YND1, a Homologue of GDA1, Encodes Membrane-bound Apyrase Required for Golgi N- andO-Glycosylation in Saccharomyces cerevisiae

Xiao-Dong Gao,Vladimir Kaigorodov,Yoshifumi Jigami

doi:10.1074/jbc.274.30.21450

Abstract

The gene for the open reading frame YER005w that is homologous to yeast Golgi GDPase encoded by the GDA1 gene was cloned and named YND1. It encodes a 630-amino acid protein that contains a single transmembrane region near the carboxyl terminus. The overexpression of the YND1 gene in the gda1 null mutant caused a significant increase in microsomal membrane-bound nucleoside phosphatase activity with a luminal orientation. The activity was equally high toward ADP/ATP, GDP/GTP, and UDP/UTP and approximately 50% less toward CDP/CTP and thiamine pyrophosphate, but there was no activity toward GMP, indicating that the Ynd1 protein belongs to the apyrase family. This substrate specificity is different from that of yeast GDPase, but similar to that of human Golgi UDPase. The Deltaynd1 mutant cells were defective in O- and N-linked glycosylation in the Golgi compartments. The overexpression of the YND1 gene complemented some glycosylation defects in Deltagda1 disruptants, suggesting a partially redundant function of yeast apyrase and GDPase. From these results and the phenotype of the Deltaynd1Deltagda1 double deletion showing a synthetic effect, we conclude that yeast apyrase is required for Golgi glycosylation and cell wall integrity, providing the first direct evidence for the in vivo function of intracellular apyrase in eukaryotic cells.

Highlights

Apyrases (EC 3.6.1.5) are known as enzymes that hydrolyze both di- and triphosphate nucleotides as well as thiamine pyrophosphate
The MNN4 gene is known to encode a positive regulator of mannosyl phosphorylation [13, 14], but the analysis of mannosylation of invertase in ⌬gda1⌬mnn4 double disruptant cells showed no effect on mannosylation when compared with that in ⌬gda1 alone
Yeast Apyrase Required for Golgi Glycosylation using a polyclonal anti-Ynd1p antibody that was not detected in the isogenic ⌬ynd1 strain (Fig. 1A)

Summary

Introduction

Apyrases (EC 3.6.1.5) are known as enzymes that hydrolyze both di- and triphosphate nucleotides as well as thiamine pyrophosphate. In ⌬gda cells, invertase is still heavily glycosylated, and in addition, a significant amount of GMP, as well as very low residual nonspecific nucleoside phosphatase activity unrelated to Gda1p, was detected in the Golgi membranes [8, 12] These observations suggested the importance of residual GDPase activity in the Golgi lumen of the ⌬gda mutant. The MNN4 gene is known to encode a positive regulator of mannosyl phosphorylation [13, 14], but the analysis of mannosylation of invertase in ⌬gda1⌬mnn double disruptant cells showed no effect on mannosylation when compared with that in ⌬gda alone This result strongly indicated that the remaining GMP did not arise from mannosyl phosphorylation in the Golgi lumen of ⌬gda cells and prompted us to investigate other GDPases involved in protein glycosylation.

Methods

Results

Conclusion