The Eukaryotic Linear Motif resource for
Functional Sites in Proteins
Functional site class:
Adaptin binding Endosome-Lysosome-Basolateral sorting signals
Functional site description:
Endocytosis and/or vesicular sorting signals for membrane proteins. Depending on organism, cell type as well as the nature of the adaptin complex bound, they can target either to cell surface or to specific, internal membrane-bound organelles (endosomes, lysosomes, melanosomes, synaptic vesicles, etc.)

All these motifs are believed to bind to the sigma subunit of activated adaptin complexes (AP-1, AP-2 and AP-3). These clathrin-associated complexes are ancient and found in most eukaryotes. Dileucine motifs are variable (especially at their negatively charged positions and at the hydrophobic residues) and the various motif subtypes tend to have slightly different functions (Mattera,2011).

One should avoid confusing the adaptin sigma-binding classical dileucine motifs discussed here, and the GGA-binding lysosomal targeting motifs (sometimes also called dileucine motifs).
ELMs with same func. site: TRG_DiLeu_BaEn_1  TRG_DiLeu_BaEn_2  TRG_DiLeu_BaEn_3  TRG_DiLeu_BaEn_4  TRG_DiLeu_BaLyEn_6  TRG_DiLeu_LyEn_5 
ELM Description:
Members of this unusual variant, also called monoleucine motifs, only contain one hydrophobic residue (Leu) at +5 instead of having two at +5 and +6 (Gephart,2011). In turn, the canonical glutamate at +1 is preceded by a second negatively charged glutamate at position -1. The monoleucine motifs are usually involved in basolateral sorting of membrane proteins in polarized epithelial cells in multicellular animals (by binding to AP-1).

While no detailed structural information is available (as of 2021), the sequence conservation is rather compelling about the existence of this dileucine motif variant. The only preserved hydrophobic amino acid (that appears to be an invariable leucine in known examples) corresponds to the first hydrophobic position of more typical dileucine motifs. The second position (that is less buried in the structures of other dileucine motifs) is replaced by a non-hydrophobic amino acid, presumably weakening the motif interaction. It might only retain functionality because of the extra Glu (-1) preceding the canonical glutamate (+1). Glu -1 is suggested to make further electrostatic contacts to adaptin sigma subunits (most notably, sigma of the AP-1 complex). The difference of charge densities across adaptin complexes likely also plays a role in the relatively restricted biological function (almost always acting as a basolateral polarity signal) of these monoleucine motifs.

Although existence of motif variants, where one of the double glutamates is replaced by an aspartate cannot be fully excluded, it is expected to be very rare. Therefore, Asp (-1 or +1) is not included in the current regular expression. On the other hand, a Pro is likely permitted at +4 without substantial alteration of function.

While not yet described in eukaryotes other than multicellular animals, proteome-wide searches and conservation analyses suggest that monoleucine motifs likely also exist in many other eukaryotic organisms, including plants.
Pattern: EE...L[^LIVMF]
Pattern Probability: 0.0004731
Present in taxon: Eukaryota
Interaction Domain:
Clat_adaptor_s (PF01217) Clathrin adaptor complex small chain (Stochiometry: 1 : 1)
o See 4 Instances for TRG_DiLeu_BaEn_4
o Abstract
Adaptin-binding acidic dileucine motifs and variants thereof occur almost exclusively on the cytosolic side of membrane proteins, mostly integral (transmembrane) proteins. In the latter, they are frequently located near the protein N- or C-termini, with relative proximity (within 10-100aa) to a transmembrane segment. These motifs bind directly to a highly conserved site located on the sigma subunits of adaptin complexes (adaptins AP1-4; Doray,2007; Kelly,2008). They serve to initiate clathrin-mediated endocytosis or protein sorting and can work synergistically with the adaptin mu subunit binding YxxPhi-type motifs (TRG_ENDOCYTIC_2). Sigma subunits of AP complexes differ slightly in their surface charge densities and binding groove geometry, allowing for both generic and selective interactions with protein partners.

In multicellular animals, AP1 targets its ligands from the trans-Golgi network to the cell membrane, mainly to the basolateral surface of polarized epithelial cells or somato-dendritic compartment of neurons (Nakatsu,2014). AP2 is chiefly involved in endocytosis of cell surface proteins and their trafficking to early or late endosomes. AP3 targets its ligands to the lysosome, late endosome or melanosome (or less commonly, to the axonal compartment of neurons), while the biological function of AP4 remains mostly unknown. In fungi and plants, dileucine motifs are often responsible for the vacuolar or tonoplast localization of proteins carrying these motifs.

Due to the similarity of the adaptin sigma subunits, variant dileucine motifs may have overlapping specificities, being capable of binding multiple adaptins. In many eukaryotes, AP3 appears to be a dominant partner, that drives permanent intracellular localization of ligands it can interact with, regardless of their binding to other adaptins. Unfortunately, the similarity of this motif to the GGA-binding dileucine motifs (that also target certain proteins to the late endosome or lysosome) has been the source of considerable confusion in the past.

The name of classical dileucine motifs stems from their preferred hydrophobic amino acids, although it is somewhat of a misnomer. In addition to the idealized ExxPL[LI] sequence, a multitude of relaxed motif variations are reported to exist, many of them still poorly characterized. The degree of relaxation seems to heavily influence the targeting properties of dileucine-like motifs (Sitaram,2012). Motifs that do not satisfy the optimal consensus tend to prefer adaptins other than AP3, hence they are more likely to be trafficked to the cell surface.
o 9 selected references:

o 12 GO-Terms:

o 4 Instances for TRG_DiLeu_BaEn_4
(click table headers for sorting; Notes column: =Number of Switches, =Number of Interactions)
Acc., Gene-, NameStartEndSubsequenceLogic#Ev.OrganismNotes
P36897 TGFBR1
161 167 HHRVPNEEDPSLDRPFISEG TP 3 Homo sapiens (Human)
Q92911 SLC5A5
578 584 ASVAPKEEVAILDDNLVKGP TP 2 Homo sapiens (Human)
P35070 BTC
156 162 KRKKKEEEMETLGKDITPIN TP 3 Homo sapiens (Human)
P15514 AREG
236 242 KYEGEAEERKKLRQENGNVH TP 3 Homo sapiens (Human)
Please cite: ELM — the eukaryotic linear motif resource in 2020. (PMID:31680160)

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