Academic journal article Genetics

Linking Gene Expression in the Intestine to Production of Gametes through the Phosphate Transporter PITR-1 in Caenorhabditis Elegans

Academic journal article Genetics

Linking Gene Expression in the Intestine to Production of Gametes through the Phosphate Transporter PITR-1 in Caenorhabditis Elegans

Article excerpt

INORGANIC phosphate (Pi) is the second most abundant mineral in the human body, essential for prokaryotic and eukaryotic cell metabolism and structure. Due to the negative electrochemical potential across the cell membrane, Pi cannot cross the cell membrane by simple diffusion. The uptake of Pi into the cell in mammals is coupled to Na+ transport (Werner et al. 1994). Mammalian Na+-Pi cotransporters have been grouped into three families NaPi-I, NaPi-II, and NaPi-III, based on their sequence and structural similarity. The physiological role of NaPi-I remains to be fully established. NaPi-II plays a major role in renal Pi reabsorbtion (Werner et al 1998). NaPiIII transporters were originally identified as retroviral receptors for rat amphotropic virus and gibbon ape leukemia virus (Miller et al. 1994; Miller and Miller 1994).

NaPi-III (also called the Pit family) family proteins have homologs in various organisms from bacteria to humans. The Caenorhabditis elegans genome encodes six predicted NaPi-III genes, also named phosphate permeases (Werner and Kinne 2001). None of these have been characterized on a functional level. Type III phosphate transporters share comparable membrane topologies, including 8-12 transmembrane spanning regions.

Mouse knockout of PiT-1 was reported to be embryonic lethal (Festing et al. 2009; Beck et al. 2010). There is currently no information on PiT-2 knockout phenotypes. In budding yeast, the high-affinity Pi transport system has been shown to function under Pi-starved conditions and is composed of independently regulated Pho84p and Pho89p (Pattison-Granberg and Persson 2000). Additionally Pho89p has been demonstrated to be mainly active under alkaline pH (Zvyagilskaya et al. 2008).

At the messenger RNA (mRNA) level, members of NaPi-III transporter family are ubiquitously expressed in tissues, and their expression levels respond to extracellular Pi concentration (Werner et al. 1998). Although discovered two decades ago, to date there is very limited data available regarding the tissue and subcellular distribution of NaPi-III transporters at the protein level. Importantly, recent evidence demonstrates an important role of NaPi-III transporters in bone Pi metabolism and vascular calcification (Lau et al. 2010).

We have previously described an in vivo assay for the trafficking of fluorescently tagged yolk protein YP170a, encoded by the vitellogenin-2 (vit-2) gene (Grant and Hirsh 1999). Like endogenous YP170, the YP170:GFP fusion protein is synthesized in the C. elegans intestine and secreted basolaterally into the body cavity. From the body cavity, it is efficiently endocytosed by the oocytes using the oocyte-specific RME-2 yolk receptor (Grant and Hirsh 1999). Here, we report on a mutant isolated in our forward genetic screen for abnormally high levels of YP170:GFP accumulation in the body cavity, a phenotype usually associated with poor endocytosis of yolk by the oocytes. However, the unusual mutant described here does not affect yolk endocytosis, but rather increases expression of yolk protein genes in the intestine. Molecular cloning showed that this mutation impairs the function of the C. elegans NaPi-III transporter gene pitr-1. Surprisingly, only expression of pitr-1 in the germline, but not the intestine, restores intestinal yolk protein gene expression to normal levels, implying the existence of a feedback mechanism linking gamete production in the germline to gene expression in the intestine needed to promote embryo production.

Materials and Methods

General methods and strains

Maintenance and genetic crosses of C. elegans strains were performed according to standard protocols (Brenner 1974). All strains of C. elegans were derived from wild-type (WT) Bristol strain N2. All strains were grown at 20°, unless otherwise stated. The following C. elegans strains were obtained from the Caenorhabditis Genetics Center: unc-24(e448)IV, unc-44(e362)IV, C48A7.2(ok2116) IV/nT1[qIs51](IV;V), ppw-1(pk2505)I, and ppw-1(pk1425). …

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