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:
Motifs of this subtype tend to carry hydrophobic amino acids other than leucines at +5 and +6, and make up for their suboptimal consensus by containing a consecutive pair of negatively-charged residues, typically two glutamates at the +1 and -1 positions. Proteins carrying this variant are typically targeted to vesicles (including synaptic vesicles) through endocytosis from the cell surface, or less commonly to the basolateral surface of polarized epithelia (Santos,2013).

This subtype was proposed based on both sequence information (two charged positions instead of a single glutamate characteristic of other dileucine motifs) as well as functional data (with many vesicular examples in addition to basolaterally-targeting instances). Although no structural information is available (as of 2021), the extra glutamic acid preceding the canonical one is expected to make additional electrostatic contacts to the sigma subunits of adaptin complexes. This motif strengthening feature would allow further relaxations to the hydrophobic core, where – in addition to phenylalanine - even isoleucine and methionine are now allowed at the first hydrophobic residue (at +5) instead of leucine. Leucine +5 is excluded in the ELM pattern as it would switch functionality.

Although the existence of examples, where one of the double glutamates are changed to aspartate cannot be completely excluded, no such motif has been described yet. Thus, they are not included in the regular expression.

These variant dileucine motifs (with Phe, Ile or Met at the first hydrophobic position) have not yet been described in eukaryotes other than multicellular animals and it is doubtful if they exist in fungi or plants.
Pattern: EE...[FIM][LIVM]
Pattern Probability: 0.0000841
Present in taxon: Metazoa
Interaction Domain:
Clat_adaptor_s (PF01217) Clathrin adaptor complex small chain (Stochiometry: 1 : 1)
o See 4 Instances for TRG_DiLeu_BaEn_3
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_3
(click table headers for sorting; Notes column: =Number of Switches, =Number of Interactions)
Acc., Gene-, NameStartEndSubsequenceLogic#Ev.OrganismNotes
Q01827 Slc18a2
478 484 RSPPAKEEKMAILMDHNCPI TP 3 Rattus norvegicus (Norway rat)
Q9JI12 Slc17a6
513 519 DPEETSEEKCGFIHEDELDE TP 3 Rattus norvegicus (Norway rat)
Q62634 Slc17a7
505 511 EPEEMSEEKCGFVGHDQLAG TP 3 Rattus norvegicus (Norway rat)
Q9Y5Y6 ST14
33 39 EKVNGLEEGVEFLPVNNVKK TP 3 Homo sapiens (Human)
Please cite: ELM — the eukaryotic linear motif resource in 2020. (PMID:31680160)

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