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. 2019 Jan:2:1-21.
doi: 10.1177/2515256419883136. Epub 2019 Oct 30.

Systematic prediction of FFAT motifs across eukaryote proteomes identifies nucleolar and eisosome proteins with the predicted capacity to form bridges to the endoplasmic reticulum

John A Slee  1 , Timothy P Levine  2
Affiliations

Systematic prediction of FFAT motifs across eukaryote proteomes identifies nucleolar and eisosome proteins with the predicted capacity to form bridges to the endoplasmic reticulum

John A Slee et al. Contact (Thousand Oaks). 2019 Jan.

Abstract

The endoplasmic reticulum (ER), the most pervasive organelle, exchanges information and material with many other organelles, but the extent of its inter-organelle connections and the proteins that form bridges are not well known. The integral ER membrane protein VAMP-associated protein (VAP) is found in multiple bridges, interacting with many proteins that contain a short linear motif consisting of "two phenylalanines in an acidic tract" (FFAT). The VAP-FFAT interaction is the most common mechanism by which cytoplasmic proteins, particularly inter-organelle bridges, target the ER. Therefore, predicting new FFAT motifs may both find new individual peripheral ER proteins and identify new routes of communication involving the ER. Here we searched for FFAT motifs across whole proteomes. The excess of eukaryotic proteins with FFAT motifs over background was ≥0.8%, suggesting this is the minimum number of peripheral ER proteins. In yeast, where VAP was previously known to bind 4 proteins with FFAT motifs, a detailed analysis of a subset of proteins predicted 20 FFAT motifs. Extrapolating these findings to the whole proteome estimated the number of FFAT motifs in yeast at approximately 50-55 (0.9% of proteome). Among these previously unstudied FFAT motifs, most have known functions outside the ER, so could be involved in inter-organelle communication. Many of these can target well-characterised membrane contact sites, however some are in nucleoli and eisosomes, organelles previously unknown to have molecular bridges to the ER. We speculate that the nucleolar and eisosomal proteins with predicted motifs may function while bridging to the ER, indicating novel ER-nucleolus and ER-eisosome routes of inter-organelle communication.

Keywords: BOP1; NOP2/Nsun6; UBR4; VPS13; eisosome; endoplasmic reticulum; lipid transfer protein; membrane contact sites; nucleolus; short linear motif.

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Figures

Figure 1
Figure 1. The conserved VAP-FFAT interaction.
(a) A FFAT motif forms an extended loop that binds to a conserved basic face of the MSP domain of VAP. VAP also has a linker of ~100 residues that includes a coiled-coil section in some species and can maximally extend to ≥20 nm (not shown), and a tail-anchoring transmembrane helix (TMH) in the ER membrane (Murphy and Levine, 2016). The basic face of VAP’s MSP domain contains two pockets, one of which binds an aromatic residue (F/Y) and the other binds a small non-charged residue (A, C, G, S, and T). These are the 2nd and 5th residues respectively of the 7 residue core of the FFAT motif. Peptide from ORP1L FFAT peptide crystallised in complex with VAP in PDB:1Z9O (Kaiser et al., 2005). (b) The weights of the 7 elements scored by the FFAT motif. An ideal motif (6 upstream = acids, then 7 core residues =EFFDAXE) has FFAT score =0. Sub-optimal substitutions at each position in the motif are assessed by the PWM: six elements are all the positions in the core except one, the seventh element is the overall acidity of the upstream residues (Murphy and Levine, 2016).
Figure 2
Figure 2. Three percent of eukaryotic proteins have FFAT scores ≤2.5
(a) Distributions of FFAT scores in human and yeast (S. cerevisiae). FFAT scores are assigned as described in Methods. The means and standard deviations are yeast: 4.6±1.1, human 4.5±1.1; the distributions are statistically similar (Chi2 0.998). B. Proportion of 6 eukaryotic proteomes at different levels of low FFAT score, from highly optimal (top, range 0.0 – 1.0) to less optimal (bottom, =2.5), including the average, un-weighted for number of proteins in each proteome, and standard deviation. C. Proportions of yeast and human proteins with motifs with highly optimal FFAT scores (range 0.0 – 1.0) showing if they are known interactors of VAP. D. Proportion of proteins identified as binding VAP in yeast and human according to their FFAT scores (range 0.0 – 6.0). Inset shows detail in the range FFAT scores 1.5 – 2.5. Dotted lines indicate the average across the two species. Identification of proteins as VAP interactors declines rapidly as FFAT score increases: 59% for FFAT score ≤1.0 (human 11/17, yeast 2/5); 8% for 1.0<FFAT score≤2.0 (human 17/180, yeast 3/62); and 3% for 2.0<FFAT score≤3.0 (human 11/440, yeast 5/146).
Figure 3
Figure 3. Comparison of the observed level of motifs with low FFAT scores with expected levels.
A. Levels of motifs with low FFAT scores in groups of bacteria and archaea, and VAP-negative eukaryotes across a range of low FFAT scores, from highly optimal (0.0 – 1.0, top left) to less optimal (=2.5, bottom right). In each case, average levels in eukaryotes are included for comparison. For species names see Methods. All data points are the average across all proteins in that class. B. Levels of motifs with low FFAT scores in randomised yeast proteins (10 randomisations, showing means and standard deviations) compared to observed levels in yeast, from highly optimal (0.0 – 1.0, left) to less optimal (=2.5, right). C. Predicted total number of FFAT motifs in yeast and human proteomes, based on the background of false positives (numbers in red as “xyzFP”) calculated from randomisation data. Motifs are subdivided into those already known (dark shading) and those predicted (light shading, black numbers).
Figure 4
Figure 4. Cellular distribution of proteins with FFAT motifs
Likely location of 24 yeast proteins with FFAT motifs, including 4 known, all 11 with FFAT scores ≤1.5, and a sample of 9 others with FFAT scores 2.0 or 2.5. Each protein is coloured by the significance of the Chi2 test of the number of motifs in its orthologue family over background (Table 2, and see Key): pink = borderline significance, increasingly dark grey = increasing significance. To reflect known localisation to multiple contact sites, Ent3, Epo1, Osh1, Sec2 and Vps13 appear more than once. Contacts between the ER and other cellular components are coloured according to the level of prior knowledge (see Key): light yellow (VAP-FFAT bridge known in yeast); dark yellow (VAP-FFAT bridge known but not in yeast); orange (contact known, but no VAP-FFAT bridge in any cell type); red (contact unknown). Abbreviation: MVE – multivesicular endosome. Note that no yeast FFAT proteins have been predicted yet that target lipid droplets or peroxisomes.
Figure 5
Figure 5. The FFAT motif in Nop2p is located in a helix
A. FFAT motifs occur at three sites in the fungal family of Nop2 orthologues. TOP: overall domain map, showing the positon of motifs relative to the central methyl-transferase domain and to the intrinsically disordered N-terminus, with its conserved basic (blue) and acidic (red) IDRs. The three locations for the 13 residues of motifs with FFAT scores ≤2.5 are indicated with yellow bars, showing the proportion of orthologues they are found there., including one motif found to the C terminus of the folded domain. BOTTOM: the methyl-transferase domain is divided into 3 parts: N-terminal extension (“N”, grey, dashed border) ending in the FFAT motif core (7 residues, dark grey), RRM motif (blue) and catalytic core (rainbow green to red); the domain associates with a PUA domain (rainbow blue to green, dotted border). In yeast the PUA domain is in a separate protein, encoded by Nip7p, while animal homologues contain a PUA domain in cis. B. Side and top views of methyl-transferase domain of NSun6, a human homologue of Nop2, shown with colours and borders as in A, from crystal structure 5WWQ (Liu et al., 2017). Four residues of the FFAT motif are folded into a single turn of a helix and then 3 in a linker to the RRM motif.
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