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

ATP13A2 deficiency disrupts lysosomal polyamine export

ATP13A2 (PARK9) is a late endolysosomal transporter that is genetically implicated in a spectrum of neurodegenerative disorders, including Kufor-Rakeb syndrome—a parkinsonism with dementia1—and early-onset Parkinson’s disease2. ATP13A2 offers protection against genetic and environmental risk factors of Parkinson’s disease, whereas loss of ATP13A2 compromises lysosomes3. However, the transport function of ATP13A2 in lysosomes remains unclear. Here we establish ATP13A2 as a lysosomal polyamine exporter that shows the highest affinity for spermine among the polyamines examined. Polyamines stimulate the activity of purified ATP13A2, whereas ATP13A2 mutants that are implicated in disease are functionally impaired to a degree that correlates with the disease phenotype. ATP13A2 promotes the cellular uptake of polyamines by endocytosis and transports them into the cytosol, highlighting a role for endolysosomes in the uptake of polyamines into cells. At high concentrations polyamines induce cell toxicity, which is exacerbated by ATP13A2 loss due to lysosomal dysfunction, lysosomal rupture and cathepsin B activation. This phenotype is recapitulated in neurons and nematodes with impaired expression of ATP13A2 or its orthologues. We present defective lysosomal polyamine export as a mechanism for lysosome-dependent cell death that may be implicated in neurodegeneration, and shed light on the molecular identity of the mammalian polyamine transport system

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

TRP protein alignment near <i>gon-2(dx60)</i>.

<p>TRP protein alignment near <i>gon-2(dx60)</i>.</p