporate purified CST into lipid vesicles, the lipids must first be transformed into small unilamellar vesicles (SUVs). Since the lipid stocks are supplied dissolved in chloroform, the first step in this process involves evaporating away the chloroform before the lipids can be resuspended in an aqueous buffer.
   2. A typical reconstitution uses 40 mg of lipids (yeast polar lipid extract). To aliquot this amount, use a glass Hamilton syringe with a Teflon plunger to measure 1.6 ml of a 25 mg/ml solution of lipids dissolved in chloroform. Place the lipid solution into a glass test tube and evaporate the chloroform using a gentle stream of argon gas. Then, re-dissolve the dried lipids with ~2 ml pentane and then evaporate the pentane using a gentle stream of argon. Repeat this pentane wash once more and then place the dried lipids in a vacuum desiccator overnight to remove any residual solvent.
   3. Re-suspend the dried lipids in Buffer C (Recipe 4) at 11.1 mg/ml and sonicate for 2-3 h in a bath sonicator. Use a ring stand and clamp to suspend the glass test tube in the bath such that the lipid suspension is completely submerged. Run the sonicator in 5-10 min intervals to prevent overheating (above ~40 °C). During this time, the solution should change from opaque to translucent indicating the formation of SUVs.
   4. Add DDM to a final concentration of 5 mM, using a 20% stock solution, to partially solubilize the SUVs. Vortex gently and then incubate for 10-15 min at room temperature.
   5. Sonicate again for up to 1 h, in 5-10 min intervals, until the solution becomes semitransparent.
   6. Add 200 μg of purified CST from the previous section. The protein should be sufficiently concentrated using a 50k MWCO concentrator such that the addition of protein to the lipids does not dilute the lipids below 10 mg/ml. For protein-free vesicle controls, substitute CST for an equivalent volume of Buffer B. For filling the vesicles with a substrate (e.g., 300 μM CMP), add the desired substrate at this step.
      Note: This protein:lipid mass ratio of 1:200 was empirically determined to give the best signal to noise in our assay. Other proteins or lipid compositions may require different protein:lipid ratios.
   7. Bring the final lipid concentration to 10 mg/ml by diluting with Buffer C, if necessary, then incubate for 1 h at 4 °C.
   8. To form proteoliposomes, remove DDM by adding Bio-Beads to 100 mg/ml. Incubate for 2 h at 4 °C under gentle rotation, then briefly pellet Bio-Beads and transfer supernatant to fresh Bio-Beads at 100 mg/ml. Incubate again for 2 h at 4 °C under gentle rotation, then transfer to a third fresh batch of Bio-Beads at 100 mg/ml. Incubate this overnight at 4 °C under gentle rotation. The finished proteoliposomes should be noticeably less translucent at the end of this incubation.
      Notes:
      
      1. Prepare Bio-Beads by adding 30 ml methanol to ~2 g of Bio-Beads in a 50 ml Falcon tube. Rotate beads for 15 min, then remove methanol by applying a vacuum to a small hole created in the bottom of the Falcon tube using a needle. Wash the beads four times in 50 ml water, removing water by applying a vacuum. Wash the beads once in 10 ml of Buffer C, and then store under Buffer C until needed.
      2. To obtain the desired amount of beads, briefly drain the washed beads by vacuum and weigh out the desired amount using a balance.
   9. Aliquot the liposomes and flash freeze in liquid nitrogen. Store at -80 °C.
      Notes:
      
      1. The liposomes can be stored at -80 °C for at least several months.
      2. We can typically incorporate about 60% of the starting amount of protein into the vesicles.
      3. To determine the efficiency of protein incorporation, solubilize a small amount of vesicles using DDM, and run on a gel filtration column. Then compare the peak height to an equivalent protein sample that was not incorporated into vesicles.
      
      
3. CMP-Sia Ligand Purification
   Notes:
   
   1. Commercial stocks of CMP-Sia contain approximately 10% CMP and will further hydrolyze to form free CMP and Sia over time. For transport assays, all free CMP must first be converted to cytidine–which has a very low affinity for CST–using Antarctic phosphatase (AnP).
   2. We screened a number of commercially-available phosphatases. Most were able to effectively convert CMP to cytidine, but we ultimately selected AnP because it retains high enzymatic activity in acidic pHs, which is the pH range where most of our crystallization hits were obtained.
   3. The hydrolysis of CMP-Sia is temperature and pH dependent (faster at higher temperature and/or lower pH), so keep AnP-treated CMP-Sia solutions in neutral pH buffers and on ice during use and store at -80 °C.
   4. We determined that the rate of hydrolysis of CMP-Sia in the transport assay buffer (Buffer C) is 0.005%/min at room temperature. This should be a factor to consider when designing and interpreting data from transport assay experiments. However, given that most of our transport experiments used relatively low concentrations of CMP-Sia and short incubation times, we did not consider the small amount of CMP generated to be significant.
   1. The Antarctic phosphatase (AnP) stock from NEB is 5,000 U/ml. This is not concentrated enough to adequately treat a concentrated (100 mM) stock of CMP-Sia; therefore, the AnP must be concentrated. To do this, first dilute the AnP to 100 U/ml in AnP Buffer (Recipe 5) (to reduce glycerol concentration), then concentrate to 10,000 U/ml with a 10k MWCO concentrator.
   2. Dissolve CMP-Sia powder into the 10,000 U/ml AnP solution to a final concentration of 100 mM. Add ZnCl₂ to a final concentration of 0.2 mM and MgCl₂ to a final concentration of 2 mM.
   3. Incubate this mixture for 8 h at room temperature.
   4. Remove AnP by filtering the mixture through a 3k MWCO concentrator, which retains the 70 kDa AnP.
      Note: We confirmed that we do not observe any AnP activity after this step.
   5. If desired, confirm the elimination of CMP form the CMP-Sia stock by HPLC analysis.
   6. Aliquot and freeze at -80 °C. Aliquots can be stored for at least several months.
      
      
4. Transport Assay
   1. Thaw the lipid vesicles from Section B and extrude 10 times through a 0.4 μm Whatman Nuclepore filter using an Avanti mini extruder.
   2. Remove external CMP by pelleting the vesicles via ultracentrifugation at 194,800 x g for 60 min at 4 °C, using high-g rated 1.5 ml Beckman tubes. 45 μl vesicles are needed per assay point.
   3. Discard supernatant and rinse pellet twice with 500 μl of Buffer C. Then resuspend the pellet in Buffer C. The final volume should be 0.444 times the starting volume; however, it is helpful to initially only add a small volume (~50 μl) of Buffer C to make it easier to completely homogenize the vesicles before bringing the sample up to the final volume. Store on ice until needed.
   4. For each assay point, initiate transport by taking 20 μl of the resuspended vesicles and adding 30 μl of Buffer C with the desired concentration of AnP-treated CMP-Sia containing 30-50 nM [³H]CMP-Sia (20 Ci/mmol).
      Note: We have not evaluated the purity of the [³H]CMP-Sia that we use for transport assays, but we do not anticipate the presence of any CMP impurity to be a significant problem for the following reasons: 1) the tritium is on the Sia moiety so we don’t have to worry about detecting [³H]CMP transport, and free Sia has a non-detectable affinity for CST and is most likely not transported; 2) the [³H]CMP-Sia is only included as a tracer and is typically diluted at least 100x with cold CMP-Sia for the assay; therefore, the relative concentration of any CMP will be very low.
   5. Incubate the mixture for the desired time and temperature depending on the experiment. We typically incubate at 23 °C for convenience and to minimize CMP-Sia hydrolysis. At this temperature, the vesicles appear to reach equilibrium by 10 min, with the rate of uptake being linear up until 30 s.
   6. Stop transport by adding 0.6 ml of ice-cold Buffer C and store on ice.
   7. To separate the vesicles from the external solution, filter the mixture through a 0.22 μm mixed cellulose ester membrane. To reduce background counts (non-specific adherence of [³H]CMP-Sia to the filters), it helps to first pre-wash the filters with 2 ml ice-cold Buffer C before applying the vesicles. After the vesicles have filtered through, wash the filters three times with 2 ml of ice-cold Buffer C. We typically use a 12-well filter vacuum manifold (MilliporeSigma) that accommodates 25 mm diameter filters.
   8. Place filters in 7 ml scintillation vials, add 5 ml scintillation cocktail, and count in a liquid scintillation counter.
      

Data analysis

1. To determine background counts, use values from protein-containing vesicles mixed with the hot substrate and then immediately quenched by adding 0.6 ml ice-cold Buffer C. Alternatively, protein-free vesicles that undergo the same experimental conditions as protein-containing vesicles can be used. We have found that these approaches yield similar results.
2. Subtract the background counts from the total counts for each sample to determine specific counts and convert to mol/min for substrate transport.
3. Fit data to a Michaelis-Menten model to determine K_m and V_max (see Figure 1 for example data).
   
   
   Figure 1. Functional properties of mouse CST. A. Time course of 30 μM CMP-Sia uptake for purified mouse CST (mCST) reconstituted into lipid vesicles with (black) or without (red) 300 μM CMP inside the vesicles. (Inset), the first minute of CMP-Sia uptake for mCST reconstituted into CMP-filled vesicles is shown to illustrate the linear relationship between uptake and time for up to 30 s, which we used to determine the initial velocity of transport for a given concentration of CMP-Sia. B. The initial velocity of CMP-Sia uptake for mCST reconstituted into CMP-filled vesicles is plotted as a function of substrate concentration to determine the K_m and V_max for transport. In all panels, the symbols show the mean ± standard error of the mean (SEM) for n = 2 (A) or n = 4 (B). Adapted from Ahuja and Whorton, 2019.
   

Recipes

1. Lysis Buffer
   50 mM HEPES, pH 7.5
   150 mM NaCl
   0.01 mg/ml deoxyribonuclease I
   0.7 μg/ml pepstatin
   1 μg/ml leupeptin
   1 μg/ml aprotinin
   1 mM benzamidine
   0.5 mM phenylmethylsulfonyl fluoride
   2% (w/v) DDM (solgrade)
2. Buffer A
   50 mM HEPES, pH 7.5
   150 mM NaCl
   0.1% (w/v) DDM (solgrade)
3. Buffer B (Gel Filtration Buffer)
   25 mM HEPES, pH 7.5
   150 mM NaCl
   0.1% (w/v) DDM (anagrade)
   5 mM DTT
   1 mM EDTA
4. Buffer C (Reconstitution Buffer)
   20 mM HEPES, pH 7.5
   0.1 M KCl
5. AnP Buffer
   50 mM Bis-Tris-Propane-HCl, pH 6.0
   0.1 mM ZnCl₂
   1 mM MgCl₂
   

Acknowledgments

This work was supported by NIH grant R01GM130909, and by Oregon Health and Science University. This protocol has been expanded from previous work (Ahuja and Whorton, 2019).

Competing interests

The authors declare no competing interests.

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