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)

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)

Novel Alleles of gon-2, a C-elegans Ortholog of Mammalian TRPM6 and TRPM7, Obtained by Genetic Reversion Screens

TRP (Transient Receptor Potential) cation channels of the TRPM subfamily have been found to be critically important for the regulation of Mg2+ homeostasis in both protostomes (e.g.,the nematode, C. elegans, and the insect, D. melanogaster) and deuterostomes (e.g.,humans). Although significant progress has been made toward understanding how the activities of these channels are regulated, there are still major gaps in our understanding of the potential regulatory roles of extensive, evolutionarily conserved, regions of these proteins. The C. elegans genes, gon-2, gtl-1 and gtl-2, encode paralogous TRP cation channel proteins that are similar in sequence and function to human TRPM6 and TRPM7. We isolated fourteen revertants of the missense mutant, gon-2(q338),and these mutations affect nine different residues within GON-2. Since eight of the nine affected residues are situated within regions that have high similarity to human TRPM1, 3, 6 and 7, these mutations identify sections of these channels that are potentially critical for channel regulation. We also isolated a single mutant allele of gon-2 during a screen for revertants of the Mg2+-hypersensitive phenotype of gtl-2(-) mutants. This allele of gon-2 converts a serine to phenylalanine within the highly conserved TRP domain, and is antimorphic against both gon-2(+) and gtl-1 (+). Interestingly, others have reported that mutation of the corresponding residue in TRPM7 to glutamate results in deregulated channel activity

Overlapping expression patterns and functions of three paralogous P5B ATPases in Caenorhabditis elegans

P5B ATPases are present in the genomes of diverse unicellular and multicellular eukaryotes, indicating that they have an ancient origin, and that they are important for cellular fitness. Inactivation of ATP13A2, one of the four human P5B ATPases, leads to early-onset Parkinson's disease (Kufor-Rakeb Syndrome). The presence of an invariant PPALP motif within the putative substrate interaction pocket of transmembrane segment M4 suggests that all P5B ATPases might have similar transport specificity;however, the identity of the transport substrate(s) remains unknown. Nematodes of the genus Caenorhabditis possess three paralogous P5B ATPase genes, catp-5, catp-6 and catp-7, which probably originated from a single ancestral gene around the time of origin of the Caenorhabditid clade. By using CRISPR/Cas9, we have systematically investigated the expression patterns, subcellular localization and biological functions of each of the P5B ATPases of C. elegans. We find that each gene has a unique expression pattern, and that some tissues express more than one P5B. In some tissues where their expression patterns overlap, different P5Bs are targeted to different subcellular compartments (e.g., early endosomes vs. plasma membrane), whereas in other tissues they localize to the same compartment (plasma membrane). We observed lysosomal co-localization between CATP-6::GFP and LMP-1::RFP in transgenic animals;however, this was an artifact of the tagged LMP-1 protein, since anti-LMP-1 antibody staining of native protein revealed that LMP-1 and CATP-6::GFP occupy different compartments. The nematode P5Bs are at least partially redundant, since we observed synthetic sterility in catp-5(0);catp-6(0) and catp-6(0) catp-7(0) double mutants. The double mutants exhibit defects in distal tip cell migration that resemble those of ina-1 (alpha integrin ortholog) and vab-3 (Pax6 ortholog) mutants, suggesting that the nematode P5Bs are required for ina-1and/or vab-3 function. This is potentially a conserved regulatory interaction, since mammalian ATP13A2, alpha integrin and Pax6 are all required for proper dopaminergic neuron function

Dosage Testing of Intragenic Revertant Mutations.

<p>Allele configuration is for animals derived from selfing or crosses, as described in Materials and Methods. Full genotypes of parental strains are listed in Materials and Methods. Vulvaless (Vul) animals have a severe gonadogenesis (Gon) phenotype, whereas Everted vulva (Evul) animals have a less severe defect in gonad development. Animals scored as wild type (WT) based on vulva morphology were also usually fertile.</p><p>¹ EJ1190</p><p>² EJ556</p><p>³ EJ959</p><p>⁴ EJ922</p><p>⁵ EJ1193</p><p>⁶ EJ557</p><p>⁷ EJ1021</p><p>Dosage Testing of Intragenic Revertant Mutations.</p

Topology map of GON-2 (2032aa) with locations and aa changes of mutations.

<p>The inactivating mutations, <i>q388</i> and <i>dx87</i>, are shown in bold.</p

TRP protein alignment from <i>gon-2(dx96)—gon-2(dx150)</i>.

<p>TRP protein alignment from <i>gon-2(dx96)—gon-2(dx150)</i>.</p

Alignment of representative TRP channel protein sequences near the site of the <i>gon-2(dx146)</i> mutation.

<p>The following GenBank accession numbers (or UniProt identifiers) were used to retrieve amino acid sequences of proteins shown in alignments. Human proteins: TRPV1 Q8NER1, TRPV3 Q8NET1, TRPC5 Q9UL62, TRPM1 NP_001238949, TRPM2 XP_011528036, TRPM3 NP_060132, TRPM4 NP_060106, TRPM5 NP_055370, TRPM6 NP_060132, TRPM7 NP_060142, TRPM8 NP_076985. <i>C</i>. <i>elegans</i> proteins: GON-2 CAB02303, GTL-1 CAA92726, GTL-2 CAB00861. <i>Drosophila melanogaster</i> protein: DTRPM A8DYE2.</p

TRP protein alignment near <i>gon-2(dx99)</i>.

<p>TRP protein alignment near <i>gon-2(dx99)</i>.</p

Effects of <i>gon-2(dx87)</i> on <i>gon-2(+)</i> Activity and in Response to Different Divalent Cation Concentrations.

<p>Crossing schemes, full parental genotypes and culture conditions are described in Materials and Methods. Supplemental ion concentrations (in mM) are indicated. It should be noted that all strains were propagated on living <i>E</i>. <i>coli</i>, which contains at least trace amounts of Ca²⁺ and Mg²⁺, even when grown on divalent cation depleted medium.</p><p>Effects of <i>gon-2(dx87)</i> on <i>gon-2(+)</i> Activity and in Response to Different Divalent Cation Concentrations.</p