Zinc is an essential nutritional factor involved in many key biological processes. However, the physiological function of zinc transporters at the organismal level is not well characterized. Early embryonic lethality of Znt1 knockout mice precludes functional analysis of the role of ZnT1 in dietary zinc absorption. Here, we report the identification and characterization of the Drosophila ZnT1 orthologue, dZnT1, for its role in Drosophila dietary zinc absorption. In cell culture, dZnT1 promoted zinc transport to reduce cytoplasmic zinc levels. Ubiquitous RNA interference of dZnT1 in Drosophila resulted in developmental arrest under restriction of dietary zinc, while dZnT1-overexpressing flies exhibited hypersensitivity to zinc. dZnT1 was prominently expressed in restricted regions of the midgut and exhibited a distribution on the basolateral membrane of the enterocytes. Gut-specific silencing of dZnT1 was sufficient to evoke lethality under zinc scarcity. Human ZnT1, but not ZnT7 or ZnT4, could rescue the zinc-acquiring defects caused by dZnT1 silencing. Taken together, our results proved that dZnT1 is a key zinc transporter in dietary zinc absorption, functioning by pumping zinc out of the enterocytes across the basolateral membrane. This study will be helpful in understanding the fundamental process of acquiring dietary zinc in higher eukaryotes.

Eukaryotic organisms typically express multiple type IV P-type ATPases (P4-ATPases), which establish plasma membrane asymmetry by flipping specific phospholipids from the exofacial to the cytosolic leaflet. Saccharomyces cerevisiae, for example, expresses five P4-ATPases, including Neo1, Drs2, Dnf1, Dnf2, and Dnf3. Neo1 is thought to be a phospholipid flippase, although there is currently no experimental evidence that Neo1 catalyzes this activity or helps establish membrane asymmetry. Here, we use temperature-conditional alleles (neo1 ts ) to test whether Neo1 deficiency leads to loss of plasma membrane asymmetry. Wild-type (WT) yeast normally restrict most of the phosphatidylserine (PS) and phosphatidylethanolamine (PE) to the inner cytosolic leaflet of the plasma membrane. However, the neo1-1 ts and neo1-2 ts mutants display a loss of PS and PE asymmetry at permissive growth temperatures as measured by hypersensitivity to pore-forming toxins that target PS (papuamide A) or PE (duramycin) exposed in the extracellular leaflet. When shifted to a semi-permissive growth temperature, the neo1-1 ts mutant became extremely hypersensitive to duramycin, although the sensitivity to papuamide A was unchanged, indicating preferential exposure of PE. This loss of asymmetry occurs despite the presence of other flippases that flip PS and/or PE. Even when overexpressed, Drs2 and Dnf1 were unable to correct the loss of asymmetry caused by neo1 ts . However, modest overexpression of Neo1 weakly suppressed loss of membrane asymmetry caused by drs2⌬ with a more significant correction of PE asymmetry than PS. These results indicate that Neo1 plays an important role in establishing PS and PE plasma membrane asymmetry in budding yeast.

In eukaryotic microbes, little is known about signals that inhibit the proliferation of the cells that secrete the signal, and little is known about signals (chemorepellents) that cause cells to move away from the source of the signal. Autocrine proliferation repressor protein A (AprA) is a protein secreted by the eukaryotic microbe Dictyostelium discoideum. AprA is a chemorepellent for and inhibits the proliferation of D. discoideum. We previously found that cells sense AprA using G proteins, suggesting the existence of a G protein-coupled AprA receptor. To identify the AprA receptor, we screened mutants lacking putative G protein-coupled receptors. We found that, compared to the wild-type strain, cells lacking putative receptor GrlH (grlH¯ cells) show rapid proliferation, do not have large numbers of cells moving away from the edges of colonies, are insensitive to AprA-induced proliferation inhibition and chemorepulsion, and have decreased AprA binding. Expression of GrlH in grlH¯ cells (grlH¯/grlHOE) rescues the phenotypes described above. These data indicate that AprA signaling may be mediated by GrlH in D. discoideum.

NEO1 is an essential gene in budding yeast and belongs to a highly conserved subfamily of P-type ATPase genes that encode phospholipid flippases. Inactivation of temperature sensitive neo1ts alleles produces pleiomorphic defects in the secretory and endocytic pathways, including fragmented vacuoles. A screen for multicopy suppressors of neo1-2ts growth defects yielded YPT7, which encodes a Rab7 homolog involved in SNARE-dependent vacuolar fusion. YPT7 suppressed the vacuole fragmentation phenotype of neo1-2, but did not suppress Golgi-associated protein trafficking defects. Neo1 localizes to Golgi and endosomal membranes and was only observed in the vacuole membrane, where Ypt7 localizes, in retromer mutants or when highly overexpressed in wild-type cells. Phosphatidylethanolamine (PE) has been implicated in Ypt7-dependent vacuolar membrane fusion in vitro and is a potential transport substrate of Neo1. Strains deficient in PE synthesis (psd1Δ psd2Δ) displayed fragmented vacuoles and the neo1-2 fragmented vacuole phenotype was also suppressed by overexpression of PSD2, encoding a phosphatidylserine decarboxylase that produces PE at endosomes. In contrast, neo1-2 was not suppressed by overexpression of VPS39, an effector of Ypt7 that forms a membrane contact site potentially involved in PE transfer between vacuoles and mitochondria. These results support the crucial role of PE in vacuole membrane fusion and implicate Neo1 in concentrating PE in the cytosolic leaflet of Golgi and endosomes, and ultimately the vacuole membrane.

Background: GABA promotes terminal differentiation during late development of Dictyostelium discoideum. Results: GABA metabolism is tightly controlled and regulates early development. Conclusion: Distinct genes regulate GABA signaling during different developmental stages. Significance: This is the first systematic study of GABA metabolism in Dictyostelium discoideum and broadens our knowledge of the evolution and function of GABA metabolism.