NIH Mock Proposal
1. Specific Aims
2. Background and Significance
3. Research Design and Methods
   1. Yeast Genetics
   2. Immunopre- cipitation
   3. Yeast Two-Hybrid
   4. Future Experiments
4. References

3.1 Determination of protein interactions with YFH1 using yeast genetics

3.1.1 Phenotype of MFT1/2 and YFH1 deletion mutant

The phenotype of null MFT1/2 mutants is poor growth on iron limited medium, and null YFH1 mutants are unable to grow on non-fermentable medium such as glycerol and ethanol. If YFH1 interacts in a regulatory manner with MFT1/2, the deletion of YFH1 and MFT1/2 may show a MFT1/2 null phenotype instead of the more severe phenotype of the YFH1 deletion. Additionally, it is possible that YFH1 plays a larger role in iron regulation by interacting with MFT1/2 and other, unknown regulatory proteins; if this is the case a mutant with null YFH1/MFT1/2 may have an improved phenotype compared to the null MFT1/2 phenotype. This will be tested by constructing a deletion strain of all three mutations. Growth will then be assessed and compared to strains with YFH1 or MFT1/2 deletions on medium containing normal or low iron and glucose or glycerol/ethanol (non-fermentable) as the main carbon source.

3.1.1.1 Interpretation

Although there may be other proteins that play a role in mitochondrial iron regulation, it will be helpful to know the phenotype of yeast with null yfh1 and null MFT1/2. If so, this experiment provides evidence that YFH1 may interact with MFT1/2. If there is no complementation, it is likely that other proteins may interact with YFH1. There may be other unknown mechanisms of iron regulation in the mitochondria that may make a negative result hard to interpret, but the phenotype may be informative in this respect as well.

3.1.1.2 Protocol

Yeast strains (null yfh1::HIS3) containing the YFH1 deletion and the MFT1/2 deletion (null MFT1:URA3, null MFT2::HIS2) will be obtained from the Kaplan lab (U Utah SOM), or they can be reconstructed as described.^13,14 A triple deletion strain can then be made by replacing the MFT genes with URA3 and HIS2 in the YFH1 deletion strain as described.¹⁴ Briefly, PCR using the end sequences of MFT1/2 as primers will be used to replace the MFT genes with URA3 and HIS2. Yeast will then be transformed with the PCR products and then sporulated and dissected, and transformants will be selected by growth on His- and Ura- media. Correct integration will be tested by PCR. Growth of the triple deletion mutant will then be compared to yeast with the YFH1 deletion or yeast with the MFT1/2 deletion. YPDGE (containing 0.1% glucose, 3% glycerol, and 2% ethanol as carbon sources) will distinguish cells capable of respiration²³ and growth in iron limited conditions will be assessed by the addition of banthophenanthroline (BPS) to the media.

3.1.2 Complementation of MFT1/2 Overexpressors

Yeast frataxin was initially identified by partial complementation of a mutant with a low growth phenotype on iron deficient media.⁸ The ability to have multicopy plasmids in yeast also allows for a test of the hypothesis that YFH1 interacts with MFT1 or MFT2 to down-regulate iron transport into the mitochondria. Yeast null mutants of MFT1, MFT2, and both MFT1/2, and yeast overexpressing MFT1 and MFT2 have already been constructed, so these mutants can be easily reconstructed or obtained from the Kaplan lab (University of Utah SOM). A mulitcopy plasmid containing YFH1 and a selectable marker could be introduced into 1) MFT1/2 double deletion strains, 2) single MFT1 or MFT2 deletion strains, 3) MFT1 or MFT2 overexpressing strains and 4) wild-type strains to assess growth and on low, normal, and high iron media as compared to the controls of each type receiving plasmid only. Mitochondrial iron concentration will be determined by atomic absorbtion spectroscopy.

3.1.2.1 Interpretation

Since YFH1 can partially complement strains unable to grow well in low iron conditions, this experiment will be very informative. If the proposed model is correct, overexpression of YFH1 will not rescue a double deletion strain of MFT1/2 when grown on low iron medium; if it did, this would provide strong evidence that YFH1 works through proteins other than MFT1/2. Additionally, overexpression of YFH1 may complement strains overexpressing MFT1/2; that is, there should be less iron accumulation and thus less mitochondrial damage in yeast that received the YFH1-plasmid than in the controls. If inconclusive or ambiguous results are obtained, other experiments will help clarify the results.

3.1.2.2 Protocol

Construction of the plasmids containing YFH1 will be accomplished using standard procedures. Briefly, restriction sites appropriate will be introduced by PCR, and care will be taken to ensure that YFH1 is inserted in-frame. Then, YFH1 and the plasmid will be digested by the appropriate enzymes and ligated together. Restriction analysis will be used to ensure that YFH1 is inserted in the appropriate orientation. Bacteria will be made competent through a standard calcium chloride protocol, and plasmid DNA with and without the insert will be transformed into the bacteria. Selection for transformants is accomplished by the use of an antibiotic since antibiotic resistance is conferred by the plasmid. The plasmid DNA will then be prepared for use in the yeast transformation. Transformation of yeast will be accomplished using the lithium acetate/single-stranded carrier DNA/polyethylene glycol protocol as previously published.¹⁷ This method generally produces up to 2.2 � 10⁷ transformants / �g DNA.¹⁷ Since Ura3 and His2 are the selection markers for MFT1 and MFT2, respectively, the plasmid used will employ a different marker such as Leu2 to select for YFH1 yeast transformants. Growth on iron-rich and iron-deficient media will then be assessed. Mitochondrial iron accumulation will be assessed as previously described.⁸ Briefly, the mitochondrial fraction will be separated by differential centrifugation, and the fractions will then be applied to a 15% percoll gradient and centrifuged. Afterwards, the enriched mitochondrial fractions will be separated from the percoll by centrifugation. Then, atomic absorption analysis will be performed, and protein concentration will be determined using BSA as a standard. The ratio of iron content to total protein content will be used to compare different constructs. Use of the instrumentation to measure absorption spectra will be aided by instruction from Bill Paplawsky (SIO).

3.1.3 Complementation of a YFH1 point mutation

In rare cases of FRDA, the diseased individuals have only one expanded copy of frataxin and a point mutation in the second copy. The most common point mutation changes an isoleucine to a phenylalanine at residue 154 in the conserved C-terminal domain. The corresponding isoleucine at residue 130 in yeast (YFH1-I130F) has also been shown to be important in YFH1 function, but the phenotype is not as severe as in the deletion.¹² The point mutation shows consistently slower growth on non-fermentable medium and a 20% reduced respiratory capacity when compared to wild type YFH1.¹² This provides a means to screen for mutants that complement the YFH1 point mutation. Once a cell is isolated with a complementary mutation, the yeast DNA can be prepared, amplified, and reintroduced into null yfh1 yeast via a plasmid to locate the complementary gene. Once a plasmid that suppresses the null yfh1 phenotype is isolated, the plasmid DNA can be sequenced and the gene identified. Confirmation of the interaction can be achieved through altering the mutations, and immunoprecipitation experiments will be used as well.

3.1.3.1 Interpretation

Yeast allows one to screen through thousands of cells in search of a complementary mutation to YFH1-I130F. Restoration to wild-type growth would be expected as a result of reversion of the YFH1-I130F mutation, or a complementary mutation in an interacting protein. It is possible that other mutations affecting iron transport in some other way might be identified.

3.1.3.2 Protocol

The mutated form of YFH1 will be introduced via a plasmid into null yfh1::HIS3 haploids before loss of mitochodrial DNA occurs. Construction of the I130F mutation will be accomplished through PCR-based site-directed mutagenesis. Yeast containing a complementary mutation will be selected based upon growth, and then the genome can be digested, amplified, and reintroduced into plasmids. Then plasmids that suppress the I130F mutation can be identified by sequence. Once a gene is identified in this manner, the corresponding mutation can be reverted to test that it no longer rescues yeast with mutated YFH1.

3.1.4 Yeast notes

The severity of the null yfh1 phenotype depends upon the genetic background of the yeast strain used. In backgrounds that are less sensitive to the YFH1 deletion, the addition of FeSO₄ to the medium causes loss of the mitochondrial genome.²⁶ In some of the proposed experiments, too severe a phenotype may lead to yeast that are unable sporulate, and in other experiments, it is critical that the phenotype be deleterious so complementary mutations can be quickly identified. To determine growth defects, strains can be used that exhibit uniform colony size.²⁵ Additionally, YFH1 mutants show more severe growth defects at high (37�C) and low (20�C) temperatures, so this will be utilized as well in the selection process.

Next Section: 3.2 Determination of other protein interactions with frataxin through immunoprecipitation

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