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:
AP-3 interacting dileucine motifs almost inevitably lead to lysosomal or late endosomal targeting for many membrane proteins bearing this motif. In neurons, however, AP-3 complexes can also support axonal cell surface localization. AP-3 binding motifs tend to be more conserved than other dileucine motifs, with some vertebrate instances even matching with their distant fungal or plant counterparts (Larisch,2012; Llinares,2015). The latter dileucine motifs were implicated in membrane protein sorting to vacuoles or tonoplasts.

These canonical lysosomal targeting signals represent the most restrictive type of all dileucine motifs. They require all four key positions to be optimal (Glu at +1, Pro or Arg at +4, Leu at +5 and Leu or Ile at +6) for full functionality. In addition, they also display a strong preference for a Gln/Glu at +3 and also for Arg/Gln/Thr at +2. Even the less strict dileucine motif subtypes (mismatching at position +1 or +4) can sometimes act as lysosomal targeting signals if they obey these additional preferences. In addition to lysosomal sorting, these specialized motifs also play a key role in melanosome assembly in certain tissues.

Unfortunately, no structure of a dileucine motif in complex with AP-3 or its sigma subunits has so far been determined (as of 2021), thus the structural reasons behind its striking preferences are currently completely unknown. However, biologically the AP-3 complexes are quite dominant, forcing lysosomal trafficking on all ligands they are capable of recruiting.

Whereas these lysosomal targeting signals also exist in many eukaryotes outside multicellular animals, their function is often slightly different. In fungi and plants, such motifs are functionally often equivalent to the less strict dileucine motif variants: All these motifs perform a similar function, localizing proteins to the central vacuole or tonoplast.
Pattern: [E]..[RP]L[LI]
Pattern Probability: 0.0001029
Present in taxons: Fungi Metazoa Viridiplantae
Interaction Domain:
Clat_adaptor_s (PF01217) Clathrin adaptor complex small chain (Stochiometry: 1 : 1)
o See 18 Instances for TRG_DiLeu_LyEn_5
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 10 selected references:

o 12 GO-Terms:

o 18 Instances for TRG_DiLeu_LyEn_5
(click table headers for sorting; Notes column: =Number of Switches, =Number of Interactions)
Acc., Gene-, NameStartEndSubsequenceLogic#Ev.OrganismNotes
P46032 NPF8.3
7 12 MGSIEEEARPLIEEGLILQE TP 3 Arabidopsis thaliana (Thale cress)
P38279 RTC2
131 136 VLQDVFNEYEPLLPRIEEED TP 3 Saccharomyces cerevisiae S288c
Q12010 YPQ1
124 129 VLHDVFNEQQPLLNSQGQPN TP 3 Saccharomyces cerevisiae S288c
Q12241 VAM3
155 160 YISIKVNEQSPLLHNEGQHQ TP 5 Saccharomyces cerevisiae S288c
9 14 AGLRNESEQEPLLGDTPGSR TP 3 Homo sapiens (Human)
57 62 GSPGHDRERQPLLDRARGAA TP 5 Homo sapiens (Human)
Q6ZP29 SLC66A1
284 289 QFLVYRRSTAASELEPLLPS TP 3 Homo sapiens (Human)
Q8BWC0 Tpcn2
4 9 MAAEEQPLLGRDRGSGQVHS TP 1 Mus musculus (House mouse)
P11344 Tyr
513 518 KKKQPQEERQPLLMDKDDYH TP 2 Mus musculus (House mouse)
O17470 ser-1
552 557 EASTTDEETKPLIPKSTVPA TP 3 Caenorhabditis elegans
Q9JJZ1 Slc2a8
8 13 MSPEDPQETQPLLRSPGARA TP 2 Rattus norvegicus (Norway rat)
Q9JIF3 Slc2a8
8 13 MSPEDPQETQPLLRPPEART TP 2 Mus musculus (House mouse)
Q60696 Pmel
616 621 SGLRARGLGENSPLLSGQQV TP 2 Mus musculus (House mouse)
Q5RKH7 Slc35f6
356 361 RHPTQEGEQERLLGDSRTPI TP 3 Rattus norvegicus (Norway rat)
Q8N697 SLC15A4
10 15 GSGGGAGERAPLLGARRAAA TP 3 Homo sapiens (Human)
Q14108 SCARB2
471 476 KGQGSMDEGTADERAPLIRT TP 2 Homo sapiens (Human)
O15118 NPC1
1271 1276 KSCATEERYKGTERERLLNF TP 3 Homo sapiens (Human)
O94823 ATP10B
26 31 GFPHCPSETTPLLSPEKGRQ TP 3 Homo sapiens (Human)
Please cite: ELM — the eukaryotic linear motif resource in 2020. (PMID:31680160)

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