All Phox Homology (PX) Domains from Saccharomyces cerevisiae Specifically Recognize Phosphatidylinositol 3-Phosphate

Abstract

Phox homology (PX) domains are named for a 130-amino acid region of homology shared with part of two components of the phagocyte NADPH oxidase (phox) complex. They are found in proteins involved in vesicular trafficking, protein sorting, and lipid modification. It was recently reported that certain PX domains specifically recognize phosphatidylinositol 3-phosphate (PtdIns-3-P) and drive recruitment of their host proteins to the cytoplasmic leaflet of endosomal and/or vacuolar membranes where this phosphoinositide is enriched. We have analyzed phosphoinositide binding by all 15 PX domains encoded by the Saccharomyces cerevisiae genome. All yeast PX domains specifically recognize PtdIns-3-P in protein-lipid overlay experiments, with just one exception (a significant sequence outlier). In surface plasmon resonance studies, four of the yeast PX domains bind PtdIns-3-P with high (micromolar range) affinity. Although the remaining PX domains specifically recognize PtdIns-3-P, they bind this lipid with only low affinity. Interestingly, many proteins with "low affinity" PX domains are known to form large multimeric complexes, which may increase the overall avidity for membranes. Our results establish that PtdIns-3-P, and not other phosphoinositides, is the target of all PX domains in S. cerevisiae and suggest a role for PX domains in assembly of multiprotein complexes at specific membrane surfaces.The phox homology (PX) 1 domain (1) was first identified in 1996 as a 130-amino acid region of homology present in two components (p40 phox and p47 phox ) of the phagocyte NADPH oxidase (phox) complex plus a wide variety of other proteins with diverse functions. Many PX domain-containing proteins are involved in vesicular trafficking, protein sorting, and lipid modification. The presence of a central PXXP motif in most (but not all) PX domains stimulated the initial suggestion that they represent binding partners for SH3 domains (1) and therefore may direct inter-and/or intramolecular protein-protein interactions. In support of this, the PX domain from p47 phox was recently shown to interact, albeit weakly, with the C-terminal SH3 domain from the same protein (2).A different class of PX domain ligand was recently reported by several groups who described binding of PX domains to phosphoinositides. Specifically, the PX domains from the yeast vacuolar t-SNARE Vam7p (3, 4), sorting nexin-3 (SNX3) (5), and p40 phox (6, 7) were all shown to bind selectively to phosphatidylinositol 3-phosphate (PtdIns-3-P), which is enriched in endosomal and vacuolar membranes (8). Isolated PX domains from p40 phox and SNX3 were found to be independently capable of localizing to endosomal structures in vivo in a manner that depends upon their ability to bind PtdIns-3-P and on PI-3-kinase activity (5-7). Similarly, analysis of deletion mutants indicated that the Vam7p PX domain is sufficient for localization of that protein to vacuoles and endosomes in yeast (3). Other phosphoinositides have been reported as the preferred ligands for certain PX domain...

Determining selectivity of phosphoinositide-binding domains

“…It should therefore only be used as a first-pass to determine whether binding to phosphoinositides (or other lipids) can be detected at all. As described in our genome-wide analyses of S. cerevisiae PH and PX domains [26,27], we have often detected phosphoinositide binding using this method that is too weak to be measured by any of the more quantitative approaches outlined later in this article. Other laboratories have reported similar findings [28].…”

“…First, the state of the phosphoinositides in the spots on the nitrocellulose filter is far from representative of that found in vivo. Second, experience has shown that in many cases phosphoinositide binding that appears robust in dot-blots cannot be detected using any of the other commonly used approaches to study lipid binding [26][27][28]. Conversely, the examples of dynamin's PH domain [31][32][33] and the PH domains from S. cerevisiae proteins Ynl047cp and Yil105cp [27,34] illustrate that phosphoinositide binding that is very weak (and only detectable with dot-blots) can nonetheless be physiologically relevant.…”

“…Lipid vesicles are immobilized on Pioneer L1 sensorchips (Biacore, Inc) essentially as described by Erb et al [26,59]. Dioleoylphosphatidylcholine (DOPC) from Sigma is codissolved with the phosphoinositide of interest at a molar ratio of 97:3 (DOPC:PtdInsP n ), in 1:1 chloroform: methanol containing 0.1% HCl.…”

The p21-activated Protein Kinase-related Kinase Cla4 Is a Coincidence Detector of Signaling by Cdc42 and Phosphatidylinositol 4-Phosphate

“…Surface plasmon resonance experiments were performed as described (32). Dioleoylphosphatidylcholine vesicles lacking or containing 3% (mol/mol) PI3P, PI4P, PI4,5P2, or PI3,5P2 were prepared and immobilized onto an L1 sensor chip using a Biacore X instrument.…”

“…Furthermore, Boi1 PH domain point mutants impaired in PI4,5P2 binding fail to function or localize to the bud cortex (31). The Cdc42 scaffold protein Bem1 possesses a phox homology domain that binds phosphatidylinositol 3-phosphate (PI3P (32)), which has a prominent role in protein trafficking to the vacuole (24).…”