Functionalizing a hemagglutinin epitope tag for induced mitochondrial protein degradation in saccharomyces cerevisiae

Mitochondria are organelles known for their role in many critical cellular processes including the production of metabolic energy in eukaryotic cells. In order to produce this energy, mitochondria continually transport metabolites across the impermeable mitochondrial inner membrane. The transport of metabolites into and out of mitochondria is controlled by a family of proteins called mitochondrial metabolite carriers. One carrier known as the mitochondrial oxaloacetate carrier (Oac1) in the Baker's yeast Saccharomyces cerevisiae was investigated in this thesis. It was demonstrated that the Oac1 protein remains functional when fused with small epitope tags at its N- or C-terminus. However, we noticed that when Oac1 is C-terminally fused to a hemagglutinin (HA) epitope tag it becomes destabilized and is rapidly degraded. By HA-tagging several other mitochondrial proteins and truncated forms of Oac1, it was shown that the HA-epitope tag generally destabilizes proteins localized within mitochondria. Here, we attempted to functionalize this HA-epitope tag by combining this destabilizing element with an RNA element that can be induced to form a translation-inhibiting RNA aptamer upon binding the antibiotic tetracycline (TC). By combining these two elements, we show that inhibiting Oac1-HA translation by TC addition leads to the rapid and specific turnover of the Oac1-HA protein. Thus, pairing a yeast-specific HA-epitope tag to a regulatable TC-binding RNA aptamer creates a tool that can be used to induce specific protein degradation in mitochondria. Future experiments may explore the induced degradation of various mitochondrial proteins in order to study their function and regulation.

FUNCTIONALIZING A HEMAGGLUTININ EPITOPE TAG FOR INDUCED MITOCHONDRIAL PROTEIN DEGRADATION IN SACCHAROMYCES CEREVISIAE by Bridget E. Ward A Senior Honors Thesis Submitted to the Faculty of The University of Utah In Partial Fulfillment of the Requirements for the Honors Degree in Bachelor of Science In Biochemistry Approved: ______________________________ Zachary N. Wilson, PhD Thesis Faculty Supervisor _____________________________ Matthew S. Sigman, PhD Chair, Department of Chemistry _______________________________ Thomas G. Richmond, PhD Honors Faculty Advisor _____________________________ Sylvia D. Torti, PhD Dean, Honors College December 2022 Copyright © 2022 All Rights Reserved ABSTRACT Mitochondria are organelles known for their role in many critical cellular processes including the production of metabolic energy in eukaryotic cells. In order to produce this energy, mitochondria continually transport metabolites across the impermeable mitochondrial inner membrane. The transport of metabolites into and out of mitochondria is controlled by a family of proteins called mitochondrial metabolite carriers. One carrier known as the mitochondrial oxaloacetate carrier (Oac1) in the Baker’s yeast Saccharomyces cerevisiae was investigated in this thesis. It was demonstrated that the Oac1 protein remains functional when fused with small epitope tags at its N- or C-terminus. However, we noticed that when Oac1 is C-terminally fused to a hemagglutinin (HA) epitope tag it becomes destabilized and is rapidly degraded. By HA-tagging several other mitochondrial proteins and truncated forms of Oac1, it was shown that the HA-epitope tag generally destabilizes proteins localized within mitochondria. Here, we attempted to functionalize this HA-epitope tag by combining this destabilizing element with an RNA element that can be induced to form a translationinhibiting RNA aptamer upon binding the antibiotic tetracycline (TC). By combining these two elements, we show that inhibiting Oac1-HA translation by TC addition leads to the rapid and specific turnover of the Oac1-HA protein. Thus, pairing a yeast-specific HA-epitope tag to a regulatable TC-binding RNA aptamer creates a tool that can be used to induce specific protein degradation in mitochondria. Future experiments may explore the induced degradation of various mitochondrial proteins in order to study their function and regulation. ii TABLE OF CONTENTS ABSTRACT ii INTRODUCTION 1 METHODS 7 RESULTS AND DISCUSSION 13 CONCLUSION 25 REFERENCES 26 iii 1 INTRODUCTION Mitochondria are membrane-bound organelles known to play a key role in providing fuel for cells through the creation of metabolic energy. These organelles are known to regulate many cellular functions including cell growth and differentiation, cell cycle regulation, and cell death.1 Mitochondria house many metabolic processes essential to the function of cells including ATP production, the citric acid (TCA) cycle, and many others.2 While these processes are imperative for cell growth and survival, they may also promote cellular alterations in certain diseases. This point is evident in cancer cells which are known to differ greatly in their metabolic processes from normal cells and contain alterations in their mitochondrial metabolic pathways.3 One such example is evident in the increased glutamine reliance and catabolism in cancer cells, which works to replenish metabolites used in the TCA cycle to produce lipids and amino acids.3 To perform these metabolic roles in cells, mitochondria constantly exchange metabolites between the mitochondrial matrix and the cytoplasm across the impermeable mitochondrial inner membrane using mitochondrial metabolite carriers. Mitochondrial Metabolite Carriers Embedded within the inner membrane of mitochondria lies a family of proteins known as mitochondrial metabolite carriers. These integral membrane proteins act by transporting numerous metabolites and nutrients vital to the function of the mitochondria and the cell as a whole.2 Because these carriers act as gatekeepers across the inner mitochondrial membrane, they are essential to the regulation of cellular processes such as 2 oxidative phosphorylation, the citric acid (TCA) cycle, and amino acid and fatty acid metabolism.2 Some of these processes and many others are depicted in Figure 1. Figure 1: Diagram of several yeast mitochondrial metabolite carriers, the metabolites they transport into and out of mitochondria, and the biosynthetic pathways those metabolites are used within. Processes that occur within mitochondria are indicated in red and enzymes are pictured in green. The red box highlights the specific mitochondrial metabolite carrier studied in this thesis, Oac1. This figure has been adapted from Palmieri et al. 2016.2 3 While the importance of the role that mitochondrial metabolite carriers play in cellular metabolism is well established, very little is known about how these carriers are regulated within mitochondria. Investigation into the function and regulation of transporter proteins is a crucial step in understanding why the expression of these proteins is altered in cancer cells.1 These carriers are similar in structure, consisting of three repeated transmembrane domains and sequence features that are highly conserved across the entire family of mitochondrial metabolite carriers.4 The specific carrier used to examine mitochondrial metabolite carriers in this study was the mitochondrial oxaloacetate carrier, Oac1, which is involved in leucine biosynthesis (See Figure 1).4 Preliminary data from the Hughes Lab showed specific growth conditions in which Oac1 protein levels were depleted, providing a starting point for examining how the protein levels of mitochondrial metabolite carriers are regulated within cells. However, they obtained this result by following the protein levels of an Oac1 protein C-terminally tagged with a hemagglutinin (HA)-epitope tag. 4 Figure 2: Western blot showing the specific depletion of Oac1-HA protein levels after yeast cultures were switched to a growth medium lacking either uracil (-Ura) or methionine (-Met). Tom70 and Por1 are mitochondrial proteins unaffected by the switch in growth conditions. Pgk1 is a protein involved in glycolysis and serves as a loading control. Data provided by the Hughes Lab. Epitope Tagging of Recombinant Proteins Epitope tagging is a method of expressing proteins carrying short amino acid sequences that are known epitopes for specific antibodies.5 This method allows for the detection of proteins where no antibody is available to detect these proteins. This can include cases of newly discovered proteins, proteins with an antibody known to be inefficient, or cases where the endogenous antibody is not readily available in a lab setting.5 Epitope tagging of a protein is accomplished by fusing an epitope for a specific monoclonal antibody to a target protein, often on the N- or C-terminus, using recombinant DNA techniques.6 This process is pictured in Figure 3. Figure 3: Epitope tagging of a gene of interest in a plasmid. The plasmid is shown to be inserted into the nucleus of a cell and is translated into the protein of interest. Figure adapted from novusbio.com.7 5 This thesis examines two epitope tags and the differences between the two: a hemagglutinin (HA) tag and a Myc tag. Epitope tag sequences used in this research are described in Longtine et.al 1998.8 Methods of Targeted Protein Degradation The ability to regulate protein activity within cells is a powerful tool in studying protein function. Targeted protein degradation is a process by which a protein of interest is purposely degraded to study its function and regulation.9 There are several methods of targeted protein degradation commonly used in research, most of which utilize the ubiquitin-proteasome system (UPS) which is responsible for the degradation of shortlived regulatory proteins and proteins that are damaged or misfolded.9 A common feature of these methods of targeted protein degradation is that the proteins being degraded reside in the cytoplasm or the nucleus, where the proteasomes are generally located.9 While these methods are commonly used for cytoplasmic and nuclear proteins, there is currently no efficient method of targeted protein degradation for proteins located within other organelles, such as mitochondria. This thesis will discuss our strategy for developing a method of targeted protein degradation for proteins located within mitochondria using a destabilizing HA-epitope tag paired with a regulatable promoter. One regulatable promoter system utilizes tetracycline (TC) binding to an RNA aptamer to act as an expression shut-off system in yeast. This method works by introducing this TC-binding RNA aptamer into the 5’UTRs of mRNA.10 Upon binding of TC to this aptamer, translation is prevented due to the stabilization of hairpin structures that inhibit ribosome function (Figure 4). This method 6 will be examined due to its ability to be utilized on proteins that are located within mitochondria. Figure 4: TC-binding RNA aptamer causing inhibition of translation. (A) Depiction of mRNAs containing the TC-binding aptamer (hairpin structure depicted upstream from GFP. GFP is used as a reporter gene to monitor gene expression. (B) Secondary structure of the TC-binding aptamer in its hairpin form. TC binding happens at B1-2 and L3. This figure has been adapted from Kötter et al. 2009.10 7 METHODS Protocols used for multiple experiments in this thesis were adapted from methods described in Goodrum et al., 2019 as well as standard protocols used in the Adam Hughes Lab.11 High-Efficiency Yeast Transformation 3mL of cell cultures of the yeast strains of interest in YPAD medium (1% yeast extract, 2% peptone, 0.005% adenine, 2% glucose) were inoculated overnight and allowed to grow to saturation. The next day, cultures were diluted back to a 1:100 ratio in fresh YPAD medium and grown for 3-4 hours at 30°C until the cultures were in the midlog phase determined by reaching an optical density (OD600) of ~0.5-0.7 OD600/mL. Cell cultures were then centrifuged for 5 minutes at 3000 rpm. Cell pellets were then washed with 1mL sterile ddH2O and spun again for 5 minutes at 8000 rpm. The cell pellets were then resuspended in 100mM LiOAc and pelleted at 8000 rpm for 1 minute. 335µL of PEG transformation mix (240µL 50% PEG 3350, 36µL 1M LiOAc, 10µL 10mg/mL ssDNA, 49µL sterile ddH2O) was added to the cell pellets. 1 µg of DNA plasmid required for each transformation was then added, and samples were incubated in a 42°C water bath for 40 minutes. Cells were then pelleted at 8000 rpm for 5 minutes and resuspended in 200µL sterile ddH2O and plated on growth medium to select for the specific plasmid. Spot Growth Assays Cells of interest were cultured overnight in a growth medium varying by experiment and strain and subsequently diluted back in a 1:100 ratio in fresh media and 8 allowed to grow at 30°C until they were in the mid-log phase (~0.5-0.7 OD600/mL). Cultures were then set to a concentration of 1 OD600/mL by spinning down cells and resuspending them in the proper volume of sterile ddH2O. Cultures were then serially diluted in a 1:10 ratio on a 96-well plate and 3µL of each culture was plated onto control and selection plates. Growth was then analyzed at 30°C for 1-4 days depending on the growth rate. Western Blotting Experiments Cell cultures from the yeast strains of interest were grown overnight at 30°C in a growth medium varying by experiment and subsequently diluted back into fresh media at the appropriate concentration to have the secondary overnight cultures reach the mid-log phase (~0.5-0.7 OD600/mL) in the morning. Cell cultures were then pelleted and split into various flasks containing different growth media. These cultures were then grown at 30°C and 4OD equivalents of cells per sample were collected at every indicated time interval through the growth period. The cell samples were subsequently snap-frozen in liquid nitrogen (LN2). To prepare the proteins, cell pellets were then resuspended in 100µL of sterile ddH2O. 100µL of 0.2M NaOH was then added to each sample and incubated at RT for 5 minutes. Samples were then spun at full speed in a 4°C microcentrifuge for 10 minutes. The pellets were then resuspended in 200µL of SDS-lysis buffer (10mM Tris-HCl pH 6.8, 100mM NaCl, 1% SDS, 1mM EDTA, 1mM EGTA, 1X protease inhibitor solution) and heated at 95°C for 5 minutes. 50µL of Laemmli buffer containing 1:100 beta- 9 mercaptoethanol was added to each sample and spun at full speed in a microcentrifuge for 3 minutes to pellet any unnecessary cell debris. 0.16 OD equivalents of the cell samples were then loaded into and run through an SDS-PAGE mini gel at a constant voltage of 160V for ~40 minutes. Using a Power Blotter-Semi-dry transfer system (ThermoFisher Scientific), the contents of the protein gel were transferred onto a nitrocellulose membrane using a constant Amp of 2.5A/gel for 10 minutes. The membrane was removed and rinsed twice with TBS to remove any excess SDS. The blot was then covered in 10% milk solution made in TBST and incubated at RT for 1 hour with constant rocking. The blot was then incubated in a varying diluted concentration of the primary antibody of interest in 10% TBST milk solution overnight at 4°C with constant rocking. The primary antibodies used in this study were anti-HA (1:750), anti-Myc (1:10000), anti-Por1 (1:1000), anti-Pgk1 (1:1000), anti-Tom70 (1:1000), anti-Ilv2 (1:1000), anti-FLAG (1:1000), and anti-Aco1 (1:1000). The following day, the blot was washed 4 times with TBST for 10 minutes each while rocking. The blot was then incubated in a 1:5000 dilution of the indicated secondary antibody in 10% TBST milk solution for 1 hour at RT. The blot was then washed twice with TBST for 10 minutes, then twice more with TBS, all with constant rocking. The blot was then developed for 5 minutes in Pierce ECL solutions, using ~2mL of mixture per blot. Images were then taken of the blots for analysis. Cloning Tagged Oac1 Alleles into pCSJ95 The pCSJ95 plasmid from the overnight bacterial culture was prepared using the QIAprep Miniprep Kit from Qiagen. Phusion High Fidelity Polymerase was then used to 10 amplify various Oac1-tagged alleles with their corresponding primers. A master mix (30µL 5X HF Phusion Buffer, 30µL 5X HF Phusion Buffer, 3µL 10mM dNTPs, 12µL 5µM forward primer, 12µL 5µM reverse primer, 6µL gDNA, 1.5µL Phusion Polymerase, and 85.5µL sterile ddH2O) was created for each different reaction and was then aliquoted into 3 PCR tubes of 50µL each. These tubes were placed in the PCR machine and ran with the conditions listed below: a. 3 min 98°C for initial denaturation b. 10s @ 98°C c. 30s @ 57°C d. 60s @ 72°C (extension 30s per kb) e. Repeat b-d 30x f. 5 min @ 72°C for final extension For the cloning process, two different methods shown in Figure 5 and Figure 6 were utilized. Oac1-3xHA and Oac1-13xMyc were cloned into pCSJ95 using the traditional cloning workflow (Figure 5). 11 Figure 5: Traditional cloning workflow overview. Restriction sites are added to both ends of a DNA segment using PCR and are then digested by the corresponding restriction enzymes. The cleaved DNA is then ligated to a plasmid vector with compatible ends. Assembled DNA is then transformed into yeast. This figure has been adapted from “Foundations of Molecular Cloning - past, present and future.” 12 Restriction sites located on pCSJ95 were digested by restriction enzymes AscI and NotI-HF. A PCR was performed on a master mix (2µL 10X NEB’s CutSmart™ Buffer, 8.3µL 600ng/µL pCSJ95 plasmid, 1µL AscI, 1µL NotI-HF, 7.7µL sterile ddH2O) at 37°C for 3 hours. The same process was performed on the Oac1-3xHA and Oac113xMyc fragments using another master mix (7µL 10X NEB’s CutSmart™ Buffer, 48µL 100ng/µL Oac1 DNA fragment, 2µL AscI, 2µL NotI-HF, 11µL sterile ddH2O). Compatible ends from the pCSJ95 vector and Oac1-3xHA and Oac1-13xMyc DNA fragments were then ligated using PCR with a master mix (1µL T4 DNA Ligase Buffer, 50ng pCSJ95 vector, 10ng Oac1 DNA fragment, 7µL ddH2O, 1µL T4 DNA Ligase) that was then incubated for 16 hours at 16°C. The newly assembled DNA was then 12 transformed using the high-efficiency yeast transformation protocol into a wild-type yeast strain and a leu4∆ oac1∆ strain. 9xMyc-Oac1 was cloned into pCSJ95 using the Gibson Assembly method shown in Figure 6. Figure 6: Gibson Assembly overview. DNA is first prepared and is subsequently assembled using the process explained in the figure. Assembled DNA is then transformed into yeast. DNA analysis is then performed using RE digest, colony PCR, or sequencing. This figure has been adapted from “Foundations of Molecular Cloning - past, present and future.” 12 In the single-tube reaction, a Gibson Assembly master mix (50ng pCSJ95 vector, 14ng 9xMyc-Oac1 DNA fragment, 5µL Gibson Assembly Buffer, 5µL sterile ddH2O) was incubated at 50°C for 1 hour. The newly assembled DNA was then transformed using the high-efficiency yeast transformation protocol into a wild-type yeast strain and a leu4∆ oac1∆ strain. 13 RESULTS AND DISCUSSION Epitope Tagging of Oac1 The oxaloacetate carrier, Oac1, is involved in leucine biosynthesis.4 In order to study Oac1, a strain of yeast (Saccharomyces cerevisiae) containing a LEU4 deletion (leu4∆) was utilized to force leucine biosynthesis to require Oac1 function. Subsequently, using the yeast transformation protocol, Oac1 was tagged with a Myc-epitope tag at either the N-terminus or the C-terminus. The correct tagging of transformation products was confirmed via PCR. The functionality of these tagged Oac1 proteins was then confirmed by a growth assay that compared the growth of all strains on nutrient-rich media as well as media lacking leucine (Figure 7). This was done because if the created Myc-Oac1 was still functional, it would be able to grow on growth media lacking leucine.13 Figure 7: Growth assay showing the growth of Oac1-Myc and Myc-Oac1 strains after plating on nutrient-rich media (SD-Complete) and media lacking leucine (SD-Leu). The positive control (leu4Δ) grows in both conditions, while the negative control (leu4Δ oac1Δ strain) does not grow without leucine present. 14 The positive growth outcome of all Myc-tagged strains suggests that Oac1 is functional in all of these strains, due to their ability to grow on media that lacks leucine, a nutrient essential to cell survival. Depletion of Oac1-HA in Certain Growth Conditions Once the functionality of the Myc-tagged yeast strains was confirmed, these strains along with an HA-tagged Oac1 strain were used in an experiment testing protein levels in different metabolic conditions. Yeast strains containing either Oac1-HA, Oac1Myc, or Myc-Oac1 were grown in nutrient-rich media and then transferred back into the same media, media lacking leucine, media lacking uracil, or media lacking methionine and allowed to continue incubating for 3 hours. Notably, in all conditions except in the nutrient-rich media, cell growth ceases because the yeast strains used are auxotrophic for leucine, methionine, and uracil. Subsequently, cell samples were collected at one-hour intervals beginning immediately after the media transfer, and protein levels at the indicated time points were analyzed by Western blotting (Figure 8). 15 Figure 8: Western blot showing the decrease in Oac1-HA protein levels after yeast cultures were switched from nutrient-rich media (SD-Comp) to media lacking either uracil (SD-Ura) or methionine (SD-Met). Both Oac1-Myc and Myc-Oac1 protein levels remain steady over the 3 hours. Por1 and Pgk1 serve as loading controls. While both the N- and C-terminally Myc-tagged strains showed steady levels of the Oac1 protein in all conditions, Oac1-HA levels decreased when switched into media lacking uracil and media lacking methionine. This finding suggested a difference in the stability of the proteins that were HA-tagged versus those that were Myc-tagged, with the Oac1-HA protein appearing less stable. This data also reproduces the preliminary data shown in Figure 2 where the specific depletion of Oac1-HA protein levels in media lacking either uracil or methionine was apparent. Depletion of Oac1-HA in Various HA-tagged Mitochondrial Proteins 16 Due to the destabilizing effect of the HA-epitope tag on Oac1, other HA-tagged proteins in mitochondria were analyzed. These proteins are depicted in Figure 9. Figure 9: Diagram of a mitochondrion and the proteins examined with HA-epitope tags. Ilv2 is located in the mitochondrial matrix. Mir1, Oac1, and three truncated versions of Oac1 (Oac1(1-267)-HA, Oac1(1-223)-HA, and Oac1(1-121)-HA) are located in the mitochondrial inner membrane. Tom70 is located on the outer mitochondrial membrane. These proteins are all tagged with an HA-epitope tag at their C-termini. 17 Ilv2 is a protein involved in the biosynthesis of branch-chained amino acids.14 Oac1 and its truncated versions, as previously discussed, are proteins involved in leucine biosynthesis.4 Mir1 is a phosphate carrier protein in mitochondria and Tom70 serves as a mitochondrial import receptor.15,16 These HA-tagged proteins were analyzed by examining the turnover of each protein when cells were switched into media lacking methionine, a condition previously shown to deplete Oac1-HA. Cell samples were collected and prepared at the 0-hour and 2-hour time points after the media transfer and analyzed with Western blotting (Figure 10). Figure 10: Western blot showing several HA-tagged protein levels over 2 hours after swapping media from nutrient-rich growth media (comp) to growth media lacking methionine (-met). Pgk1 is a protein involved in glycolysis and serves as a loading control for all 8 strains. 18 The protein levels of Oac1-HA and the three truncated versions of Oac1 were all depleted when switched to media lacking methionine. The same was found for Mir1-HA, the other protein located in the mitochondrial inner membrane, and Ilv2, the protein found in the mitochondrial matrix. The only protein that was not depleted in the SDmethionine condition was Tom70-HA, a protein located on the mitochondrial outer membrane (Figure 9). This data supports the previous findings that the HA-epitope tag appears to be destabilizing proteins in certain metabolic conditions, in this case, growth media lacking methionine. Analyzing Tom70-HA and Ilv2-HA Protein Levels Using Endogenous Antibodies In order to further demonstrate that the HA-epitope tag destabilizes proteins as the previous results suggest, a similar Western blot experiment was performed using endogenous Ilv2 and Tom70 antibodies to track protein levels. An untagged wild-type yeast strain was compared to both a strain containing Ilv2-HA and a strain containing Tom70-HA in an experiment where the turnover of each protein was examined when cells were switched into media lacking methionine, the condition previously shown to deplete various HA-tagged proteins in mitochondria. Cell samples were collected at onehour intervals beginning immediately after cells were switched from nutrient-rich media to media lacking methionine. Subsequently, protein levels at the indicated time points were analyzed by Western blotting (Figure 11). 19 Figure 11: Western blot showing protein levels of wild-type versus yeast strains containing the proteins Tom70-HA and Ilv2-HA during an experiment where growth media was switched from nutrient-rich (SD-Comp) to media lacking methionine (SDMet). Pgk1 was used as a loading control for all yeast strains. Both the wild-type strain and Tom70-HA showed steady levels of the Tom70 protein in each growth condition, which is consistent with the data shown previously of Tom70-HA being the only HA-tagged mitochondrial protein that was not specifically depleted when switched into media lacking methionine (Figure 10). While the wild-type strain also showed steady levels of the Ilv2 protein in both growth conditions, Ilv2-HA protein levels were very low in both conditions when analyzed with an endogenous antibody detecting Ilv2. This result suggests that the HA tag destabilizes Ilv2 to the extent that the protein level is barely detectable when using the Ilv2 antibody. 20 Cloning of Epitope Tagged Oac1 into the pCSJ95 Vector Because the HA-tag has been shown to destabilize proteins in specific conditions, a series of experiments were conducted to functionalize this HA-tag to induce protein degradation in mitochondrial proteins. This was done by first separately cloning Oac13xHA, Oac1-13xMyc, and 9xMyc-Oac1 into pCSJ95, the vector shown in Figure 12. Oac1-3xHA and Oac1-13xMyc were cloned into pCSJ95 using the traditional cloning workflow shown in Figure 5, and 9xMyc-Oac1 was cloned into pCSJ95 using the Gibson Assembly method shown in Figure 6. Through these processes, the FBP1 gene in pCSJ95 was replaced with the OAC1 gene containing the epitope tag of interest. This places the OAC1 open reading frame under the control of a constitutive expression promoter (GAP promoter) and creates an mRNA product that contains the TC-binding RNA aptamer within the 5’UTR (Figure 4). The plasmid products from this cloning process were confirmed with a PCR reaction as well as Sanger sequencing through GENEWIZ (Azenta Life Sciences). An example of this process is depicted in Figure 12, with FBP1 being replaced by Oac1-13xMyc. 21 Figure 12: Unaltered pCSJ95 vector containing various genes and sequence components including the original FBP1 (top). Altered form of the pCSJ95 vector containing various genes and sequence components with Oac1-13xMyc in place of FBP1 (bottom). This figure was created using Snap Gene software. 22 Confirmation of the TC-Binding Aptamer Function Through Tetracycline Addition In order to determine if the TC-binding aptamer was functioning properly, an experiment was conducted in which different concentrations of tetracycline were added to different yeast cell cultures. The leu4∆ oac1∆ strain transformed with the unaltered pCSJ95 plasmid was compared to the leu4∆ oac1∆ strain transformed with the pCSJ95 plasmid altered to contain the Oac1-3xHA DNA fragment. Each of these two strains was grown in media lacking histidine in order to select for the plasmid of interest and subsequently split into three different flasks where they were treated with different amounts of tetracycline. Differing concentrations of tetracycline solution were added to different flasks to reach a final concentration of 200µM TC for one and 500µM TC for the other. The same volume of H2O was added to the final flask as a control, and all three cultures were allowed to grow for 3 hours. Cell samples were collected at one-hour intervals beginning immediately after the TC or water addition. Levels of HA for the strain containing Oac1-3xHA and levels of Flag for the strain containing the pCSJ95 plasmid with Fbp1-Flag were then analyzed by Western blotting (Figure 13). Figure 13: Western blot showing protein levels of HA and Flag in yeast strains containing Oac1-HA and Fbp1-Flag, respectively, during an experiment where growth media was altered to contain various levels of tetracycline (0µM, 200µM, or 500µM). Aco1 was utilized as a loading control for this experiment. 23 The data from these Western blots indicate the successful functioning of the TCbinding aptamer. This is because, in all cases of tetracycline addition for both strains, protein levels decrease which is to be expected when the RNA aptamer binds tetracycline because this action inhibits the translation of new proteins.10 Importantly, Fbp1 is involved in gluconeogenesis and is known to be a short-lived protein that is rapidly degraded upon tetracycline addition when expressed from the pCSJ95 plasmid.17 Remarkably, the Oac1-HA protein appears to be more rapidly degraded than Fbp1 (Figure 13). Induced Degradation in Oac1 via Activation of the TC-Binding Aptamer in Oac1-HA In order to examine differences between TC-binding aptamer function in the leu4∆ oac1∆ yeast strain transformed with the pCSJ95 plasmid altered to contain either the Oac1-3xHA sequence or the Oac1-13xMyc sequence, the same experiment above was conducted using these two strains. The same procedure was followed, again using tetracycline concentrations of 0µM, 200µM, and 500µM. Cell samples were again collected at one-hour time points over a 3-hour time course. Levels of HA for the strain containing Oac1-3xHA and levels of Myc for the strain containing the pCSJ95 plasmid with Oac1-13xMyc were then analyzed by Western blotting (Figure 14). 24 Figure 14: Western blot showing protein levels of HA and Myc in yeast strains containing Oac1-HA and Oac1-Myc expressed from the modified pCSJ95 plasmids, respectively, during an experiment where tetracycline was added to final concentrations of 0µM, 200µM, or 500µM. Aco1 (a mitochondrial protein) and Pgk1 (a cytoplasmic protein) were used as loading controls for this experiment. While the Oac1-Myc strain showed steady levels of the Oac1 protein in all conditions regardless of tetracycline addition, Oac1-HA levels decreased when either 200µM TC or 500µM TC was added. This data re-demonstrates the successful functioning of the TC-binding aptamer to induce Oac1 protein degradation in Oac1-HA. This data also supports the previous finding that the HA-tagged proteins are inherently more unstable than the Myc-tagged proteins in certain growth conditions because when translation was shut down through aptamer activation, the Myc protein levels remained steady. 25 CONCLUSION The many observed differences in yeast strains containing Oac1-HA versus Oac1Myc indicate that the HA-tagged Oac1 is more susceptible to being degraded when placed in certain altered metabolic growth conditions. The literature supports this finding with current research suggesting that proteins that are epitope tagged with the specific HA-tag used in this study may be inherently unstable.18 Utilizing these findings, this HAtag was incorporated with a regulatable promoter that contained a TC-binding aptamer as a method of induced protein degradation within mitochondria during activation of the aptamer. This method of protein degradation was successfully demonstrated through Western blots showing the depletion of proteins upon the addition of tetracycline to cells in growth media. Importantly, growth assays with cells expressing Oac1-HA demonstrated that Oac1-HA is still a functional protein capable of maintaining the growth of leu4∆ strains on media lacking leucine (Figure 7). Thus, while the HA-tag destabilizes Oac1, enough functional protein must be produced to allow for the leu4∆ strains to grow. Altogether, these results suggest that this HA-tag combined with the regulatable promoter described could be used for targeted protein degradation. It is expected that future research can utilize this HA-tag as a tool to purposely degrade other proteins of interest within mitochondria. This is an especially useful tool due to the difficulty of selectively degrading proteins in mitochondria, as discussed previously. 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