Research objectives
While the ATPase activity of Cod1p is dependent on magnesium, transport of magnesium by Cod1p has not been directly demonstrated. I plan to measure ion transport in microsomal vesicles enriched for Cod1p. Ion transport into vesicles is usually measured using radioisotopes of the substrate being studied. The ability of Cod1p to transport calcium was assayed in this manner using 45Ca. However, the utility of this type of assay for studying Cod1p at an undergraduate university is limited since radioisotopes of most other metals emit gamma and x-rays. Furthermore, radioisotopes of the prime candidate substrate for Cod1p, magnesium, are not commercially available. To circumvent this limitation, I intend to detect the transport of ions into vesicles using mass spectrometry. I have already used this technique to measure ion amounts in whole yeast cells (Cronin 2002), and am currently working on applying it to measure the ion content of purified ER vesicles. While we do not have the inductively coupled plasma mass spectrometry (ICP-MS) needed for the work here at Ave Maria University, the samples can be analyzed off-site. I have been able to collaborate with analytical chemists at Florida International University to analyze the samples.
In addition to COD1/SPF1 the yeast genome contains a gene second type 5 P-type ATPase currently labeled yor291w. While its function in yeast is obscure, human homologs have recently been implicated in brain function. During my graduate work, I deleted yor291w in yeast but found no obvious phenotypes related to the deletion (Cronin 2002). Currently the only known yeast phenotype known is decreased transposition of the ty3 transposon in the null mutant (Aye 2004). Two human homologs of yor291w have recently been linked to neural function. One human homolog (ATP13A2) has been implicated in hereditary parkinsonism with dementia (Ramirez 2006), and a second (ATP13A4) was found to be disrupted in an individual with language delay (Kwasnicka-Crawford 2005). I am currently on characterizing the yeast homolog yor291w using the same techniques used to characterize COD1/SPF1. The first step toward characterizing yor291w will be to generate a epitope-tagged version of the protein for expression and localization studies. Localization can be followed by purification of the protein followed by ATPase assays.
Ultimately, I would like to use transgenic yeast to study the mammalian homologs of COD1 and yor291w. While this is a long term goal, the initial cloning of the mammalian cDNAs into yeast vectors can proceed concurrently with the other research objectives and provide ample opportunity for undergraduate participation.
Aye M, et al. (2004) Host Factors That Affect Ty3 Retrotransposition in Saccharomyces cerevisiae. Genetics 168(3):1159-76
Cronin SR, et al. (2002) Cod1p/Spf1p is a P-Type ATPase involved in calcium homeostasis and ER function. J. Cell Biol. 151:69-82
Kwasnicka-Crawford DA, (2005) Characterization of a novel cation transporter ATPase gene (ATP13A4) interrupted by 3q25-q29 inversion in an individual with language delay. Genomics. 86(2):182-94.
Ramirez A, et al. (2006) Hereditary parkinsonism with dementia is caused by mutations in ATP13A2, encoding a lysosomal type 5 P-type ATPase. Nat Genet. 38(10):1184-1191.