Abstract
Extracellular UDP-sugars promote cellular responses by interacting with widely distributed P2Y(14) receptors, but the mechanisms by which these molecules are released from cells are poorly understood. Given the active role of UDP-sugars in glycosylation reactions within the secretory pathway, we hypothesized that UDP-sugar release includes an exocytotic component. This hypothesis was tested by assessing the contribution of endoplasmic reticulum (ER)/Golgi-resident UDP-GlcNAc transporters to the cellular release of their cognate substrates. A sensitive and highly selective assay for UDP-GlcNAc mass was developed using purified AGX2, an isoenzyme of human UDP-GlcNAc pyrophosphorylase. Robust constitutive release of UDP-GlcNAc was observed in yeast as well as in well differentiated human airway epithelial cells. The human UDP-GlcNAc transporter HFRC1 was overexpressed in human bronchial epithelial cells and was shown to localize in the Golgi and to enhance the surface expression of N-acetylglucosamine-rich glycans. HFRC1-overexpressing cells also displayed increased constitutive and hypotonic stress-stimulated release of UDP-GlcNAc. Yeast mutants lacking Yea4 (the ER UDP-GlcNAc transporter endogenously expressed in Saccharomyces cerevisiae) showed reduced UDP-GlcNAc release. Yea4-deficient cells complemented with Yea4 showed UDP-GlcNAc release rates at levels similar to or higher than wild type cells. Our results illustrate that ER/Golgi lumen constitutes a significant source of extracellular UDP-sugars and therefore plays a critical role in nucleotide sugar-promoted cell signaling.
Highlights
Grant P01-HL034322. □S The on-line version of this article contains supplemental Fig. S1. 1 To whom correspondence should be addressed: Cystic Fibrosis/Pulmonary phates [2]
Nucleotide release was associated with vesicle exocytosis and/or was impaired in cells treated with Golgi-disrupting agents or by conditions resulting in impaired vesicle trafficking/fusion (15, 16, 19 –23)
Quantification of UDP-GlcNAc Using Recombinant UDPGlcNAc Pyrophophorylase—UDP-GlcNAc pyrophosphorylase catalyzes the reversible pyrophosphorolysis of UDP-GlcNAc, according to the following reaction
Summary
Grant P01-HL034322. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. 1 To whom correspondence should be addressed: Cystic Fibrosis/Pulmonary phates [2]. The calculated rates of mucosal UDP-GlcNAc release from resting vector- and HFRC1-transformed cells were 60 and 200 fmol/min, respectively (Fig. 5A).
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