Molecular Mechanism of Membrane Docking by the Vam7p PX Domain

Abstract

The Vam7p t-SNARE is an essential component of the vacuole fusion machinery that mediates membrane trafficking and protein sorting in yeast. Vam7p is recruited to vacuoles by its N-terminal PX domain that specifically recognizes PtdIns(3)P in the bilayers, however the precise mechanism of membrane anchoring remains unclear. Here we describe a molecular basis for membrane targeting and penetration by the Vam7p PX domain based on structural and quantitative analysis of its interactions with lipids and micelles. Our results derived from in vitro binding measurements using NMR, monolayer surface tension experiments and mutagenesis reveal a multivalent membrane docking mechanism involving specific PtdIns ( The inositol headgroup of PIs is reversibly phosphorylated at three positions yielding seven isoforms, which cluster in distinct intracellular membranes. The apparent segregation of PIs is important for the fine-tuned regulation of protein and membrane traffic and also provides reliable and handy markers of organelles, such as the plasma membrane, trans-Golgi network, early endosomes, and multivesicular bodies. PIs were first recognized to function as precursors of secondary messengers in various signal transduction cascades and later as docking sites for modular effectors. A number of cytosolic protein effectors that are recruited to membranes and activated through interactions with PIs have recently been identified and structurally or biochemically characterized. The major PI binding modules include the ANTH (AP180 N-terminal homology), ENTH (epsin N-terminal homology), FERM (four-point one, ezrin, radixin, moesin), FYVE (Fab1, YOTB, Vac1, EEA1), PH (pleckstrin homology), and PX (Phox homology) domains (reviewed in Refs. 2-5, 50).The PX domain was first identified within a set of eukaryotic proteins in 1996 and named after the two phagocyte NADPH oxidase subunits, p40 phox and p47 phox (7). Since then, it has been found in at least 69 human and 15 yeast signaling proteins, protein kinases, PtdIns kinases and phospholipases (SMART). These proteins are implicated in a variety of physiological events playing fundamental roles in endocytosis, protein sorting, membrane trafficking, transcription, signaling, cell growth, differentiation, and proliferation (5, 7-9). The PX domain consists of ϳ130 residues that are folded in a highly conserved three-dimensional structure despite little sequence similarity between the family members. A proline-rich region (PXXP), involved in the interaction with SH3 domains, and a set of basic residues, shown to coordinate PIs, comprise the most con-

Structural Basis for Interactions of thePhytophthora sojaeRxLR Effector Avh5 with Phosphatidylinositol 3-Phosphate and for Host Cell Entry

“…The latter can be explained by the fact that the RFLR motif is present and may contribute to PtdIns ( for PtdIns(3)P to be 2.3 µM. This value is within the range of other PtdIns(3)P-binding protein domains such as the EEA1 FYVE and the Vam7p PX domains (131,132), but is 3-10-fold higher to that estimated for Avr1b and AvrL567 using effector binding to cells and liposome binding assays (11); this higher affinity value may be explained by the presence of additional tags in the proteins tested in these assays that may contribute to the binding (134). Nonetheless, a modest affinity of a protein to phosphoinositides, such as Avh5, may be required for its further release from intracellular membranes to target other subcellular compartments (135).…”

Structural and functional characterization of the two phosphoinositide binding sites of PROPPINs, a β-propeller protein family

“…This model further predicts the insertion of two loops into the membrane, which would synergistically enhance the membrane association of PROPPINs. Participation of membrane insertion loops and nonspecific electrostatic interactions in membrane binding has already been demonstrated for several phosphoinositide binding proteins (1,43), for example, the PtdIns3P binding PX domain of Vam7p (44) and FYVE domains (45).…”

Yeast vacuole fusion: A model system for eukaryotic endomembrane dynamics

“…49,50,90 Vam7 lacks a transmembrane domain, but contains a phosphoinositide-3-phosphate (PI(3)P) binding module, a PX domain, at its N terminus, which is required for membrane binding. [91][92][93] Following its release, Vam7 is specifically recruited to the docking site. 50 Moreover, Vam7 initiates docking of vacuoles in conjunction with Ypt7, 90 while the free Q-SNARE Vam3 engages in binding to the HOPS complex, 89,94 which subsequently leads to the docking of vacuoles.…”

“…96 Lipid rearrangements are important for these events to proceed. 40,70,93,[97][98][99] SNARE pairing between vacuoles can be detected using SNARE deletion strains or assays with tagged SNAREs. 53,57,59 Each of these assays shows a Sec17-Sec18-, ATP-and Ypt7-dependent accumulation of SNARE complexes, which are also detectable when fusion inhibitors are added to the reaction.…”