Systematic Prediction of FFAT Motifs Across Eukaryote Proteomes Identifies Nucleolar and Eisosome Proteins With the Predicted Capacity to Form Bridges to the Endoplasmic Reticulum

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.

FFAT motif phosphorylation controls formation and lipid transfer function of inter‐organelle contacts

“…In order to identify the human proteins possessing a Phospho‐FFAT, we used an in silico approach. We designed a position weight matrix strategy which has previously served to identify conventional FFATs in many proteins (Mikitova & Levine, 2012; Murphy & Levine, 2016; Slee & Levine, 2019). It allows the identification of motifs with variations around an ideal FFAT sequence.…”

“…This analysis suggests that Phospho-FFATs and conventional ones are equally distributed in the human proteome. To further exploit this in silico approach, we sought for the presence of novel proteins containing a Phospho-FFAT motif which were not identified by the original FFAT-prediction algorithm (Mikitova & Levine, 2012;Murphy & Levine, 2016;Slee & Levine, 2019). We chose to describe three proteins of the list of 110 VAP-A/VAP-B/ MOSPD2 partners (Table EV2) that are already characterized as being involved or potentially involved in the formation of MCSs (Fig 1F).…”

VPS13D bridges the ER to Miro containing membranes

“…S3C). Three FFAT motifs are predicted with low score(Murphy and Levine, 2016; Slee and Levine, 2019) in this portion of VPS13D, but the combined mutation of all three sites did not abolish VAP-dependent recruitment to the ER, raising the possibility of an alternative, possibly indirect, way of binding (Fig. S3 ) .…”

“…(B) Sequence and score of each of the 3 predicted FFAT motifs contained in the first 1576 residues of VPS13D. *Score was calculated using a previously described algorithm; scores 3.5 and 4 are considered weak FFAT motifs (Slee and Levine, 2019). (C) Co-expression of an EGFP-tagged N-terminal fragment of VPS13D truncated at residue 1576 with the ER marker mRFP-Sec61β shows no ER recruitment (first column from the left).…”

Interactions by Disorder – A Matter of Context

“…Communication between cell compartments also depend on context. Proteins with FFAT motifs (Loewen et al, 2003) communicate between the ER and other compartments by bridging (Murphy and Levine, 2016;Costello et al, 2017;Slee and Levine, 2019). The core of the FFAT motif, EFFDAxE, found in, e.g., oxysterol-binding protein (OSBP) (Furuita et al, 2010), is an extended region of seven residues (Table 1), the second and fifth of which bind into pockets in the integral ER membrane protein VAMP-associated protein (VAP) (Kaiser et al, 2005).…”