Directed research project with Dr. Stephen Cronin investigating the substrate specificity of type V P-type ATPases

Background: All eukaryotes have Type V ATPases, but what do they do?

You would think that if humans, yeast, rice, small worms and all other eukaryotic organisms all shared a common feature, it would be pretty easy to figure out what the common feature does, and why it is important to the organism. Unfortunately, this is not the case, especially when the object of inquiry is one of the many proteins made by a cell. The purpose of this project is to characterize the function of two proteins produced by baker’s yeast (Saccharomyces cerevisiae). One protein is named Cod1p (or Spf1p) and the corresponding gene is named COD1 (or SPF1). The other is produced by the gene yor291w but almost nothing is known about it other than it is related to Cod1p.

Cod1p

Genes homologous to COD1 have been found in every eukaryotic organism that has been sequenced, including humans. However, the precise function of the gene and the protein it produces is unknown.

A few things are known about Cod1p:

Cod1p is a P-type ATPase By comparisons with other proteins, Cod1p belongs to a family of proteins called P-type ATPases. In general, P-type ATPases use the energy gained from hydrolyzing ATP to pump a substrate across a membrane. The ATPase activity of the protein has been experimentally demonstrated.
Cod1p is a type V P-type ATPase Within the family of P-type ATPases different ATPases pump different substrates. By comparison with other P-type ATPases, it has been determined that Cod1p belongs to a group of ATPases (the type V group) for which no substrate is known.
Yeast cells lacking Cod1p have problems with protein folding in the endoplasmic reticulum (ER) The gene encoding Cod1p was first identified as being needed for proper regulation of sterol production and sensitivity to a killer toxin. These phenotypes appear to result from defective protein folding and glycosylation in the secretory pathway.
Cod1p is located in the ER I determined the localization of Cod1p experimentally. Presumably it pumps something into the ER.

While a few things are known about Cod1p, a very important question remains.

What does Cod1p pump? Without knowing what Cod1p pumps, it is hard to make sense of the other information known about the protein.

The purpose of the proposed investigations is to identify the substrate pumped by Cod1p.

Experimental approach

To identify the substrate being pumped across the endoplasmic reticulum, we need to be able to detect it. To detect the substrate it is helpful to have an idea as to what the substrate might be. A reasonable hypothesis is that Cod1p pumps a metal ion. This hypothesis is reasonable because most other P-type ATPase pump metal ions, although some transport protons and another group transports phospholipids.

We’ll look for metal ions transported by Cod1p indirectly by measuring the amount of metal ions in membrane vesicles containing Cod1p compared to vesicles lacking Cod1p. The vesicles will be obtained by breaking open yeast cells (with and without Cod1p) and by collecting the resulting membranes. The vesicles will be suspended in an assay mixture: a solution with ATP to power the Cod1p ATPase and various metal ions. The substrate for Cod1p should be pumped into the vesicles and therefore the amount of the substrate in the vesicles should increase. The amount of metal present in the vesicles will be measured using a technique borrowed from analytical chemistry: ICP-MS or inductively coupled plasma mass spectrometry. ICP-MS identifies metals by sending them through a plasma torch and detecting the mass/charge ratio of the traveling ions.. Preparing the vesicles for analysis is simple in concept. The vesicles are separated from the assay mixture by filtration. The vesicles are then digested with sulfuric acid or hydrochloric acid. After digestions the samples are diluted and sent to a lab at Florida International University for analysis by ICP-MS. After the results are returned to us, the data is analyzed and we plan the next experiment.

While the experimental plan is relatively straightforward, there will be many difficulties encountered along the way. A number of things must be done before any results can be expected.

The efficacy of the assay needs to be proven by measuring the pumping activity of a pump with a known substrate. In this case we will use Pmr1p, which is known to pump calcium and manganese. Working out the conditions by which we can detect pumping by Pmr1p will help us design assays for detecting pumping by Cod1p.
The assay mixture needs to be developed, since the optimal conditions for pumping are not known.
The ability of vesicles to retain metal ions needs to be confirmed. The approach will not work if the vesicles do not retain the substrate that is pumped in.
A method for isolating vesicles containing Cod1p must be developed. Measuring ion transport of vesicles lacking Cod1p will not help us unless we can also measure ion transport in vesicles with Cod1p.

Some progress towards these goals has made by two students Kaitlyn Humphrey and Su Li Lee who carried out a number of assays in the summer of 2005.

yor291w

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, its function does not overlap with COD1 and 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). The same techniques I used in graduate school to characterize COD1 can be used to characterize yor291w. The first step toward characterizing yor291w will be to generate an epitope-tagged version of the protein for expression and localization studies. An epitope tag is a small peptide added to a protein that allows it to be detected by commercially available antibodies. For instance the HA tag allows recognition of a protein by antibodies to the hemaglutinin (HA) protein from influenza virus. To add an eppitope tag to a protein, the gene encoding the protein is modified so it produces an altered protein. Having an eppitope tag allows us to determine where the protein is localized, purify the protein to investigate biochemical activity, and carry out other studies. Perhaps the information learned about yor291w in yeast will be useful in identifying the functions of similar proteins in humans.

Required work

Biological research can be rewarding as well incredibly frustrating. To successfully obtain BIOL497 credit and participate in research into the biochemical substrate of Cod1p you need to consider the following:

Biological research takes lots of time. In order to make progress in biological research there is simply no way around spending lots of time on the problem. Unlike the labs for regular biology courses in which everything has been tested ahead of time, biological research always involves planning and executing experiments that have never been tried and might not work. Plan on spending at least 3 hours a week for each BIOL497 credit hour you wish to attain.
Biological research takes persistence. Research experiments rarely produce the desired data the first time they are attempted. Successful researchers need to remain motivated and continue to work even though experiments are not working as planned.
Biological research involves studying. Planning and carrying out a successful BIOL497 requires spending time studying and thinking about the known processes that may be influencing the system that you are working on. Since Cod1p is needed for the normal function of the ER, you will need to read up on the many processes and proteins that function in the ER.
Biological research is a collaboration. Very few researchers have been successful working on their own. Other researchers will almost always be able to offer suggestions concerning experiments and experimental techniques that you wouldn’t have thought of. At the same time, a successful directed research project requires that you take the initiative whether dealing with problems, seeking assistance, carrying out experiments or analyzing data. For this project you will need to work closely with Dr. Cronin and other students involved in the project, as well as talking frequently with the other professors about your project.

Evaluation

Students participating in research will be evaluated on the following:

Commensurate work for credit hours requested. Was the time spent on the project adequate given the number of credit hours requested? Plan on spending at least 3 hours a week for each BIOL497 credit hour you wish to attain.
Technical proficiency. Did the student keep a useful notebook? Was proper experimental procedure followed? Was the project carried out in a safe manner?
Initiative and organization. When the next steps in the project were within the technical competence of the student, did he or she take the initiative to proceed in a reasonable time frame. Was the student able to organize their work efficiently to be able to carry out multi-day procedures. When in need of information, assistance or guidance did the student seek out the needed information, assistance and guidance, without being prompted.
Communication. Is the student able to successfully communicate the purpose of the project, how the project was carried out, and what the results mean?

Ready to start researching?

Talk to Dr. Cronin. You can start as soon as you are ready.

References for Cod1p/Spf1p and homologous proteins

Jakobsen MK, Poulsen LR, Schulz A, Fleurat-Lessard P, Moller A, Husted S, Schiott M, Amtmann A, Palmgren MG. Pollen development and fertilization in Arabidopsis is dependent on the MALE GAMETOGENESIS IMPAIRED ANTHERS gene encoding a type V P-type ATPase.

Genes Dev. 2005 Nov 15;19(22):2757-69

Yoshida SH, Nakamura T, Shimoda C. The cation-transporting P-type ATPase Cta4 is required for assembly of the forespore membrane in fission yeast.

Genes Genet Syst. 2005 Oct;80(5):317-24.

Ando A, Suzuki C. Cooperative function of the CHD5-like protein Mdm39p with a P-type ATPase Spf1p in the maintenance of ER homeostasis in Saccharomyces cerevisiae.

Mol Genet Genomics. 2005 Jul;273(6):497-506.

Vashist S, Frank CG, Jakob CA, Ng DT. Two distinctly localized p-type ATPases collaborate to maintain organelle homeostasis required for glycoprotein processing and quality control.

Mol Biol Cell. 2002 Nov;13(11):3955-66

Cronin SR, Rao R, Hampton RY. Cod1p/Spf1p is a P-type ATPase involved in ER function and Ca2+ homeostasis.

J Cell Biol. 2002 Jun 10;157(6):1017-28.

Facanha AL, Appelgren H, Tabish M, Okorokov L, Ekwall K.

The endoplasmic reticulum cation P-type ATPase Cta4p is required for control of cell shape and microtubule dynamics.

J Cell Biol. 2002 Jun 10;157(6):1029-39.

Tipper DJ, Harley CA. Yeast genes controlling responses to topogenic signals in a model transmembrane protein.

Mol Biol Cell. 2002 Apr;13(4):1158-74.

Suzuki C. Immunochemical and mutational analyses of P-type ATPase Spf1p involved in the yeast secretory pathway.

Biosci Biotechnol Biochem. 2001 Nov;65(11):2405-11.

Suzuki C, Kawano M, Kashiwagi T, Arata Y, Kawasumi T, Kashiwagi Y. Lethal effect of the expression of a killer gene SMK1 in Saccharomyces cerevisiae.

Protein Eng. 2000 Feb;13(2):73-6.

Cronin SR, Khoury A, Ferry DK, Hampton RY. Regulation of HMG-CoA reductase degradation requires the P-type ATPase Cod1p/Spf1p.

J Cell Biol. 2000 Mar 6;148(5):915-24.

Suzuki C, Shimma YI. P-type ATPase spf1 mutants show a novel resistance mechanism for the killer toxin SMKT.

Mol Microbiol. 1999 May;32(4):813-23.

Kwasnicka-Crawford DA Characterization of a novel cation transporter ATPase gene (ATP13A4) interrupted by 3q25-q29 inversion in an individual with language delay. Genomics. 2005; 86(2):182-94.

Ramirez A, et al. Hereditary parkinsonism with dementia is caused by mutations in ATP13A2, encoding a lysosomal type 5 P-type ATPase. Nat Genet. 2006; 38(10):1184-1191.

Aye M, et al. Host Factors That Affect Ty3 Retrotransposition in Saccharomyces cerevisiae. Genetics; 2004 168(3):1159-76