Phosphatidylinositol 3-Phosphate Induces the Membrane Penetration of the FYVE Domains of Vps27p and Hrs

Abstract

The FYVE domain mediates the recruitment of proteins involved in membrane trafficking and cell signaling to phosphatidylinositol 3-phosphate (PtdIns(3)P)-containing membranes. To elucidate the mechanism by which the FYVE domain interacts with PtdIns(3)P-containing membranes, we measured the membrane binding of the FYVE domains of yeast Vps27p and Drosophila hepatocyte growth factor-regulated tyrosine kinase substrate and their mutants by surface plasmon resonance and monolayer penetration analyses. These measurements as well as electrostatic potential calculation show that PtdIns(3)P specifically induces the membrane penetration of the FYVE domains and increases their membrane residence time by decreasing the positive charge surrounding the hydrophobic tip of the domain and causing local conformational changes. Mutations of hydrophobic residues located close to the PtdIns(3)P-binding pocket or an Arg residue directly involved in PtdIns(3)P binding abrogated the penetration of the FYVE domains into the monolayer, the packing density of which is comparable with that of biological membranes and large unilamellar vesicles. Based on these results, we propose a mechanism of the membrane binding of the FYVE domain in which the domain first binds to the PtdIns(3)P-containing membrane by specific PtdIns(3)P binding and nonspecific electrostatic interactions, which is then followed by the PtdIns(3)P-induced partial membrane penetration of the domain.FYVE domains are small (70 -80 amino acids) cysteine-rich domains that bind two zinc ions (1, 2). They are named for the first letters of the first four proteins in which they were identified: Fablp, YOTB, Vac1p, and early endosomal antigen 1 (EEA1).1 A functional role for the FYVE domain was first recognized when it was found to facilitate the localization of EEA1 to endosomes (3). Thereafter, it has been demonstrated that the FYVE domain specifically binds phosphatidylinositol 3-phosphate (PtdIns(3)P) in vitro (4, 5). PtdIns(3)P is constitutively present in eukaryotic cells, and the majority of PtdIns(3)P in mammalian cells is produced by class III phosphoinositide 3-kinase (6). PtdIns(3)P is thought to be involved in vesicle trafficking as mutations and inhibition of phosphoinositide 3-kinase have caused mis-sorting of vacuolar proteins, changes in vacuole morphology, and defects in the endocytic pathway (7). As expected from proposed roles of PtdIns(3)P, it has been found in specific subcellular locales, including the cytoplasmic face of early endosomes and internal vesicles of multivesicular bodies (6). Consistent with its in vitro PtdIns(3)P specificity, the FYVE domain has been also shown to be localized to these membrane sites in vivo (8, 9). Recent structural analyses of several FYVE domains (10 -13), a crystallographic structure analysis of EEA1-FYVE in particular (13), have elucidated the mechanism by which FYVE domains specifically recognize the PtdIns(3)P molecule. However, less is known about the mechanism by which FYVE domains interact with and are targeted to P...

Determining selectivity of phosphoinositide-binding domains

“…Similarly, estimates for PtdIns3P binding by the Hrs1 FYVE domain are within 2-fold when comparing results from SPR (K D = 4.8 µM [57]) and centrifugation assays (K D = 2.5 µM [46]). By contrast, using k off /k on ratios, Cho and colleagues report a 100-fold higher apparent affinity for the Hrs1/PtdIns3P interaction [58] than we have measured, the origin (or validity) of which is not clear. Indeed, SPR-based K D estimates for phosphoinositide binding by PH and other domains that rely upon determination of kinetic parameters tend, in general, to overestimate affinities by 10-100 (or more) fold compared with our equilibrium-based determinations.…”

pH-dependent Binding of the Epsin ENTH Domain and the AP180 ANTH Domain to PI(4,5)P2-containing Bilayers

“…Concurrently a decrease of k a and an increase of k d were observed, most likely due to the reduced electrostatic interactions and a lesser degree of membrane penetration by the ENTH domain. 19,20 A similar enhancement of the membrane binding affinity upon acidification was seen for the AP180 ANTH domain. We monitored the ANTH domain association with PtdIns(4,5)P 2 -containing vesicles at pH 6.0, 7.4, and 8.0.…”

Phosphoinositides in Constitutive Membrane Traffic

“…Thus, in EEA1, specificity of the head groupbinding pocket is supplemented by nonspecific hydrophobic interactions as well as strengthened by being bivalent. A similar mode of binding has been reported for the FYVE domains of Vps27p and Hrs (335), and it is likely that FYVE domains, like PH and PX domains, use multiple contacts to achieve proper membrane location.…”