Saccharomyces cerevisiae Models of Alzheimer's Disease to Screen Genes, Mutations, and Chemicals Affecting Amyloid Beta Production by γ-Secretase

Summary

Here, we describe a yeast system reconstituting human γ-secretase. This system allows for the identification and study of mutations affecting activity and for screening of γ-secretase inhibitors (GSIs). Utilizing the loss of function properties of familial Alzheimer's disease mutants, it is possible to screen for γ-secretase modulators (GSMs).

Abstract

γ-Secretase is a protease complex embedded in the cell membrane, consisting of the catalytic presenilin subunits (PS1 or PS2) and three additional co-factors: nicastrin, Aph-1, and Pen2. It cleaves the transmembrane domains of type-I transmembrane proteins, such as the amyloid precursor protein (APP) and Notch. The cleavage of APP generates the amyloid β peptides (Aβ), which accumulate in patients with Alzheimer's disease. Despite significant research, the exact mechanism of this unique proteolysis, which occurs within the lipid bilayers, is still not fully understood. To study the enzymatic properties of γ-secretase, we have established a yeast reporter system using artificial γ-secretase substrates containing APP or Notch fragments fused to the transcriptional activator Gal4. The γ-secretase activity was evaluated by transcriptional activation of reporter genes upon Gal4 release from the membrane-bound substrates, as assessed by the growth of yeast or β-galactosidase activity. Furthermore, we have developed an in vitro assay to identify the different forms of Aβ produced from yeast microsomes. These yeast models provide a platform to screen mutations, genes, and compounds that affect γ-secretase function. By studying the loss of function properties of PS1 familial Alzheimer's disease (FAD) mutants, it is possible to screen for FAD suppressor mutations and γ-secretase modulators (GSMs), in addition to γ-secretase inhibitors (GSIs). In this report, we describe the genetic and biochemical methods used to study γ-secretase activity in the yeast system with the essential steps of the protocol for the video.

Introduction

γ-Secretase is responsible for the intramembrane cleavage of over 150 type-I transmembrane proteins, including the amyloid precursor protein (APP) and Notch¹. The significance of intramembrane cleavage includes not only protein turnover but also signal transduction through the generated fragments. For example, the processing of APP produces small amyloid β peptides (Aβ). Although Aβ should be removed as a degradation product, in Alzheimer's disease, it accumulates in the brain to form senile plaques². On the other hand, the processing of Notch is essential for NOTCH signaling³, which is crucial for development. γ-Secretase consists of four transmembrane proteins: presenilin (PS1 or PS2), nicastrin (NCT), anterior pharynx-1 (Aph-1a or 1b), and presenilin enhancer 2 (Pen2)⁴. These proteins have a total of 19 transmembrane domains: there are nine in PS, seven in Aph1, two in Pen2, and one in NCT. PS serves as the catalytic subunit and contains two aspartic acid residues located in transmembrane domains TM6 and TM7⁵. During maturation of the complex, PS undergoes endoproteolytic cleavage within its hydrophilic loop HL6, leading to the formation of amino-terminal and carboxyl-terminal fragments⁴. Mutations associated with Familial Alzheimer's disease (FAD) are found in both the APP and PS genes². These pathogenic missense mutations adversely affect γ-secretase activity, reducing its ability to effectively process APP. This leads to increased production of the aggregation-prone Aβ42 forms rather than the common Aβ40⁶. The exact mechanisms by which these mutations disrupt the cleavage remain unclear. γ-Secretase modulators (GSMs), which can alter the ratio of Aβ42 to other Aβ forms, show promise as potential therapeutic agents for the treatment of Alzheimer's disease by lowering Aβ42 levels⁷.

The budding yeast Saccharomyces cerevisiae has long served as a model organism to investigate key functions in eukaryotic cells, such as cell division, protein transport, and autophagy. Researchers have employed functional complementation to study human homologs, including cell division cycle (CDC), secretion (SEC), and autophagy-related (ATG) genes. Transforming yeast mutants with human genes allows researchers to analyze cellular processes from a functional perspective. Of the five known families of intramembrane proteases, yeast possesses only three: signal peptide peptidase (Ypf1), rhomboids (Pcp1 and Rbd2), and Ras-converting enzyme (Rce1). However, there are no homologs of γ-secretase or site-2 protease in yeast⁸, making the complementation approach not suitable. Despite the absence of yeast counterparts for these proteases, S. cerevisiae remains valuable for exploring human proteins. Techniques such as yeast one-hybrid, two-hybrid, and three-hybrid assays, which use the yeast transcriptional activator Gal4, are employed to detect interactions between DNA, transcription factors, proteins, or RNA⁹. We have modified the Gal4 system to monitor intramembrane proteolysis mediated by γ-secretase.

We have successfully reconstituted the γ-secretase cleavage reaction in yeast by introducing human γ-secretase along with artificial substrates fused to Gal4 (Figure 1)¹⁰. The cleavage reaction was tracked through the Gal reporter system, either by monitoring yeast growth or measuring β-galactosidase activity. Additionally, we have identified various Aβ forms (Aβ38, Aβ40, Aβ42, Aβ43, and Aβ45) released in yeast microsomes by immunoblotting (Figure 1)¹¹.

When FAD-related PS1 or APP mutants were introduced into this system, variable degrees of loss-of-function activity were observed¹². We conducted a growth-based screen to identify suppressor mutations that restore the impaired function of these FAD mutants (Figure 2). These suppressors not only rescued the defect but also independently enhanced the trimming activity of γ-secretase. Specifically, they reduced the levels of longer forms of Aβ, particularly Aβ42, in a manner similar to Aβ42-lowering γ-secretase modulators (GSMs). The yeast reconstitution system allows screening of mutations, genes, and compounds that regulate activity, including γ-secretase inhibitor (GSI) and modulator (GSM)¹³.

The goal of this protocol is to accomplish two main objectives: i) to establish a yeast-based γ-secretase screening system and ii) to provide an example of reconstitution techniques for human enzymatic reactions in yeast, emphasizing the essence of reconstitution methods. We outline the methods in three key procedures with the following essential steps: 1) reconstituting γ-secretase in yeast, 2) performing an in vitro γ-secretase assay using yeast microsomes, and 3-5) screening γ-secretase mutations and chemical regulators in yeast.

Protocol

1. Measurement of γ-secretase activity using a yeast reporter system

1. Transform the plasmids (Table 1) into the yeast PJ-69-4A strain (Table 2) using the standard lithium acetate method to express γ-secretase and its substrate¹⁴. To reconstitute the cleavage reaction, transform three plasmids: two for γ-secretase and one for the substrate. Select transformants on SC agar medium lacking Leu, Trp, and Ura (SC-LWU).
2. To assess substrate cleavage by growth, streak yeast strains onto SC-LWU medium also lacking histidine and adenine (SC-LWHUAde) and incubate at 30 °C for 3 days.
3. Use colony size to assess the expression levels of HIS3 and ADE2. Evaluate colony size as follows: +++ represents wild-type growth with colonies larger than 1 mm, - represents no growth, and ++ or + represents partial growth with colonies larger than 0.5 mm or smaller than 0.5 mm, respectively. Expression of these genes is an indirect measurement of γ-secretase activity (Figure 1).
4. To assess substrate cleavage by β-galactosidase activity, i.e., the expression level of lacZ (Figure 1), grow strains in SC-LWU medium at 30 °C until the culture reaches a concentration of 1 x 10⁷ cells/mL, corresponding to OD₆₀₀ of 0.8-1.0.
   1. Harvest cells from a 1.5 mL culture by centrifugation in 1.5 mL tube at 21,100 x g for 3 min at 4 °C, wash with 100 µL of ice-cold water and resuspend in 30 µL of D-buffer (20 mM Tris/Cl (pH 8.0), 10 mM MgCl₂, 50 mM KCl, 1 mM EDTA, 5% glycerol) with 1 mM dithiothreitol (DTT) and protease inhibitor cocktails PIs/water (1000x solution: 1 M p-aminobenzamidine (pABA), 1 mg/mL antipain, 1 mg/mL aprotinin, 1 mg/mL leupeptin) and PIs/DMSO (1000x solution: 1 M phenylmethylsulfonyl fluoride (PMSF), 0.1 M N-p-Tosl-L-phenylalanine chloromethyl ketone (TPCK), 1 mg/mL Chymostatin, 1 mg/mL Pepstatin).
   2. Add an acid-washed glass bead just below the surface of the solution and lyse the cells by vortexing the mixture 10x at maximum speed for 1 min while placing it on ice at 1 min intervals.
   3. Centrifuge lysed cells at 21,100 x g for 10 min at 4 °C and collect the supernatant as crude lysate.
   4. Prepare the reaction mixture with 5-10 µL of crude lysate and 0.4 mL of Z-buffer (40 mM NaH₂PO₄, 60 mM Na₂HPO₄, 10 mM KCl, 1 mM MgSO₄). Start the reaction by adding 80 µL of 0.4% o-nitrophenyl-β-D-galactopyranoside (ONPG) solution, then incubate at 37 °C until the solution turns pale-yellow due to the formation of nitrophenol.
   5. Stop the reaction by adding 0.2 mL of 1 M Na₂CO₃ solution and measure the absorbance at 420 nm to calculate β-galactosidase activity.
   6. Quantify the protein concentrations in crude lysates by using a protein assay kit, based on the method of Bradford using bovine serum albumin (BSA) as a standard. Prepare the dye reagent by diluting 1 part of the dye stock with 4 parts of Double-distilled deionized (DDI) water.
   7. Mix 1-5 µL of lysate or BSA solution with 100 µL of diluted dye in a microtiter plate well. Incubate at room temperature for 5 min and measure absorbance at 595 nm. Determine the protein concentration from the BSA standard curve.
      β-galactosidase unit = (1,000 x A₄₂₀) / (t x c x V)
      ​where t = time of incubation, c = protein concentration (mg/mL), V = volume of lysate in the assay (mL). One unit of β-galactosidase is defined as the amount of enzyme required to hydrolyze 1 µmol of ONPG to produce o-nitrophenol and D-galactose per min per mg of protein.
5. To confirm the expression of γ-secretase subunits and the substrates, grow the strains in SC-LWU medium at 30 °C until the culture reaches OD₆₀₀ 0.8-1.0.
   1. Harvest cells from a 10 mL culture by centrifugation in a 50 mL tube at 2,100 x g for 10 min at 4 °C, wash with 1 mL of ice-cold water followed by centrifugation in a 1.5 mL tube at 21,100 x g for 3 min at 4 °C, and resuspend in 100 µL of B88-buffer (20 mM Hepes/KOH (pH 6.8), 250 mM Sorbitol, 150 mM KOAc, 5 mM Mg(OAc)₂) with 1 mM DTT and the protease inhibitor cocktails, PIs/water and PIs/DMSO.
   2. Add glass beads just below the surface of the solution and lyse the cell by vortexing 10x at maximum speed for 1 min while keeping the mixture on ice for 1 min intervals.
   3. Centrifuge lysates at 845 x g for 2 min at 4 °C and collect the supernatant as total lysate which contains the membrane fraction.
   4. Mix 100 µL of total lysate with 96 mg of urea and 40 µL of 6x SDS-sample buffer (0.4 M Tris/Cl (pH 6.8), 12% SDS, 0.6M DTT, 0.05% Bromophenol Blue, 50% glycerol) and incubate at 65 °C for 10 min. The addition of urea is necessary to prevent the aggregation of presenilin.
   5. Analyze the expression of γ-secretase and its substrates by running samples on 12.5% polyacrylamide Tris/Glycine gels and immunoblotting.
   6. Detect γ-secretase and its substrates by antibodies against presenilin, nicastrin, Aph1, Gal4, FLAG, HA, or Pgk1 (loading control), and the horseradish peroxidase (HRP)-conjugated secondary antibody against mouse IgG (for FLAG, HA, and Pgk1), rabbit IgG (for presenilin, Aph1, and Gal4), or guinea pig IgG (for nicastrin).
   7. For chemiluminescent detection, mix solution A (50 mM Tris/Cl (pH 8.5), 1.25 mM luminol, 0.02 mM p-coumaric acid) with solution B (50 mM Tris/Cl (pH 8.5), 0.009% H₂O₂) immediately before use. Incubate the blot in a sufficient volume of the mixture for 1 min. The required volume depends on the blot size; for a 10 cm x 6 cm blot, 4 mL is used. Develop the blot and quantify the signal intensity using the luminescent image analyzer.

2. Measurement of Aβ production by in vitro microsome assay

1. Transform APP C55, APP C99, or γ-secretase plasmids (Table 1) into yeast strain PJ-69-4A pep4Δ as described in step 1.1. to express the γ-secretase and its substrate separately (Table 2; γ311, γ312, and γ313 strains)¹⁴. Select the C55 and C99 strains on the SC-U agar medium and the γ-secretase strain on the SC-LW agar medium.
2. Isolate microsomes from yeast strains expressing APP C55, APP C99, or γ-secretase according to the standard protocol¹¹. Separate microsomes by sucrose density gradient centrifugation and recover at 1.2 M-1.5 M sucrose interface using a disposable plastic pipette. Determine the protein concentration in microsomes by measuring absorbance at 260 nm after solubilizing them in 0.2% SDS.
3. Prepare the reaction mixture with two microsomes, i.e., APPC55 or γ-secretase, containing 40 µg of protein each and γ-buffer (50 mM PIPES/KOH (pH 7.0), 250 mM Sucrose, 1 mM EGTA) in a final volume of 12.5 µL. Solubilize by mixing with an equal volume (12.5 µL) of 2% CHAPSO in γ-buffer and incubate in 1% CHAPSO for 1 h.
4. To start the reaction, dilute the mixtures four-fold to a final volume of 100 µL using γ-buffer containing 1x protease inhibitor (PI) mix: 50 µM diisopropyl fluorophosphate (DIFP), 50 µM PMSF, 0.1 µg/mL N^α-p-tosyl-L-lysine chloromethyl ketone (TLCK), 0.1 µg/mL antipain, 0.1 µg/mL leupeptin, 0.1 mM EGTA, 1 mM thiolphan, and 5 mM phenanthroline (PNT) to 0.25% CHAPSO with 400 µg/mL proteins. Incubate the reaction mixture at 37 °C for 24 h. If necessary, add 0.1% phosphatidylcholine (PC), 10 µM L685,458, or DAPT (γ-secretase inhibitors).
5. Stop the reaction by adding 500 µL of chloroform/methanol (2:1) and incubate for 1 h with vortexing intermittently. Add 900 µL of methanol, mix, and centrifuge at 21,100 x g for 15 min to collect the Aβ peptides in the pellet.
6. Wash the pellet with 500 µL of chloroform/methanol/water (1:2:0.8), allow it to air-dry, suspend it in 40 µL of SDS-sample buffer, and boil the mixture for 10 min. Analyze the Aβ production by running samples on a 16.5% Tris/Tricine polyacrylamide gel. To identify the Aβ forms, use a 10% Tris/Tricine polyacrylamide gel with 8 M urea (pH 8.45).
7. Detect Aβ by immunoblotting using the anti-antibody 82E1 specific to the Asp-1 site of human Aβ and the HRP-conjugated secondary antibody against mouse IgG. For chemiluminescent detection, mix equal amounts of solution A and B immediately before use as described in step 1.5.7, and incubate the blot in a sufficient volume of the mixture for 1 min. The required volume depends on the blot size; for a 10 cm x 6 cm blot, 4 mL is used.
8. Develop the blot and quantify signal intensity using the luminescent image analyzer.

3. Screening for γ-secretase or APP mutations with modulatory effect

1. To generate random mutations by error-prone PCR, design primers to incorporate 40 bp overlapping regions of the gene of interest at the 5' end of each primer.
2. Prepare 50 µL of error-prone PCR reaction in 1x Taq buffer with 50 ng of template DNA, 0.2 mM dGTP, 1 mM dATP/dTTP/dCTP, 3 mM MgCl₂, 0.5 mM MnCl₂, 1.25 U Taq polymerase, and 400 nM primers. Use the following PCR protocol: 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 120 s, ending with 72 °C for 10 min. This setting produces an approximate mutation rate of 1.9 mutations per kilobase¹⁰.
3. Transform the mutagenized DNA fragments (4 µg) with linearized plasmid DNA (4 µg) into PJ69-4A yeast strain containing FAD APP or PS1 mutations¹⁴. The 40 bp overlap in primers facilitates homologous recombination between the fragments in yeast, leading to the reconstruction of the plasmid. Select transformants on SC-LWU plates as described in step 1.1.
4. Count transformants on SC-LWU plates and replicate them onto SC-LWHUAde plates using velvet. To replicate, press the transformant plate onto a velvet-covered cylindrical block, then press a fresh SC-LWHUAde plate onto the velvet to transfer the colonies. Strains expressing APP or PS1 FAD mutants grow poorly on SC-LWHUAde, while those with modulatory mutations exhibit robust growth and form colonies.
5. To recover plasmid DNA from positive clones, culture the strains in 3 mL of SC-LWU medium overnight at 30 °C until OD₆₀₀ >1.0. Centrifuge the cells in a 15 mL tube at 2,100 x g for 10 min at 4 °C, wash with 0.8 mL ice-cold water followed by centrifugation in a 1.5 mL tube at 21,100 x g for 3 min at 4 °C, and then resuspend in 0.3 mL of lysis solution (10 mM Tris/Cl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 2% Triton X-100, 1% SDS).
6. Lyse cells by adding 0.3 mL of phenol:chloroform:isoamyl alcohol (25:24:1) and 0.3 g glass beads. Vortex at maximum speed for 10 min, then centrifuge at 21,100 x g for 10 min at room temperature.
7. After centrifugation, the sample separates into an upper aqueous phase and a lower phenol phase. Recover the aqueous phase and mix with 30 µL of 3 M sodium acetate and 700 µL of 100% ethanol. Avoid taking materials on the interphase.
8. Centrifuge the solution at 21,100 x g for 10 min at 4 °C, wash the pellet with 70% ethanol, air-dry, and resuspend in 15 µL of TE buffer.
9. Since the concentration of plasmid DNA is typically too low for further analysis, it is necessary to amplify it in E. coli. Transform the plasmid DNA into E. coli DH5α by high-efficiency transformation methods¹⁵ and purify for further analysis. Identify mutations by DNA sequencing using the Sanger method¹⁶.
10. This method is applicable to mutagenized plasmids prepared by different methods (as in steps 3.1 and 3.2). If targeting specific protein regions, design inverse PCR primers with random nucleotides. After PCR and ligation to circularize the plasmid DNA, a library of mutagenized plasmids is obtained. Introduce specific mutations using the site-specific mutagenesis kit.

4. Screening for γ-secretase inhibitor (GSI) in yeast

1. Use the high-throughput chemical screening of GSI developed based on yeast growth or β-galactosidase assay, as described below.
2. Conduct GSI screening with yeast strains, γ124, γ126, and γ190 (Table 2). Select GSI candidates as compounds that reduce the growth or β-galactosidase activity of wild-type γ-secretase on APP (γ124) and/or Notch (γ126) (Table 3).
3. Select substrate-specific APP-sparing or Notch-sparing GSIs. Exclude toxic compounds by counter-screening with the soluble Gal4 expression. GSI candidates should not affect the growth or β-galactosidase activity of the Gal4 control strain (γ190) (Table 3).
4. For the growth assay, grow strains in SC-LWHUAde medium containing potential GSIs in 384-well plates from OD₆₀₀ of 0.03. Incubate overnight at 30 °C without shaking. Monitor growth after 24 h by OD₆₀₀.
5. For β-galactosidase assays in a 384-well format, grow strains to a concentration of 1 x 10⁷ cells/mL in SC-LWU medium containing potential GSIs. Lyse the cells using the Beta-Glo assay system and measure β-galactosidase activity using 6-o-β-galactopyranosyl-luciferin as a substrate according to the manufacturer's protocol.
6. For GSI candidates, measure the Aβ production by in vitro microsome assay to confirm the inhibitory effect.

5. Screening for γ-secretase modulator (GSM) in yeast

1. Use the high-throughput chemical screening of GSM, which has been developed based on yeast growth or β-galactosidase assay, as described below.
2. Conduct GSM screening with yeast strains, γ106, γ112, and γ5 (Table 2). Select GSM candidates as compounds that enhance the growth or β-galactosidase activity of FAD PS1 (γ106 and γ112) or FAD APP mutant strains (Table 4). Exclude non-specific reporter activator chemicals by counter-screening with the strain without γ secretase catalytic subunit. GSM candidates should not affect the growth or β-galactosidase activity of the control strain without PS1 (γ5) (Table 4).
3. For the growth assay, grow strains in SC-LWHUAde medium containing potential GSMs in 384-well plates from OD₆₀₀ of 0.03. Incubate overnight at 30 °C without shaking. Monitor growth after 48 h by OD₆₀₀.
4. For β-galactosidase assays in a 384-well format, grow strains to a concentration of 1 x 10⁷ cells/mL in SC-LWU medium containing potential GSMs. Lyse the cells using the Beta-Glo assay system and measure β-galactosidase activity using 6-o-β-galactopyranosyl-luciferin as a substrate according to the manufacturer's protocol.
5. For GSM candidates, measure the Aβ production by in vitro microsome assay to confirm the modulatory effect, i.e., the reduction of toxic Aβ forms Aβ42 and Aβ43.

Results

Using the techniques described so far, we have analyzed genes, mutations, chemicals, and peptides as factors that affect the activity of γ-secretase^{10,11,12,13,17,18,19}. Growth-based screening of genes and mutations allows testing of a large number of clones (100,000-1,000,000). The large si...

Discussion

Critical steps in the protocol and troubleshooting
The reconstitution of γ-secretase in yeast using genetic and biochemical methods is relatively straightforward, but there are some critical steps to ensure success.

During yeast transformation (steps 1.1, 2.1, and 3.3), multiple plasmids should be introduced: two for the γ-secretase components and one for the substrates. Sequential transformation (one at a time) can also be performed, but it is time-consuming. ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Antibodies
anti Amyloidβ (N) (82E1) antibody	IBL	Cat# 10323
anti Aph1a, 245-265 (C terminus) antibody	BioLegend	Cat# 823101
anti FLAG (M2) antibody	Sigma	Cat# F3165
anti Gal4 antibody	Sigma	Cat# G9293
anti HA (12CA5) antibody	Sigma	Cat# 11 583 816 001
anti Nicastrin antibody	Chemicon	Cat# AB5890
anti PGK1 (22C5D8) antibody	abcam	Cat# ab197960
anti PS1 N-terminus (G1Nr) or loop (GIL) antibodies	Takasugi et.al., J. Biol. Chem., 277, 50198-50205
anti rabbit  IgG, HRP-linked antibody	Cell Signaling	Cat# 7074
anti-guinea pig IgG-HRP	Santa Cruz Biotechnology	Cat# sc-2438
anti-mouse IgG, HRP-linked antibody	Cell Signaling	Cat# 7076
Bacterial and yeast strains
E. coli DH5α	Invitrogen	Cat# 18265017
S. cerevisiae PJ-69-4A	James P. et. al., Genetics 144:1425-1436 (1996)
Chemicals, peptides, recombinant proteins
(3,5-Difluorophenylacetyl)-Ala-Phg-OBut (DAPT)	Peptide Institute	Cat# 3219-v
Amyloid β-protein (Human, 1-38)	Peptide Institute	Cat# 4484-v
Amyloid β-protein (Human, 1-40)	Peptide Institute	Cat# 4307-v
Amyloid β-protein (Human, 1-42)	Peptide Institute	Cat# 4349-v
Amyloid β-protein (Human, 1-43)	Peptide Institute	Cat# 4370-v
antipain, dihydrochloride	Peptide Institute	Cat# 4062
aprotinin	Sigma	Cat# A1153
CHAPSO	Dojindo	Cat# 343-05163
Chymostatin	Sigma	Cat# C7268
Diisopropyl fluorophosphate (DIFP)	Wako chemical	Cat# 049-18921
L685,458	Peptide Institute	Cat# 4394-v
leupeptin	Peptide Institute	Cat# 4041
N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK)	Sigma	Cat# T4376
Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK)	Sigma	Cat# T7254
o-nitrophenyl-β-D-galactopyranoside (ONPG)	Sigma	Cat# N1127
p-aminobenzamidine (pABA), dihydrochloride	Sigma	Cat# 857661
Pepstatin A	Sigma	Cat# P5318
Phenanthroline (PNT)	Sigma	Cat# P9375
phenylmethylsulfonyl fluoride (PMSF)	Sigma	Cat# P7626
phosphatidyl choline (PC), egg yolk, Type XVI-E	Sigma	Cat# P3556
Taq DNA polymerase	Takara	Cat# R001A
thiolphan	Sigma	Cat# T6031
Commercial assay kits, other materials, and Instruments
Beta-Glo Assay System	Promega	Cat# E4720
Bio-Rad Protein Assay kit	Bio-Rad	Cat# 5000001
Glass beads, acid-washed (425-600 μm)	Sigma	Cat# G8772
ImageQuant LAS4000	GE Healthcare	ImageQuant LAS4000
QuikChange Site-Directed Mutagenesis Kit	Agilent	Cat# 200518