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. 2012;7(1):e30455.
doi: 10.1371/journal.pone.0030455. Epub 2012 Jan 19.

Analysis of the key elements of FFAT-like motifs identifies new proteins that potentially bind VAP on the ER, including two AKAPs and FAPP2

Veronika Mikitova  1 , Timothy P Levine
Affiliations

Analysis of the key elements of FFAT-like motifs identifies new proteins that potentially bind VAP on the ER, including two AKAPs and FAPP2

Veronika Mikitova et al. PLoS One. 2012.

Abstract

Background: Two 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.

Results: We 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).

Conclusion: All 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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The FFAT-like motif of Rab3GAP1 targets VAP.
(A) 26 amino acids including the FFAT-like motif 1EFFEC-S7 from Rab3GAP1 (see Table 1) were tagged with GFP and expressed in TLY251, which was grown in galactose to induce maximal yeast VAP (Scs2p) expression. Confocal sections through the plane of the nuclei showed the GFP construct interact with Scs2p, as fluorescence was found on the nuclear envelope (NE), in patches in the cell cortex, and in occasional strands in the cytoplasm, a pattern highly characteristic of the ER in yeast. Arrows indicate cells likely where the nucleus has been optically sectioned off the mid-line, so increasing central fluorescence and underestimating targeting. (B) As A, except GFP was tagged with the Px domain of Bem1p. This construct did not target membranes. (C) As A, except the FFAT-like sequence was altered at position 4: 1EFFAC-S7. ER targeting is barely detectable, but much weaker. (D/E/F) Fluorescence was measured across nuclear profiles from (A) (B) and (C) respectively, (examples shown in the insets, above). Line scans were re-plotted to normalise for different nuclear widths, and profiles from five nuclei are shown.
Figure 2
Figure 2. FFAT-like motifs in two AKAPs.
(A) 29 amino acids from AKAP220 including the FFAT-like motif 1EFFDS-D7 (see Table 1) were expressed as in Figure 1A, together with variants at position 5: 1EFFDE-D7 and 1EFFDA-D7. The wild-type (WT) sequence showed moderate targeting, which was lost with E5 and enhanced by A5. (B) 32 amino acids from AKAP110 including the FFAT-like motif 1DFLTA-E7 (see Table 1) were expressed as (A), together with variants at position 4: 1 DFLDA-E 7 and 1 DFLAA-E 7. The wild-type (WT) sequence showed weak targeting, which was enhanced by D4 and lost with A4.
Figure 3
Figure 3. FFAT-like motifs in plant ORPs.
(A–D) Aligned FFAT-like motifs from (A) the subfamily of StART proteins related to Edr2 in Arabidopsis thaliana (At); as shown in the diagram, this region is between the StART domains and DUF1336, which are domains of unknown function structurally related to galectins. (B) ORPs. FFAT-like motifs, which occur upstream of ORP domains. Top: in At and homologues in other plants: Gm – Glycine max; Os – Oryza sativa; Zm – Zea mays; Vv – Vitis vinifera; Pi – Petunia integrifolia; bottom: in other species, as listed. (C) homologues of rabphilin-11from human, fish (one each) and At (x7). These motifs are at the extreme amino-termini. (D) Opi1p in the fungus Ashbya gossypii. (E) 34 amino acids from Orp1c (Gm) including the FFAT-like motif 1AFFDT-D7 were expressed as in Figure 1A, together with variants 1AAADT-D7 and 1 KFFDT-D 7. The wild-type (WT) sequence showed moderate targeting, which was lost with 2AA3 and reduced with K1. (F) 32 amino acids from Orp2a (At) including the FFAT-like motif 1SFHDT-E7 were expressed as in (E) (left hand panel). We also expressed the dimer of this motif, and a dimer of a mutant: 1SAADT-E7 (middle and right panels, images are at 2-fold lower magnification. The wild-type (WT) monomer failed to target, but moderate targeting was obtained upon dimerization, which was lost with 2AA3.
Figure 4
Figure 4. FAPP2 in mammals has a weak FFAT motif.
(A) An alignment of ≤60 a.a. from the region at the amino-terminus of 7 divergent GLTP sequences and the related region near the middle of 7 divergent FAPP2 sequences (see diagram, top). Three motifs are shown where the core 2FFD4 is partly conserved. (B) Diagram of the aligned sequences from (A), where the FFAT-like motifs are shown as logos, and those individual motifs that meet minimal criteria are shown as boxes, where darker shading indicates closer resemblance to the optimal motif (see Tables S2 and S3). The best FFAT-like motif lies at different positions in different species. The most amino-terminal motif in human FAPP2 is quite weak. (C) 32 amino acids from FAPP2 in the opossum M. domestica including the FFAT-like motif 1TFFSA-N7 were expressed as in Figure 1A, together with variants 1 TFFDA-N 7 and 1 EFFDA-N7. While the wild-type (WT) sequence did not target, the variants with D4 targeted weakly. (D) The dimer of 37 amino acids including the FFAT-like motif from human FAPP2 with two substitutions 1 EFFDT-N7 (see Table 1) were expressed as in (C), together with a dimer carrying a third change 1 EFFDA-N7, and a tetramer of 1TFFDT-N7. While the dimer with E1/D4/T5 failed to target, the E1/D4/A5 version targeted moderately, and the tetramer with D4 targeted weakly.
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References

    1. Behnia R, Munro S. Organelle identity and the signposts for membrane traffic. Nature. 2005;438:597–604. - PubMed
    1. Singer SJ, Nicolson GL. The fluid mosaic model of the structure of cell membranes. Science. 1972;175:720–731. - PubMed
    1. Carlton JG, Cullen PJ. Coincidence detection in phosphoinositide signaling. Trends Cell Biol. 2005;15:540–547. - PMC - PubMed
    1. Miller JD, Tajima S, Lauffer L, Walter P. The beta subunit of the signal recognition particle receptor is a transmembrane GTPase that anchors the alpha subunit, a peripheral membrane GTPase, to the endoplasmic reticulum membrane. J Cell Biol. 1995;128:273–282. - PMC - PubMed
    1. Biederer T, Volkwein C, Sommer T. Degradation of subunits of the Sec61p complex, an integral component of the ER membrane, by the ubiquitin-proteasome pathway. Embo J. 1996;15:2069–2076. - PMC - PubMed

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