Identification and Characterization of a Novel Drosophila 3′-Phosphoadenosine 5′-Phosphosulfate Transporter

Akiko Kinoshita-Toyoda,Shin Kamiyama,Takaaki Uno,Hideki Yoshida,Ryu Ueda,Emi Goda,Shoko Nishihara,Hidenao Toyoda,Morio Ueyama

doi:10.1074/jbc.m605045200

Akiko Kinoshita-Toyoda, Shin Kamiyama

Open Access

https://doi.org/10.1074/jbc.m605045200

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Abstract

Sulfation of macromolecules requires the translocation of a high energy form of nucleotide sulfate, i.e. 3'-phosphoadenosine 5'-phosphosulfate (PAPS), from the cytosol into the Golgi apparatus. In this study, we identified a novel Drosophila PAPS transporter gene dPAPST2 by conducting data base searches and screening the PAPS transport activity among the putative nucleotide sugar transporter genes in Drosophila. The amino acid sequence of dPAPST2 showed 50.5 and 21.5% homology to the human PAPST2 and SLALOM, respectively. The heterologous expression of dPAPST2 in yeast revealed that the dPAPST2 protein is a PAPS transporter with an apparent K(m) value of 2.3 microm. The RNA interference of dPAPST2 in cell line and flies showed that the dPAPST2 gene is essential for the sulfation of cellular proteins and the viability of the fly. In RNA interference flies, an analysis of the genetic interaction between dPAPST2 and genes that contribute to glycosaminoglycan synthesis suggested that dPAPST2 is involved in the glycosaminoglycan synthesis and the subsequent signaling. The dPAPST2 and sll genes showed a similar ubiquitous distribution. These results indicate that dPAPST2 may be involved in Hedgehog and Decapentaplegic signaling by controlling the sulfation of heparan sulfate.

Highlights

Sulfation of proteins, proteoglycans, and lipids is involved in a variety of biological phenomena
We found that the putative transporter gene SLC35B3 encodes a phosphoadenosine 5Ј-phosphosulfate (PAPS) transporter in humans, and we named it PAPST2 (24)
DPAPST2 Genetically Interacts with the dHS6ST, dHs3st-B, and dOXT Genes in the Eye—To confirm the involvement of dPAPST2 in the sulfation of GAGs, we examined the genetic interaction between the dPAPST2 gene and another gene that is involved in the synthesis of heparan sulfate (HS); we focused on the sulfotransferase gene dHS6ST

Summary

Introduction

Proteoglycans, and lipids is involved in a variety of biological phenomena. A mutation in a gene encoding Drosophila N-deacetylase/N-sulfotransferase (sulfateless, sfl ) causes defects in Wingless (4) and fibroblast growth factor signaling (5). The sll gene is required for the determination of the embryonic dorsal/ventral axis, possibly for the activation of the signaling cascade that is initiated by the product of the putative sulfotransferase gene pipe (7, 10). In the Drosophila embryonic salivary glands, both sll and the PAPS synthase gene (papss) are required for the production of sulfated macromolecules (7, 11, 12). This study aimed at identifying a novel Drosophila gene involved in the sulfation pathway. The dPAPST2 aminoglycan; GMR, glass multiple reporter; HA, influenza hemagglutinin epitope; HS, heparan sulfate; IR, inverted repeat; mAb, monoclonal antibody; papss, PAPS synthase; RpL32, ribosomal protein L32; RNAi, RNA interference; S2, Schneider 2; sll, slalom; dsRNA, double-stranded RNA. We demonstrated the possibility that dPAPST2 plays a role in the sulfation of HS

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