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. Author manuscript; available in PMC: 2021 Apr 12.
Published in final edited form as: Methods Mol Biol. 2020;2159:3–15. doi: 10.1007/978-1-0716-0676-6_1

Isolation and Analysis of Mitochondrial Fission Enzyme DNM1 from Saccharomyces cerevisiae

Nolan W Kennedy 1,#, Lora K Picton 1,#, R Blake Hill 1
PMCID: PMC8040746  NIHMSID: NIHMS1669950  PMID: 32529359

Abstract

Mitochondrial fission, an essential process for mitochondrial and cellular homeostasis, is accomplished by evolutionarily conserved members of the dynamin superfamily of large GTPases. These enzymes couple the hydrolysis of guanosine triphosphate to the mechanical work of membrane remodeling that ultimately leads to membrane scission. The importance of mitochondrial dynamins is exemplified by mutations in the human family member that causes neonatal lethality. In this chapter, we describe the subcloning, purification, and preliminary characterization of the budding yeast mitochondrial dynamin, DNM1, from Saccharomyces cerevisiae, which is the first mitochondrial dynamin isolated from native sources. The yeast-purified enzyme exhibits assembly-stimulated hydrolysis of GTP similar to other fission dynamins, but differs from the enzyme isolated from non-native sources.

Keywords: Mitochondrial dynamics, Mitochondrial fission, Dynamin, Drp1, Membrane scission

1. Introduction

Proteins in the dynamin superfamily are large GTPases thought to be mechanoenzymes that harness the energy of GTP hydrolysis to remodel intracellular membranes [1-9]. The dynamins can be classified as either fission or fusion enzymes [10], and studies with recombinant proteins have revealed that a common feature of the fission dynamins is self-assembly, which stimulates GTP hydrolysis [11-23]. Mutations that impair self-assembly and hydrolysis in the human fission dynamins cause severe phenotypic defects, such as, neurological abnormalities and neonatal lethality [2, 24-26]. For example, in the human mitochondrial fission dynamin-1-like protein (DNM1L) (also known as DRP1, DLP1), a spontaneous heterozygous mutation in DNM1L causes neonatal lethality [27] and was subsequently shown to impair protein localization, assembly, and hydrolysis [28]. In this latter study, as in many others, the mitochondrial fission dynamin was heterologously expressed. This has led to detailed mechanistic and structural insights into this important enzyme superfamily [13, 18, 29-31]. In this chapter, we describe the cloning, purification, and preliminary characterization of the mitochondrial dynamin DNM1 from Saccharomyces cerevisiae that is the first description of this family member not heterologously expressed. GTP hydrolysis of DNM1 purified from native S. cerevisiae compared to that purified from Escherichia coli suggests differences in their activities that support isolation from native sources may be important.

2. Materials

2.1. Yeast Strains and Plasmids

  1. A modified pEG(KT) plasmid for protein overexpression in Saccharomyces cerevisiae [32].

  2. Saccharomyces cerevisiae strain SEY6210 (MATα, leu2-3, 112, ura3-52, his3-Δ200, trp1-Δ901, suc2-Δ9, lys2-801; GAL).

  3. A protease-deficient strain of S. cerevisiae, DDY1810 (MATa, leu2Δ, trp1Δ, ura3-52, prb1-1122, pep4-3, pre1-451) (Shang 2003).

2.2. Chemicals and Reagents

  1. Drop-out Mix Synthetic Medium Minus Uracil w/o Yeast Nitrogen Base (US Biologicals, Cat #D9535).

  2. Yeast Nitrogen Base w/o AA, Carbohydrate & w/o AS (YNB) (US Biologicals, Cat #Y2030).

  3. Carbohydrates: dextrose, galactose, raffinose, maltose.

  4. Tryptophan.

  5. Bacto™ Peptone.

  6. Yeast extract.

  7. 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF).

  8. Phenylmethylsulfonyl fluoride (PMSF).

  9. NaCl.

  10. KCl.

  11. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES).

  12. Piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES).

  13. Phosphoenolpyruvate (PEP).

  14. NaH2PO4.

  15. MgCl2.

  16. Dithiothreitol (DTT).

  17. Dry ice.

  18. Ethanol.

  19. cOmplete, ULTRA, Mini, EDTA-free protease inhibitors (EASYpack from Roche, Cat. No. 05 892 791 001).

  20. Enzymes: DNase (Sigma-Aldrich), Tobacco Etch Virus (TEV) protease (in-house, see Note 1), pyruvate kinase (Sigma-Aldrich), lactate dehydrogenase (Sigma-Aldrich).

  21. Nucleotides: guanosine 5′-triphosphate (GTP), guanosine 5′-[γ-thio]triphosphate (GTP-γ-S) (Sigma-Aldrich).

  22. Mdivi-1 ≥98% (HPLC) (Sigma-Aldrich, M0199-25MG).

  23. Dynasore hydrate ≥98% (HPLC) (Sigma-Aldrich, D7693-25MG).

  24. Bottle-top filter units with 0.2 μm cutoff, 500 mL, for filtering buffer and solutions.

  25. Acrodisc® 25 mm syringe filters, 0.45 μm, low-protein binding.

  26. 50 kDa MWCO dialysis tubing (Spectra/Por®).

2.3. Buffers and Media

All buffers are made at 25 °C using analytical grade reagents or better with distilled and deionized water (>18 MΩ-cm). For protein isolation and purification, all buffers are stored and used at 4 °C. All buffers should be filtered and degassed.

  1. Complete synthetic medium lacking uracil with dextrose (CSM +D−U):
    1. 2 g Drop-out Mix Synthetic Medium.
    2. g Yeast Nitrogen Base (w/o AA, carbohydrate, and AS).
    3. 20 g Glucose (dextrose).
    4. 5 g Ammonium sulfate.
    5. pH 6.0.
    6. Bring to ~900 mL of ddH2O, stir until dissolved, adjust pH to ~6, and bring to 1 L total volume.
    7. Sterile filter (0.2 μm).

  2. Complete synthetic medium lacking uracil and leucine (CSM +R−UL), CSM with 2% raffinose plus 40 mg/L tryptophan.

  3. Induction medium: 100 mL of 20% galactose, 20 g Bacto™ Peptone, and 10 g yeast extract supplemented to each liter of cell growth.

  4. Lysis buffer: 50 mM NaH2PO4, pH 7.4, 500 mM NaCl, 5 mM MgCl2, 2 mM DTT.

  5. Elution buffer: Lysis buffer plus 20 mM maltose, pH 7.4.

  6. Reaction buffer: 125 mM HEPES pH7, 125 mM PIPES pH 7.0, 5 mM MgCl2, 37.5 mM KCl, 5 mM PEP, 100 U/mL pyruvate kinase/lactose dehydrogenase, 1.5 mM NADH, and varying NaCl and 5× GTP as indicated.

  7. 5× assay buffer: 125 mM HEPES, 125 mM PIPES, 37.5 mM KCl, 25 mM MgCl2, 5 mM PEP, 3 mM NADH, 100 U/mL pyruvate kinase/lactose dehydrogenase, (pH 7.0). These are stored as single-use aliquots at −20 °C.

  8. 4 M NaCl, stored at room temperature.

  9. 100× GTP stocks that are stored at various concentrations (0.1 mM to 100 mM) at −20 °C. This range of substrate concentrations is sufficient for determining enzyme kinetics (see Note 2).

  10. DNM1 stocks flash–frozen in a dry ice and ethanol bath and are stored at −80 °C until use. Stocks are not used for multiple freeze-thaw cycles and are not stored for more than a month before use.

2.4. Chromatography Columns

Following columns are used for protein purification and analysis: Dextrin Sepharose® High Performance column (MBTrap, GE Healthcare) and Superose™ 6, 16/60 column (GE Healthcare) (see Note 3).

2.5. Instrument

  1. New Brunswick Shaker incubator.

  2. EmulsiFlex-C3 homogenizer (Avestin).

  3. Avanti J-20XP centrifuge with 6-L rotor JLA-8.1000 and rotor JA-25.50 (Beckman Coulter) or equivalent.

  4. ÄKTA FPLC chromatography system (GE Healthcare).

  5. Molecular Devices FlexStation 3 plate reader.

  6. Eppendorf Multichannel Pipettes (P2, P200) and pipette tips.

3. Methods

3.1. Subcloning of DNM1 in S. cerevisiae

DNM1 was expressed as a fusion construct with E. coli maltosebinding protein (MBP) separated by a Tobacco Etch Virus (TEV) protease-cleavage site (MBP–TEV–DNM1) [33]. A gene encoding MBP–TEV–DNM1 was subcloned into a modified pEG(KT) backbone by homologous recombination in yeast. The pEG (KT) expression vector contains the glutathione S-transferase (GST) gene in the open reading frame [32], which was removed by digesting the vector with SacI followed by agarose gel purification of the pEG (KT) backbone lacking GST gene. The full-length MBP–TEV–DNM1 open reading frame was PCR amplified using MBP–TEV–DNM1 primers as a template [34] and with homology to pEG(KT) at the 5′ and 3′ ends. The MBP–TEV–DNM1 PCR fragment and SacI-digested pEG(KT) were transformed into SEY6210 yeast using the LiAc method [35]. Circularized plasmids were recovered by plasmid rescue, and successful recombination of MBP–TEV–DNM1 into the pEG(KT) backbone was verified by sequencing. Protease-deficient DDY1810 cells (MATa, leu2Δ, trp1Δ, ura3-52, prb1-1122, pep4-3, pre1-451) [36] were transformed with the pEG(KT)-MBP–TEV–DNM1 plasmid using the LiAc method.

3.2. Expression of DNM1 in S. cerevisiae

  1. Inoculate starter cultures of MBP–TEV–DNM1 yeast cells in a 250 mL baffled Erlenmeyer flask containing 50 mL of synthetic complete medium lacking uracil and with 2% glucose for 36–48 h at 30 °C with shaking at 200 rpm until reaching OD600 of ~2.

  2. Dilute the 25 mL starter culture into 1L of complete synthetic medium lacking uracil and leucine, with 2% raffinose and 2× tryptophan. Cells are grown at 26 °C with shaking at 250 rpm until OD600 of ~2 (~24 h).

  3. Induce protein expression by addition of 100 mL of 20% w/v galactose. The medium is also supplemented at this time with 10 g yeast extract and 20 g Bacto™ Tryptone. Before induction, freeze a 500 μL aliquot of the growth for subsequent SDS-PAGE analysis.

  4. Grow cells overnight (typically 16–18 h) at 26 °C with shaking at 250 rpm.

  5. Collect cells by centrifugation at 6000 × g for 20 min at 4 °C.

  6. Wash cell pellet by resuspending in 50 mL of ice-cold sterile water on ice, transfer to two, tared 50 mL conical tubes, and spin at 2500 × g at 4 °C for 10 min. Repeat this once and determine mass of the cell pellet and record cell weight in a laboratory notebook (see Note 4).

  7. Resuspend and pool the cell pellets to a final volume of ~80 mL (or less) in ice-cold, sterile water with 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (0.1 mM final). Dispense as 10 mL aliquots into 50 mL conical tubes, freeze on dry ice or ethanol bath, and store at −80 °C until purification.

3.3. Lysis of S. cerevisiae Cells

All steps need to be at 4 °C to ensure retention of DNM1 activity.

  1. Thaw four 10 mL aliquots of frozen yeast cells on ice, and pool into a 250 mL Erlenmeyer flask using ice-cold lysis buffer to assist in quantitative transfer. Total volume should be ~60–80 mL.

  2. Dissolve one tablet of EDTA-free protease inhibitors using a spatula or glass rod. Add PMSF to a final concentration of 2 mM.

  3. Lyse the cells at 4 °C by mechanical disruption by 5–6 passes through an EmulsiFlex-C3 homogenizer (Avestin) at 21,000 psi (see Note 5). Assess 50–200 μL by microscopy to ensure cell lysis (see Note 6).

  4. Add DNase to 1 μg/mL (from 1000× stock), transfer the cell lysate into precooled centrifuge tubes, and clarify by centrifugation at 25,000 × g for 20 min.

  5. Determine protein concentration of the lysate by Bradford assay.

3.4. Purification of DNM1

All steps need to be at 4 °C including chromatography to ensure retention of DNM1 activity.

  1. Procure appropriate amylose affinity column chromatography. Amylose resin capacity is ~3 mg/mL bed volume (NEB), and for a typical 1 L preparation, a 10 mL column suffices.

  2. Pre-equilibrate the amylose column with 5–10 column volumes of lysis buffer (see Note 7). Record baseline A280 if using a chromatography system with a UV detector.

  3. Filter supernatant of cell lysate using 0.45 μm low-protein binding syringe filter using 80 mL syringe. This step may require more than one syringe filter.

  4. Apply filtrate onto the amylose resin at 1 mL/min.

  5. Wash the column with 10 column volumes of lysis buffer or until A280 returns to baseline value noted above.

  6. Elute with 5–10 column volumes of elution buffer (lysis buffer + 20 mM maltose) or until baseline A280 is reached. Collect 1–3 mL fractions.

  7. Analyze fractions by 10% SDS-PAGE for estimation of protein purity and UV/Vis for A280:A260 ratio. DNM1 typically gives purity at this stage of >85% with little if any nucleotide bound giving an A280:A260 ratio <1 (see Note 8). Pool desired fractions and determine protein concentration.

  8. Liberate DNM1 from MBP by transferring pooled MBP–TEV–DNM1 fractions into 50 kDa MWCO dialysis tubing (DNM1 = 85 kD, MBP = 45 kD, and TEV = 15 kD), adding TEV protease at 1:10 molar ratio (TEV:MBP–TEV–DNM1), and incubating in 1–2 L of ice-cold lysis buffer for 12–24 h with gentle stirring at 4 °C to cleave the N-terminal MBP tag (see Note 9).

  9. Concentrate the DNM1 using centrifugal concentrating devices with 50 kDa MWCO (see Note 10).

  10. Isolate the DNM1 by size-exclusion chromatography using a Superose™ 6 column (GE Healthcare), discarding protein found in the void volume. Collect 1–3 mL fractions.

  11. Assess DNM1 homogeneity of chromatographic fractions by Coomassie-stained 10% SDS-PAGE gel, which is typically found to be at least 90% homogeneous.

  12. Single-use aliquots of the DNM1 are frozen at 2–9 mg/mL using a dry ice or ethanol bath and stored at −80 °C until needed (see Note 11).

3.5. Measurement of GTP Hydrolysis by DNM1

An enzyme-coupled GTPase assay can be used to measure the rate of GTP hydrolysis by DNM1, as described previously [14, 37]. For this assay, the rate of hydrolysis is determined by monitoring NADH depletion at A340 using a plate reader. NADH is depleted as pyruvate is converted to lactate via lactate dehydrogenase (LDH). This reaction is coupled to the hydrolysis of GTP to GDP and phosphate by DNM1 and subsequent conversion of phosphoenolpyruvate (PEP) to pyruvate via pyruvate kinase (PK). This assay provides constant regeneration of GTP, maintaining the desired substrate concentration until depletion of NADH. With a multimodal plate reader, this assay can also be used to measure the degree of light scattering by recording the A450 signal, which correlates with DNM1 assembly [13]. A further adaptation of this approach has been used to assess assembly and disassembly of human DNM1L [38].

  1. Thaw stocks of DNM1 on ice-water bath mixture (see Note 12).

  2. While enzyme is thawing, mix appropriate reagents into a master mix stock so that the final reaction conditions are as follows: 25 mM HEPES, 25 mM PIPES, 7.5 mM KCl, 5 mM MgCl2, 1 mM PEP, 600 μM NADH, and 20 units/mL PK/LDH. NaCl concentration can be varied from 50 mM to 1000 mM (see Note 13).

  3. Once the enzyme stock is thawed, add it to the master mix to the appropriate concentration (usually 1 μM to 20 μM).

  4. Incubate the master mix with enzyme on ice for 15 min (see Note 14).

  5. During the incubation, thaw and aliquot GTP at necessary concentrations into a V-bottom 96-well plate (see Note 15).

  6. After the 15 min incubation, aliquot 148.5 μL of the master mix containing DNM1 into a well of a clear-bottom V-bottom 96-well plate. Repeat as necessary (see Note 16).

  7. To start the reaction, pipette 1.5 μL of the GTP stocks into the appropriate reaction wells using an Eppendorf P2 Multichannel Pipette (see Note 17).

  8. Mix the reactions by pipetting up and down with an Eppendorf P200 Multichannel Pipette (see Note 18).

  9. Place the 96-well plate into the plate reader, and set it to measure the absorbance at 340 nm and 450 nm for 1 h at the shortest-allowable intervals.

  10. Once measurements are complete, export the data as a plain text (.txt or .csv) file and open in Microsoft Excel or other data analysis software.

  11. Plot the absorbance data at 340 nm as a line graph, and determine the rate of hydrolysis for each reaction (see Note 19).

  12. Plot the absorbance data at 450 nm as a line graph, and determine the rate of assembly for each reaction. This is done by fitting the linear range of the data to a straight line over the largest possible window.

3.6. Preliminary Results

Previous studies on DNM1 used purified enzyme from either bacteria [34] or insect cells [13, 14]. In this study, we overexpressed DNM1 in native Saccharomyces cerevisiae as an N-terminal fusion construct with a TEV protease-cleavable MBP in protease-deficient strain for purification. Side-by-side GTP hydrolysis experiments confirmed that DNM1 purified from bacteria differed in enzyme properties from yeast-purified enzyme (Fig. 1). Substrate kinetics measurements at various NaCl concentrations indicate that bacterially expressed DNM1 has a higher affinity for substrate than the yeast-expressed enzyme (K0.5 of 110 ± 8 μM for bacterial versus 685 ± 47 μM for yeast expressed), but a lower Vmax (4.0 ± 0.1 nmol/min for bacterial versus 5.2 ± 0.1 nmol/min for yeast expressed). The yeast-expressed enzyme also appears to have weaker substrate affinity and activity than DNM1 derived from insect cells [14]. The basis for these differences might derive from differences in posttranslational modifications, which are known to affect activity of other dynamins [39, 40].

Fig. 1.

Fig. 1

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GTP hydrolysis by DNM1 isolated from E. coli (filled circles) and S. cerevisiae (open circles). 2 μM DNM1 was incubated with the indicated concentrations of GTP, and hydrolysis measured via NADH depletion using a coupled, regenerative assay that allows for continuous supply of substrate (see text). The linear region of the NADH depletion curve was fit to a straight line and converted to initial velocity, Vo, and plotted versus GTP. The data were fit to the generalized Michaelis–Menten equation: Vo = (Vmax*[S])/(K0.5 + [S]). For the bacterially expressed DNM1, K0.5 = 110 ± 8 μM and Vmax = 4.0 ± 0.1 nmol/min. For the yeast-expressed DNM1, KM0.5 = 685 ± 47 μM and Vmax = 5.2 ± 0.1 nmol/min. [NaCl] = 233 mM

DNM1 isolated from yeast also shows the similar salt-dependent assembly compared to other dynamins. Dynamin-related proteins are known for increasing GTP hydrolysis activity as a function of assembly, which has been stimulated by increasing concentrations of protein or decreasing salt concentrations. Increasing NaCl concentration decreased GTP hydrolysis of DNM1 (Fig. 2). At the highest salt concentration (1 M), the activity was 2.7 nmol/min and increased to 16 nmol/min at the lowest NaCl concentration tested (57 mM). These experiments indicate that DNM1 isolated from yeast is assembly competent with characteristics similar to other dynamins [14, 23, 41, 42]. Finally, the yeast-derived enzyme is also inhibited by the pharmacological inhibitors, Dynasore hydrate (not shown), and Mdivi-1 [43].

Fig. 2.

Fig. 2

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NaCl dependence on GTP hydrolysis by 10 μM DNM1 isolated from yeast. Dnm1 activity is dependent on NaCl concentration. Initial velocity Vo ± SE (n = 3) is plotted versus [NaCl]

Acknowledgement

The reagents pEG(KT), SEY6210, and DDY1810 were kind gifts from B. Wendland. This work was supported by the National Institutes of Health grant R01GM067180.

Footnotes

4 Notes

1

We express and purify the catalytic domain variant (S219V) from the Tobacco Etch Virus protease in E. coli as developed for protein expression by Waugh and coworkers [32]. This variant resists autoinactivation and is approximately twofold more active than wild type.

2

We typically make up stock solutions fresh or use within 1 month.

3

We have also used amylose resin from NEB with similar success.

4

Greater than 50% increases or decreases in cell pellet mass can signal problems with the growth that can be diagnosed by SDS-PAGE of the whole cell lysate samples taken at induction and harvest.

5

This is the maximum pressure observed during the power stroke.

6

We have not extensively investigated other common methods for yeast cell lysis that include french press, glass beads, and planetary ball mill grinders, with the latter being popularized by Dr. David Rout’s laboratory for yielding intact protein complexes.

7

Ensure that the resin is cleaned before use, especially in multiuse laboratories. For amylose resins, we wash with 5 column volumes of 20 mM maltose, 5 column volumes of water, 3 column volumes of 0.1% SDS, 10 column volumes of water, 5 column volumes of 20% EtOH.

8

DNM1 is typically purified without nucleotide bound unlike small GTPases. If nucleotide is present, procedures can generate nucleotide-free GTPase [44].

9

A 1 or 2 L graduated cylinder works well for this purpose.

10

DNM1 self-assembly can occlude the pores in centrifugal devices. This can be minimized by using 500 mM NaCl and <10 mg/mL protein concentrations that favor disassembly. The next purification step is size-exclusion chromatography, in which a minimal volume is desirable, so we typically concentrate down to 1 mL or 2 mL max.

11

The final concentration depends on the NaCl concentration and other considerations. Some mutants affect assembly of DNM1 and can be concentrated to >20 mg/mL. For DNM1 concentration determination, the protein is unfolded with 6 M GdnHCl, and A280 measured. The protein concentration is calculated from Beer’s law using the theoretical extinction coefficient (38,280 M−1 cm−1).

12

We find that rapid thawing at room temperature leads to enzyme aggregation. Once the enzyme is thawed, it degrades over time and can aggregate within 6 h. If aggregation is apparent, we discard that stock.

13

Consistent with other dynamins [11, 42], we find that low NaCl (50 mM) leads to the highest levels of hydrolysis or assembly, while high NaCl (1000 mM) leads to the lowest levels of hydrolysis or assembly (Fig. 2).

14

The rationale of the 15-min incubation is that some variation exists in how long it takes to set up the GTP stocks or salt titration plate, depending on what conditions are being tested. Regardless, this never takes more than 15 min. GTP-γ-S is used as a control for contaminating NTPases that might hydrolyze GTP.

15

V-bottom 96-well plate plates are used for rapid transfer of the substrate into the reaction plate to start the reaction. Use of the 96-well plate allows transfer with an Eppendorf Multichannel Pipette. We find that a minimum of 5 μL is necessary to ensure accurate pipetting from the V-bottom plate into the reaction plate.

16

Pipette carefully to avoid bubble formation, which can interfere with absorbance measurements. If present, use a narrow gauge needle or pipette tip to disrupt them.

17

Using multichannel pipettes is essential but can result in inaccuracies of one or more channels, which needs to be visually checked before dispensing. When this occurs, the solution is placed back into the reservoir, and all tips discarded. Inaccuracies can be minimized by ensuring a good seal between the pipette tip and pipettor.

18

Make sure to do this step as quickly as possible, as reactions under high enzyme, assembly, and/or substrate conditions can rapidly deplete NADH.

19

This is done by fitting the linear range of the data to a straight line over the largest possible window. Convert slope (ΔA340/s) to activity in nmol/min with the following equation: activity = (ΔA340/s *60 s/min) * (0.001 L) * (19 nmol/mol)/(6220 M−1 cm−1)/(0.41 cm). A buffer control for each GTP concentration is also collected, and a background hydrolysis rate calculated and subtracted from the activity.

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