A FFAT motif (FFAT being an acronym for two phenylalanines (FF) in an acidic tract [nb 1] ) is a protein sequence motif of six defined amino acids plus neighbouring residues that binds to proteins in the VAP protein family. [1]
The classic FFAT motif was defined on the basis of finding the sequence EFFDAxE in 16 different eukaryotic cytoplasmic proteins (where E = glutamate, F = phenylalanine, D = aspartate, A = alanine, x = any amino acid, according to the single letter amino acid code (see Table of standard amino acid abbreviations and properties in amino acids). In all cases, the core sequence is surrounded by regions that are rich in acids D and E (hence negatively charged), and also in residues that can acquire negative charge by phosphorylation (S and T – serine and threonine). This is the Acidic Tract of the name FFAT, and it is mainly found amino-terminal to the core motif, but also extends to the carboxy-terminal side to some extent. Also, this immediate region is almost completely devoid of basic residues (K and R – lysine and arginine).
The finding of these sequences on its own implied an important functional relationship because 13 of the 16 proteins shared the same overall function: they are lipid transfer proteins (LTPs). These include several homologs of oxysterol binding protein (OSBP, both in humans and in baker's yeast, as well as ceramide transfer protein (CERT) – previously known as Goodpasture's antigen binding protein (GPBP) or Collagen type IV alpha-3-binding protein (COL4A3BP), and Nir2/RdgB. The significance of this was enhanced by the linked finding in a proteomics study published in Nature , where all three of proteins in baker's yeast with FFAT motifs (Osh1p/Swh1p, Osh2p and Opi1p) were in protein complexes that contain Scs2p, the baker's yeast homolog of VAPA and VAPB. [2] Complexes had also been reported between OSBP and VAPA. [3]
This led to a simple hypothesis that VAP directly binds FFAT motifs, which was tested by biochemical interaction between purified components, [1] and was later confirmed by structural analysis of VAP-FFAT complexes, both by X-ray crystallography [4] and by NMR. [5] The crystallography study indicated that the parts of FFAT that interact most strongly with VAP were F2 and A5, each binding in highly conserved pockets in the major sperm protein domain of VAP, which has a large electropositive patch nearby. The NMR study indicated a “fly-casting” process, whereby a weak non-specific electrostatic interaction between VAP and the acidic tract precedes the more specific high affinity interaction with EFFDAxE.
Humans have three VAPs: VAPA, VAPB and MOSPD2. [6] All of these share a conserved major sperm protein domain in the cytoplasm anchored to the endoplasmic reticulum membrane by a largely unstructured linker leading to a transmembrane domain. MOSPD2 additionally at its amino-terminus has a lipid transfer domain in the CRAL/TRIO domain family. The main yeast homolog is Scs2p, which has the same domain architecture as VAPA and VAPB, and is also an integral membrane protein of the endoplasmic reticulum. [7]
Many of the proteins with FFAT motifs were previously not known to be targeted to the endoplasmic reticulum, with the exception of OSBP, [3] and PITPNM1 (the fly homologue of which is called RdgB). [8] Instead, they were known for their localization to other sites especially the trans Golgi network (OSBP, Osh1p and CERT) and the plasma membrane (Osh2p, Osh3p). The discovery that these proteins also targeted the endoplasmic reticulum led to a far more detailed analysis of their targeting, and revealed that all the FFAT-containing lipid transfer proteins are present at both the endoplasmic reticulum and their other target trans Golgi network or plasma membrane) at the same time, which can only be achieved by their targeting to membrane contact sites. This discovery has turned out to apply to many other lipid transfer proteins, even those that do not contain FFAT motifs. This strongly suggests that intracellular lipid traffic takes place across membrane contact sites.
At the very inception of the original, highly restricted definition (EFFDAxE), it was already evident that other amino acids could substitute at certain positions in the FFAT motifs of other homologs of OSBP, CERT and PITPNM1, in particular Y (tyrosine) in place of F at positions 2 and more so 3, also H (histidine) at position 3, and C (cysteine) or V (valine) at position 5. [1] A substituted motif was used for the crystal structure. [4] Subsequently, other proteins have been found in variants of FFAT motifs with quite divergent residues, including K (lysine) at position 3 in protrudin. [9] An attempt was made to rank FFAT-like sequences by scoring substitutions at all 6 positions of the core motif and the number of nearby acidic residues (DEST). [10] Variant, FFAT-like motifs were described in >10 new proteins, in particular in the A-kinase anchor proteins (AKAPs) AKAP3 and AKAP11 that scaffold protein kinase A and many interactors. This finding has since been confirmed by finding several members of the AKAP family and protein kinase A family in protein complexes with VAPB. [11] This indicates that cAMP signalling is yet another cellular activity involving small molecules that is regulated at membrane contact sites, along with lipid and calcium ion traffic.
Recent research revealed two new FFAT-like motifs: phospho-FFAT and FFNT (Two phenylalanines (FF) in a neutral tract). Phospho-FFAT motifs contain a serine (S) or threonine (T) at position 4 instead of aspartate (D) that is phosphorylated for interaction with VAPA and VAPB. [12] Unlike FFAT and phospho-FFAT motifs, FFNT motifs primarily interact with MOSPD1 and MOSPD3, two homologs of VAPA, VAPB and MOSPD2. [13]
The endoplasmic reticulum (ER) is a part of a transportation system of the eukaryotic cell, and has many other important functions such as protein folding. It is a type of organelle made up of two subunits – rough endoplasmic reticulum (RER), and smooth endoplasmic reticulum (SER). The endoplasmic reticulum is found in most eukaryotic cells and forms an interconnected network of flattened, membrane-enclosed sacs known as cisternae, and tubular structures in the SER. The membranes of the ER are continuous with the outer nuclear membrane. The endoplasmic reticulum is not found in red blood cells, or spermatozoa.
A transmembrane domain (TMD) is a membrane-spanning protein domain. TMDs may consist of one or several alpha-helices or a transmembrane beta barrel. Because the interior of the lipid bilayer is hydrophobic, the amino acid residues in TMDs are often hydrophobic, although proteins such as membrane pumps and ion channels can contain polar residues. TMDs vary greatly in size and hydrophobicity; they may adopt organelle-specific properties.
A signal peptide is a short peptide present at the N-terminus of most newly synthesized proteins that are destined toward the secretory pathway. These proteins include those that reside either inside certain organelles, secreted from the cell, or inserted into most cellular membranes. Although most type I membrane-bound proteins have signal peptides, most type II and multi-spanning membrane-bound proteins are targeted to the secretory pathway by their first transmembrane domain, which biochemically resembles a signal sequence except that it is not cleaved. They are a kind of target peptide.
SNARE proteins – "SNAPREceptors" – are a large protein family consisting of at least 24 members in yeasts and more than 60 members in mammalian and plant cells. The primary role of SNARE proteins is to mediate the fusion of vesicles with the target membrane; this notably mediates exocytosis, but can also mediate the fusion of vesicles with membrane-bound compartments. The best studied SNAREs are those that mediate the release of synaptic vesicles containing neurotransmitters in neurons. These neuronal SNAREs are the targets of the neurotoxins responsible for botulism and tetanus produced by certain bacteria.
Adenylylation, more commonly known as AMPylation, is a process in which an adenosine monophosphate (AMP) molecule is covalently attached to the amino acid side chain of a protein. This covalent addition of AMP to a hydroxyl side chain of the protein is a post-translational modification. Adenylylation involves a phosphodiester bond between a hydroxyl group of the molecule undergoing adenylylation, and the phosphate group of the adenosine monophosphate nucleotide. Enzymes that are capable of catalyzing this process are called AMPylators.
Ribophorins are dome shaped transmembrane glycoproteins which are located in the membrane of the rough endoplasmic reticulum, but are absent in the membrane of the smooth endoplasmic reticulum. There are two types of ribophorines: ribophorin I and II. These act in the protein complex oligosaccharyltransferase (OST) as two different subunits of the named complex. Ribophorin I and II are only present in eukaryote cells.
Vesicle-associated membrane protein-associated protein B/C is a protein that in humans is encoded by the VAPB gene. The VAPB gene is found on the 20th human chromosome. Together with VAPA, it forms the VAP protein family.
Collagen type IV alpha-3-binding protein, also known as ceramide transfer protein (CERT) or StAR-related lipid transfer protein 11 (STARD11) is a protein that in humans is encoded by the COL4A3BP gene. The protein contains a pleckstrin homology domain at its amino terminus and a START domain towards the end of the molecule. It is a member of the StarD2 subfamily of START domain proteins.
The oxysterol-binding protein (OSBP)-related proteins (ORPs) are a family of lipid transfer proteins (LTPs). Concretely, they constitute a family of sterol and phosphoinositide binding and transfer proteins in eukaryotes that are conserved from yeast to humans. They are lipid-binding proteins implicated in many cellular processes related with oxysterol, including signaling, vesicular trafficking, lipid metabolism, and nonvesicular sterol transport.
Oxysterol-binding protein-related protein 3 is a protein that in humans is encoded by the OSBPL3 gene.
VAMP-Associated Protein A is a protein that in humans is encoded by the VAPA gene. Together with VAPB and VAPC it forms the VAP protein family. They are integral endoplasmic reticulum membrane proteins of the type II and are ubiquitous among eukaryotes.
Oxysterol-binding protein 1 is a protein that in humans is encoded by the OSBP gene.
Membrane contact sites (MCS) are close appositions between two organelles. Ultrastructural studies typically reveal an intermembrane distance in the order of the size of a single protein, as small as 10 nm or wider, with no clear upper limit. These zones of apposition are highly conserved in evolution. These sites are thought to be important to facilitate signalling, and they promote the passage of small molecules, including ions, lipids and reactive oxygen species. MCS are important in the function of the endoplasmic reticulum (ER), since this is the major site of lipid synthesis within cells. The ER makes close contact with many organelles, including mitochondria, Golgi, endosomes, lysosomes, peroxisomes, chloroplasts and the plasma membrane. Both mitochondria and sorting endosomes undergo major rearrangements leading to fission where they contact the ER. Sites of close apposition can also form between most of these organelles most pairwise combinations. First mentions of these contact sites can be found in papers published in the late 1950s mainly visualized using electron microscopy (EM) techniques. Copeland and Dalton described them as “highly specialized tubular form of endoplasmic reticulum in association with the mitochondria and apparently in turn, with the vascular border of the cell”.
Radical S-adenosyl methionine domain-containing protein 2 is a protein that in humans is encoded by the RSAD2 gene. RSAD2 is a multifunctional protein in viral processes that is an interferon stimulated gene. It has been reported that viperin could be induced by either IFN-dependent or IFN-independent pathways and certain viruses may use viperin to increase their infectivity.
CXorf26, also known as MGC874, is a well conserved human gene found on the plus strand of the short arm of the X chromosome. The exact function of the gene is poorly understood, but the polysaccharide biosynthesis domain that spans a major portion of the protein product, as well as the yeast homolog, YPL225, offer insights into its possible function.
GRAM domain containing 1B, also known as GRAMD1B, Aster-B and KIAA1201, is a cholesterol transport protein that is encoded by the GRAMD1B gene. It contains a transmembrane region and two domains of known function; the GRAM domain and a VASt domain. It is anchored to the endoplasmic reticulum. This highly conserved gene is found in a variety of vertebrates and invertebrates. Homologs are found in yeast.
VAP proteins are conserved integral membrane proteins of the endoplasmic reticulum found in all eukaryotic cells. VAP stands for VAMP-associated protein, where VAMP stands for vesicle-associated membrane protein. Humans have two VAPs that consist of the essential Major Sperm Protein domain and linker plus transmembrane helix to attach to the ER: VAPA and VAPB. A third VAP-like protein is Motile sperm domain containing 2 (MOSPD2), which has all the elements of VAP, and like them binds FFAT motifs, but has at its N-terminus a CRAL-TRIO domain that can bind and transfer lipids.
StAR related lipid transfer domain containing 3(STARD3) is a protein that in humans is encoded by the STARD3 gene. STARD3 also known as metastatic lymph node 64 protein (MLN64) is a late endosomal integral membrane protein involved in cholesterol transport. STARD3 creates membrane contact sites between the endoplasmic reticulum (ER) and late endosomes where it moves cholesterol.
Phosphatidylinositol 3-phosphate-binding protein 2 (Pib2) is a yeast protein involved in the regulation of TORC1 signaling and lysosomal membrane permeabilization. It is essential for the reactivation of TORC1 following exposure to rapamycin or nutrient starvation.
Nir1 or membrane-associated phosphatidylinositol transfer protein 3 (PITPNM3) is a mammalian protein that localizes to endoplasmic reticulum (ER) and plasma membrane (PM) membrane contact sites (MCS) and aids the transfer of phosphatidylinositol between these two membranes, potentially by recruiting additional proteins to the ER-PM MCS. It is encoded by the gene PITPNM3.
This article incorporates text from the United States National Library of Medicine, which is in the public domain.