Tumor protein D54 binds intracellular nanovesicles via an amphipathic lipid packing sensor (ALPS) motif

Abstract

Tumor Protein D54 (TPD54) is an abundant cytosolic protein that belongs to the TPD52 family, a family of four proteins (TPD52, 53, 54 and 55) that are overexpressed in several cancer cells. Even though the functions of these proteins remain elusive, recent investigations indicate that TPD54 binds to very small cytosolic vesicles with a diameter of ca. 30 nm, half the size of classical transport vesicles (e.g. COPI and COPII). Here, we investigated the mechanism of intracellular nanovesicle capture by TPD54. Bioinformatical analysis suggests that TPD54 contains a small coiled-coil followed by several amphipathic helices, which could fold upon binding to lipid membranes. One of these helices has the physicochemical features of an Amphipathic Lipid Packing Sensor (ALPS) motif, which, in other proteins, enables membrane binding in a curvature-dependent manner. Limited proteolysis, CD spectroscopy, tryptophan fluorescence and cysteine mutagenesis coupled to covalent binding of a membrane sensitive probe show that binding of TPD54 to small liposomes is accompanied by large structural changes in the amphipathic helix region. TPD54 binding to artificial liposomes is very sensitive to liposome size and to lipid unsaturation but is poorly dependent on lipid charge. Cellular investigations confirmed the key role of the ALPS motif in vesicle targeting. Surprisingly, the vesicles selected by TPD54 poorly overlap with those captured by the golgin GMAP-210, a long vesicle tether at the Golgi apparatus, which displays a dimeric coiled-coil architecture and an N-terminal ALPS motif. We propose that TPD54 recognizes nanovesicles through a combination of ALPS-dependent and -independent mechanisms.

Efficient quantification of lipid packing defect sensing by amphipathic peptides; comparing Martini 2 & 3 with CHARMM36

“…First, we will study the ALPS motif that allows curvature sensing by the ArfGAP1 protein, 30 and is also found on other proteins. [31][32][33][34] Since its discovery, ALPS has served as an important model peptide in many computational studies on curvature/lipid packing sensing, [35][36][37] also in our own group. 12,13 Second, we will include an amphipathic helix (AH) that was derived from the NS5A protein of hepatitis C virus (HCV) and discovered to sense and rupture vesicles in a size-dependent manner: small vesicles (including HCV particles themselves) were more readily ruptured than bigger ones and this size range overlaps with the diameter of many enveloped viruses (50-160 nm).…”

Integrating intracellular nanovesicles into integrin trafficking pathways and beyond

“…Two studies have examined how TPD54 binds INVs. Reynaud et al [ 27 ] found that TPD54 has sequences corresponding to four amphipathic helices (AH1-4) downstream of the coiled-coil region. The third helix (AH3) corresponds to an amphipathic lipid packing sensor (ALPS) motif which is known to govern membrane binding in a curvature-dependent manner [ 28 ].…”

“…Overall, this downstream region is disordered; however, when TPD54 protein is incubated with liposomes, TPD54 becomes ordered upon binding to the membrane. Both studies showed that TPD54 has a preference for high curvature membranes in vitro [ 24 , 27 ]. The ALPS-containing AH3 is crucial for binding [ 27 ].…”

“…Both studies showed that TPD54 has a preference for high curvature membranes in vitro [ 24 , 27 ]. The ALPS-containing AH3 is crucial for binding [ 27 ]. However, this preference only partially explains the targeting of INVs in cells.…”

“…However, this preference only partially explains the targeting of INVs in cells. Mutation of hydrophobic residues in AH2 and AH3 but not AH1 or AH4 caused mislocalization of TPD54 in cells, suggesting that AH3 is not the only determinant for INV-binding [ 27 ]. Moreover, mutation of charged residues in AH1, AH3, and AH4 all caused mislocalization in cells, but none of these affected binding to high curvature vesicles in vitro [ 24 ].…”