Sphingolipids Function as Downstream Effectors of a Fungal PAQR

Abstract

The Izh2p protein from Saccharomyces cerevisiae belongs to the newly characterized progestin and adipoQ receptor (PAQR) superfamily of receptors whose mechanism of signal transduction is still unknown. Izh2p functions as a receptor for the plant PR-5 defensin osmotin and has pleiotropic effects on cellular biochemistry. One example of this pleiotropy is the Izh2p-dependent repression of FET3, a gene involved in iron-uptake. Although the physiological purpose of FET3 repression by Izh2p is a matter of speculation, it provides a reporter with which to probe the mechanism of signal transduction by this novel class of receptor. Receptors in the PAQR family share sequence similarity with enzymes involved in ceramide metabolism, which led to the hypothesis that sphingolipids are involved in Izh2p-dependent signaling. In this study, we demonstrate that drugs affecting sphingolipid metabolism, such as D-erythro-MAPP and myriocin, inhibit the effect of Izh2p on FET3. We also show that Izh2p causes an increase in steadystate levels of sphingoid base. Moreover, we show that Izh2p-independent increases in sphingoid bases recapitulate the effect of Izh2p on FET3. Finally, our data indicate that the Pkh1p and Pkh2p sphingoid base-sensing kinases are essential components of the Izh2p-dependent signaling pathway. In conclusion, our data indicate that Izh2p produces sphingoid bases and that these bioactive lipids probably function as the second messenger responsible for the effect of Izh2p on FET3.The PAQR superfamily of proteins is composed of membrane-bound receptors (Tang et al., 2005) and includes two mammalian proteins that function as receptors for the hormone adiponectin, which represents a molecular link between obesity and type II diabetes. Other members of this family sense and respond to progesterone, making them excellent candidates for the long-sought-after receptors that mediate many of the rapid nontranscriptional effects of progesterone. Because of arguable similarity to G-protein-coupled receptors, it has been proposed that PAQRs function as a new class of GPCRs (Thomas et al., 2007). However, this assignment is highly debatable, and, in reality, almost nothing is known about how these proteins transduce signals. What is needed to explore this complicated question is a simple system with which to study the functionality of PAQR receptors.We chose to use the model eukaryote, Saccharomyces cerevisiae, to study these receptors for several reasons. First, the S. cerevisiae genome encodes 4 proteins-Izh1p, Izh2p, Izh3p, and Izh4p-in the PAQR superfamily of receptors (Lyons et al., 2004), and the study of yeast orthologs as models for human proteins has been a remarkably fruitful method for characterizing poorly understood protein families

Sphingosine-1-phosphate and ceramide are associated with health and atresia of bovine ovarian antral follicles

“…Sphingolipids were extracted as described by Villa et al (2009). Briefly, 3 ml of chloroform : methanol v/v, 0.8 ml deionised water and 0.5 ml methanol were added to 500 µl of the homogenate and the mixture was maintained at 4°C overnight.…”

Membrane topology of yeast alkaline ceramidase YPC1

“…S1) indicate the exact same topology (not shown). Interestingly, Izh2p has been shown to be involved in a signaling pathway, which results in the elevation of LCBs, raising the possibility that also this group of proteins acts as ceramidases [17]. If this can be shown biochemically, it would imply that functionally and structurally similar yeast proteins are made with two opposite topologies.…”

“…To date this gene family contains 374 members in 211 species ranging from bacteria to man. A recent bioinformatics study revealed some distant similarity between alkaline ceramidases, the PAQR family of receptors, the PER1 GPI remodelase family, and several other gene families with known and unknown functions, all possessing conserved [SxxxH], [D] and [HxxxH] motifs being located at defined positions at the ends of predicted TMs 2,3 and 7 [17,21]. The members of this so-called CREST superfamily may all be metal-binding hydrolases [21].…”

“…The simultaneous presence of acyl-CoA-dependent ceramide synthases and alkaline ceramidases in the ER seems to potentially create a futile circle. Indeed, in wild type (wt) cells, the overexpression of Ypc1p and Ydc1p proteins was shown to significantly increase the levels of free LCBs and LCB-phosphates and to reduce the biosynthetic flow of long chain bases towards mature sphingolipids, whereas deletion of YPC1 caused a significant increase of mature sphingolipids as detected through metabolic labeling with [ 3 H]palmitate or [ 3 H]serine or through mass spectrometric quantification of LCBs [14,17]. The three alkaline ceramidases ACER1, ACER2 and ACER3 of humans are homologues of YPC1.…”

PAQRs: A Counteracting Force to Ceramides?: Figure 1.

“…In stark contrast, the adipose-derived factor adiponectin (Acpr30), has emerged as an insulin sensitizing protein with protective effects on the ␤-cell, cardiomyocyte and vasculature (for review, see Trujillo and Scherer, 2006). In the current issue of Molecular Pharmacology, Villa et al (2009) provide evidence that suggests that yeast homologs of adiponectin receptors may function, in part, by modulating sphingolipid metabolism.…”