Functional characterization of the three Caenorhabditis elegans orthologs of the human Parkinson's disease-associated gene PARK9/ATP13A2

P-type ATPases are an ancient family of transmembrane proteins that are found in genomes of eukaryotes as well as prokaryotes. These transporters use the energy derived from hydrolysis of ATP to actively transport substrates from one side of a lipid bilayer to the other. Missense-mutations in ATP13A2, one of the four human P5B ATPases, lead to early-onset Parkinson’s disease (Kufor-Rakeb Syndrome). The genome of the model nematode Caenorhabditis elegans encodes three P5B ATPases: CATP-5, CATP-6 and CATP-7. This dissertation aims to provide a basic characterization of all three paralogous P5B ATPases of C. elegans with regard to expression, function and phenotypic analysis of deficient animals. In addition, this dissertation describes novel revertant mutations of gon-2(lf), a genetic interactor of CATP-6. The phylogenetic analysis presented in Chapter 1 suggests that the paralogous P5B ATPases of C. elegans evolved from a common ancestral protein after the origin of the Caenorhabditis clade. The sequence alignment of all three C. elegans P5B ATPases shows a high degree of similarity in the M4 transmembrane domain, which is thought to be the putative substrate interaction region. Therefore, CATP-5, CATP-6 and CATP-7 are likely to have the same substrate specificity and thus fulfill the same transport function. In addition, Chapter 1 provides a detailed characterization of all three C. elegans P5B ATPases with regard to spatiotemporal expression pattern and subcellular localization in living animals, by using state-of-the-art CRISPR/Cas9 mediated recombination. Although each nematode P5B ATPase has a unique expression pattern, there is significant spatiotemporal overlap. In some tissues, each protein localizes to a different subcellular compartments e.g. early endosomes vs. plasma membrane. Whereas in other tissues they localize to the same compartment, e.g. the plasma membrane. Unlike its human ortholog, ATP13A2, CATP-6 does not localize to lysosomes. By using CRISPR/Cas9 to generate a KO allele of CATP-7, it became possible to construct and analyze double and triple mutant strains involving catp-5(0), catp-6(0) and catp-7(dx189). The double mutants, catp-6(0); catp-5(0) and catp-7(dx189) catp-6(0) exhibit synthetic defects in germline proliferation and are often sterile. In catp-7(dx189) catp-6(0); catp-5(0) triple mutants, this synthetic sterility is strongly enhanced, and none of the animals produce progeny. Chapter 2 describes the results of transgene complementation tests to rescue the corresponding phenotypes. The ability of each protein to undergo autophosphorylation is crucial in order to rescue the various mutant phenotypes, including the reestablishment of norspermidine sensitivity. CATP-6 and CATP-7 are redundantly required for the development of the somatic gonadal tissues, since expression of either gene product in somatic cells is sufficient to rescue sterility of catp-7(dx189) catp-6(0) double mutants. CATP-5 and CATP-6 are redundantly required for germline proliferation; the sterile phenotype of catp-6(0); catp-5(0) double mutants can be rescued by germline expression of either protein. Artificial sheath cell specific expression of CATP-5 can also bypass the requirement for CATP-6 in catp-6(0); catp-5(0) double mutants; this suggests that the putative transport substrate can probably be transferred from the somatic sheath cells to the germline via gap junctions. In addition, overexpression of CATP-7::GFP can substitute for CATP-6 in the genetic background of gon-2(lf); catp-6(lf); gem-1(gf). Therefore, all three nematode P5B ATPases can probably perform the same function(s), but in different tissues and/or subcellular compartments. Chapter 3 describes 10 revertant mutations of gon-2(lf) that affect 9 different residues within GON-2. Nine of the revertant mutations of gon-2(lf) are located in the N-terminal cytosolic domain and 1 mutation is located in the C-terminal cytosolic domain. Chapter 3 also describes a single gon-2 mutant allele that reverts the Mg2+-hypersensitive phenotype of gtl-2(0) mutant animals. This mutation is located in the cytosolic C-terminal TRP-domain. Six representative revertant mutations of gon-2(lf) were tested via scoring the phenotypes of segregants from homozygous and heterozygous animals. The suppression of the Gon phenotype by the 6 revertant mutations ranges from 85% (dx99) to 100% (dx146).