Dileucine-based sorting signals bind to the beta chain of AP-1 at a site distinct and regulated differently from the tyrosine-based motif-binding site

Heterotetrameric adaptor complexes and SNAREs play key roles in the specificity of membrane budding and fusion. Here we test the hypothesis that vesicle budding and membrane fusion are coupled by the interaction of these molecules. We investigate the role of the di-leucine motif of vesicle-associated membrane protein 4 (VAMP4) in adaptor binding and localization of VAMP4. Mutation of the di-leucine motif inhibits AP-1 binding in vitro and affects the steady state distribution of VAMP4 in vivo.Heterotetrameric adaptor complexes (AP-1 1 through AP-4) play a role in the selection of cargo and budding of vesicles from donor membranes. The adaptor complexes interact with sorting signals in the cytosolic domains of membrane proteins as well as structural components of the coat (clathrin for AP-1 and AP-2), thereby gathering appropriate cargo into the budding vesicle (1). SNARE proteins mediate the fusion of these vesicles with acceptor membranes. This is achieved through interactions between vesicle-associated SNAREs (v-SNAREs or VAMPs) and SNAREs associated with target membranes (t-SNAREs or syntaxins) (2). A combination of cargo selection and targeted fusion would provide selectivity to vesicular transport, thus it is important that both systems function together.Adaptor complexes may interact with or be involved in the trafficking of SNAREs, it is still not known whether there are direct interactions between these molecules (3-5). Such an interaction has been proposed from the analysis of SNARE protein sequences, because several of the syntaxins (syntaxins 4, 6, 7, and 13) contain di-leucine or tyrosine-based motifs that have been shown in other membrane proteins to directly interact with adaptor complexes (6). There is also evidence that VAMPs may interact with adaptor complexes (7-9). However, only VAMP4 and VAMP7 contain recognizable di-leucine motifs, suggesting that alternative or additional motifs may regulate adaptor binding to SNAREs or cargo molecules.In this study, we show that VAMP4 is enriched in clathrincoated vesicles, AP-1 specifically binds to VAMP4, and this binding is dependent on the di-leucine motif of VAMP4. Transfection of cells with VAMP4 constructs lacking the di-leucine motif results in the mislocalization of VAMP4 into peripheral structures, which overlap with AP-1. EXPERIMENTAL PROCEDURESReagents and Antibodies-␥-Adaptin hinge in pGEX3X and ␦-adaptin in pBluescript were generous gifts from Dr. Margaret S. Robinson. VAMP4-GFP was generated by PCR and cloned into pEGFP-N3 (CLONTECH Laboratories) using the XhoI and KpnI sites. Sec22, VAMP2, VAMP4, VAMP7, and VAMP8 in pGEX-KG were previously described (7, 8, 10 -12). PCR-based site-directed mutagenesis was performed as directed in the Stratagene manual.Clathrin-coated Vesicles Were Purified as Previously Described (13)-Anti-␥-adaptin hinge (amino acids 596 -702) and ␦-adaptin hinge (amino acids 608 -800) polyclonal antibodies were generated by immunization of rabbits with bacterially expressed GST fusion proteins. The sera were purified as pr...

Section: Discussionmentioning

confidence: 64%

The Di-leucine Motif of Vesicle-associated Membrane Protein 4 Is Required for Its Localization and AP-1 Binding

Peden

Park

Scheller³

2001

“…Clathrin-coated pit formation recruits a clathrin lattice and the AP-2 complex to the plasma membrane, whereas AP-1 is lured to the trans-Golgi network. In this process, dileucine motifs interact with the ␤1 subunit of AP-1 (40), directing the sorting of proteins to the trans-Golgi network. The structural determinants and the mechanisms underlying BLT1 endocytosis are still not defined, but we have previously demonstrated that BLT1 C-tail is essential for BLT1 internalization, as it is for a vast number of GPCRs, and that this phenomenon is uniquely independent of arrestins.…”

Section: Discussionmentioning

confidence: 99%

Structural Determinants Regulating Expression of the High Affinity Leukotriene B4 Receptor

Gaudreau¹,

Beaulieu

Chen³

et al. 2004

Mutational analysis of determinants located in the C-terminal (C) tail of the high affinity leukotriene (LT)BA variety of stimuli, including light, neurotransmitters, hormones, and inflammatory lipid mediators produce their effects via activation of G-protein-coupled receptors (GPCRs). 1 Taking into account the heterogeneity of ligands, it is interesting that all members of this superfamily of receptors share the same topology characterized by the presence of seven transmembrane ␣-helices. Conserved residues within subfamily A (related to rhodopsin) are localized throughout these helices, serving as reference points for sequence alignments. Biophysical and biochemical evidence indicate that upon photoactivation, relative movements of transmembrane (TM) 3 and TM6 occur in rhodopsin; similarly, rearrangement of the cytoplasmic proximal portion of TM7 relative to TM1 and of TM2 relative to the intracellular helix VIII were also observed (reviewed in Ref.

“…However, in only a very few cases have membrane proteins and adaptors co-immunoprecipitated (20). Several in vitro assays, including bead pull-down and surface plasmon resonance, had shown interaction between tyrosine-based sorting signals and clathrin adaptor complexes (21)(22)(23)(24)(25), yet surprisingly few data are available concerning whether such interactions occur in vivo or are physiologically important. Although a direct interaction between YXX⌽ internalization sequences and 2 subunits has been demonstrated, the binding of receptors to AP-2 does not necessarily correlate with the internalization capacity of proteins bearing YXX⌽ motifs.…”

mentioning

confidence: 99%

μ2 Binding Directs the Cystic Fibrosis Transmembrane Conductance Regulator to the Clathrin-mediated Endocytic Pathway

Weixel

Bradbury

2001

The cystic fibrosis transmembrane conductance regulator (CFTR) contains a conserved tyrosine-based internalization motif, 1424 YDSI, which interacts with the endocytic clathrin adaptor complex, AP-2, and is required for its efficient endocytosis. Although direct interactions between several endocytic sequences and the medium chain and endocytic clathrin adaptor complexes have been shown by protein-protein interaction assays, whether all these interactions occur in vivo or are physiologically important has not always been addressed. Here we show, using both in vitro and in vivo assays, a physiologically relevant interaction between CFTR and the subunit of AP-2. Cross-linking experiments were performed using photoreactive peptides containing the YDSI motif and purified adaptor complexes. CFTR peptides cross-linked a 50-kDa subunit of purified AP-2 complexes, the apparent molecular mass of 2. Furthermore, isolated 2 bound to the sorting motif, YDSI, both in cross-linking experiments and glutathione S-transferase pull-down experiments, confirming that 2 mediates the interaction between CFTR and AP-2 complexes. Inducible overexpression of dominant-negative 2 in HeLa cells results in AP-2 complexes that fail to interact with CFTR. Moreover, internalization of CFTR in mutant cells is greatly reduced compared with wild type HeLa cells. These results indicate that the AP-2 endocytic complex selectively interacts with the conserved tyrosine-based internalization signal in the carboxyl terminus of CFTR, YDSI. Furthermore, this interaction is mediated by the 2 subunit of AP-2 and mutations in 2 that block its interaction with YDSI inhibit the incorporation of CFTR into the clathrin-mediated endocytic pathway.