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

Organelles are physically connected in membrane contact sites. The endoplasmic reticulum possesses three major receptors, VAP-A, VAP-B, and MOSPD2, which interact with proteins at the surface of other organelles to build contacts. VAP-A, VAP-B, and MOSPD2 contain an MSP domain, which binds a motif named FFAT (two phenylalanines in an acidic tract). In this study, we identified a non-conventional FFAT motif where a conserved acidic residue is replaced by a serine/threonine. We show that phosphorylation of this serine/threonine is critical for non-conventional FFAT motifs (named Phospho-FFAT) to be recognized by the MSP domain. Moreover, structural analyses of the MSP domain alone or in complex with conventional and Phospho-FFAT peptides revealed new mechanisms of interaction. Based on these new insights, we produced a novel prediction algorithm, which expands the repertoire of candidate proteins with a Phospho-FFAT that are able to create membrane contact sites. Using a prototypical tethering complex made by STARD3 and VAP, we showed that phosphorylation is instrumental for the formation of ER-endosome contacts, and their sterol transfer function. This study reveals that phosphorylation acts as a general switch for inter-organelle contacts.

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

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.