Identification of a cDNA/Protein Leading to an Increased P i -uptake in Xenopus laevis Oocytes

Abstract

In a previous report we documented an increased Na(+)-dependent transport of inorganic phosphate (P(i)) in Xenopus laevis oocytes injected with mRNA isolated from rabbit duodenum (Yagci et al., Pfluegers Arch. 422:211-216, 1992; ref 24). In the present study we have used expression cloning in oocytes to search for the cDNA/mRNA involved in this effect. The identified cDNA (provisionally named PiUS; for P(i)-uptake stimulator) lead to a 3-4-fold stimulation of Na(+)-dependent P(i)-uptake (10ng cRNA injected, 3-5 days of expression). Na(+)-independent uptake of P(i) was also affected but transport of sulphate and L-arginine (in the presence or absence of sodium) remained unchanged. The apparent K(m)-values for the induced Na(+)-dependent uptake were 0.26 +/- 0.04 mM for P(i) and 14.8 +/- 3.0 mM for Na+. The 1796 bp cDNA codes for a protein of 425 amino acids. Hydropathy analysis suggests a lack of transmembrane segments. In vitro translation resulted in a protein of 60 kDa and provided no evidence of glycosylation. In Northern blots a mRNA of approximately 2 kb was recognized in various tissues including different intestinal segments, kidney cortex, kidney medulla, liver and heart. Homology searches showed no similarity to proteins involved in membrane transport and its control. In conclusion, we have cloned from a rabbit small intestinal cDNA library a novel cDNA encoding a protein stimulating P(i)-uptake into Xenopus laevis oocytes, but which is not a P(i)-transporter itself.

PiUS (Pi uptake stimulator) is an inositol hexakisphosphate kinase

“…2), similar to the apparent molecular weight of about 60 kDa reported for PiUS [13]. This enzyme preparation was tested for kinase activity against some of its possible substrates.…”

“…The a¤nity of the enzyme for InsP 6 is lower than that of the enzyme puri¢ed from brain by Voglmaier et al [10] (their reported K m was 0.7 þ 0.192 WM). However, as with the Ins-(1,4,5)P 3 3-kinases [3], it may be that a brain-speci¢c isoform exists and given that the source of PiUS is an intestinal epethlial library [13], we are most likely studying an enzyme from peripheral tissue.…”

“…A cDNA encoding rabbit PiUS in the pSPORT vector [13] was used as a PCR template with the following primer set: 5P-CGGGATCC-ATGAGCCCAGCCTTCAGG-3P/5P-ACGCGTCGACTCACTCCC-CACTGTCCTCAC-3P. Underlined regions correspond to BamHI and SalI restriction sites, respectively.…”

“…A few years ago, we cloned a cDNA from a rabbit enterocyte library that caused the stimulated uptake of inorganic phosphate when injected into Xenopus oocytes and called it phosphate uptake stimulator (PiUS), but its mode of action was unknown [13]. As we describe below, we have found a signi¢cant similarity between part of this protein and the catalytic domain of Ins(1,4,5)P 3 3-kinase and have therefore sought an inositol phosphate substrate for the bacterially expressed PiUS enzyme.…”

Eukaryotic Phosphate Homeostasis: The Inositol Pyrophosphate Perspective

“…The IP6K2 gene was cloned almost 20 years ago, while searching for a novel mammalian intestinal phosphate transporter and was identified as PiUS (Phosphate inorganic Uptake Stimulator) [59]. When PiUS RNA was injected into Xenopus oocytes, it stimulated the cellular uptake of radioactive phosphate.…”