Analysis of the Key Elements of FFAT-Like Motifs Identifies New Proteins That Potentially Bind VAP on the ER, Including Two AKAPs and FAPP2

Abstract

BackgroundTwo phenylalanines (FF) in an acidic tract (FFAT)-motifs were originally described as having seven elements: an acidic flanking region followed by 6 residues (EFFDA–E). Such motifs are found in several lipid transfer protein (LTP) families, and they interact with a protein on the cytosolic face of the ER called vesicle-associated membrane protein-associated protein (VAP). Mutation of which causes ER stress and motor neuron disease, making it important to determine which proteins bind VAP. Among other proteins that bind VAP, some contain FFAT-like motifs that are missing one or more of the seven elements. Defining how much variation is tolerated in FFAT-like motifs is a preliminary step prior to the identification of the full range of VAP interactors.ResultsWe used a quantifiable in vivo system that measured ER targeting in a reporter yeast strain that over-expressed VAP to study the effect of substituting different elements of FFAT-like motifs in turn. By defining FFAT-like motifs more widely than before, we found them in novel proteins the functions of which had not previously been directly linked to the ER, including: two PKA anchoring proteins, AKAP220 and AKAP110; a family of plant LTPs; and the glycolipid LTP phosphatidylinositol-four-phosphate adaptor-protein-2 (FAPP-2).ConclusionAll of the seven essential elements of a FFAT motif tolerate variation, and weak targeting to the ER via VAP is still detected if two elements are substituted. In addition to the strong FFAT motifs already known, there are additional proteins with weaker FFAT-like motifs, which might be functionally important VAP interactors.

Phosphoregulation of the Ceramide Transport Protein CERT at Serine 315 in the Interaction with VAMP-associated Protein (VAP) for Inter-organelle Trafficking of Ceramide in Mammalian Cells

“…3A and 4E). Serine/threonine residues are frequently observed in the flanking region of FFAT motifs (21). Similar scenarios on the adjacent phosphorylation may occur in various FFAT motif-containing proteins to regulate their interaction with VAP.…”

“…Mikitova and Levine (21) showed that many proteins have FFAT-like motifs, which are similar, but with one or two suboptimal substitutions, to the canonical FFAT motifs (EFFDAXE). They further suggested that, although an FFATlike motif (TFFSAN) could not interact with VAP, the replacement of neutral amino acids to acidic amino acids in the motif (e.g.…”

“…The canonical FFAT motif has been suggested to divide into two regions, a core region composed of 7 amino acids EFFDAXE and an acidic flanking region around the core region, and that also the CERT-VAP interaction may start with an initial electrostatic interaction between the acidic flanking region and basic electropositive face of VAP followed by binding of the core region with VAP in a "lock-and-key" manner (20,21). A phosphoproteome study by Olsen et al (22) revealed the phosphorylation of CERT at serine 315 (Ser-315) in an acidic flanking region of the FFAT motif (Fig.…”

STARD3/STARD3NL and VAP make a novel molecular tether between late endosomes and the ER

“…2A) revealed the presence of a conserved FFAT-like motif at the C-terminal end of the MENTAL domain. Like other FFATlike motifs, this motif is centered on two aromatic residues with an adjacent tract of acidic amino acids (Mikitova and Levine, 2012). However, this motif differs from most other FFAT motifs as the acidic tract does not precede but follows the FF.…”

Phosphoinositides: Tiny Lipids With Giant Impact on Cell Regulation

“…The recently solved structure of the FAPP1 PH domain identified separate binding sites for binding of these two regulatory components (587, 885). An important common feature of all of these lipid-transfer proteins is the presence of a FFAT [two phenylalanines (FF) in an acidic tract] motif that provides an ER-association signal via interaction with ER-localized VAP (VAMP-associated) proteins (747,931,1049). Both the FFAT domain and the PH domain is essential for the lipid transfer (but not lipid cargo binding) functions of the proteins (564, 769, 1210), although the exact mechanism of how PtdIns4P regulates this process remains obscure (1262).…”