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Tepsin is currently the only accessory trafficking protein identified in adaptor-related protein 4 (AP4)-coated vesicles originating at the trans-Golgi network (TGN). The molecular basis for interactions between AP4 subunits and motifs in the tepsin C-terminus have been characterized, but the biological role of tepsin remains unknown. We determined X-ray crystal structures of the tepsin epsin N-terminal homology (ENTH) and VHS/ENTH-like domains. Our data reveal unexpected structural features that suggest key functional differences between these and similar domains in other trafficking proteins. The tepsin ENTH domain lacks helix0, helix8 and a lipid binding pocket found in epsin1/2/3. These results explain why tepsin requires AP4 for its membrane recruitment and further suggest ENTH domains cannot be defined solely as lipid binding modules. The VHS domain lacks helix8 and thus contains fewer helices than other VHS domains. Structural data explain biochemical and biophysical evidence that tepsin VHS does not mediate known VHS functions, including recognition of dileucine-based cargo motifs or ubiquitin. Structural comparisons indicate the domains are very similar to each other, and phylogenetic analysis reveals their evolutionary pattern within the domain superfamily. Phylogenetics and comparative genomics further show tepsin within a monophyletic clade that diverged away from epsins early in evolutionary history (~1500 million years ago). Together, these data provide the first detailed molecular view of tepsin and suggest tepsin structure and function diverged away from other epsins. More broadly, these data highlight the challenges inherent in classifying and understanding protein function based only on sequence and structure., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

BEACH (named after 'Beige and Chediak-Higashi') is a conserved ∼280 residue domain, present in nine human BEACH domain containing proteins (BDCPs). Most BDCPs are large, containing a PH-like domain for membrane association preceding their BEACH domain, and containing WD40 and other domains for ligand binding. Recent studies found that mutations in individual BDCPs cause several human diseases. BDCP alterations affect lysosome size (LYST and NSMAF), apoptosis (NSMAF), autophagy (LYST, WDFY3, LRBA), granule size (LYST, NBEAL2, NBEA) or synapse formation (NBEA). However, the roles of each BDCP in these membrane events remain controversial. After reviewing studies on individual BDCPs, we propose a unifying hypothesis that BDCPs act as scaffolding proteins that facilitate membrane events, including both fission and fusion, determined by their binding partners. BDCPs may also bind each other, enabling fusion or fission of vesicles that are not necessarily of the same type. Such mechanisms explain why different BDCPs may have roles in autophagy; each BDCP is specific for the cell type or the cargo, but not necessarily specific for attaching to the autophagosome. Further elucidation of these mechanisms, preferably carrying out the same experiment on multiple BDCPs, and possibly using patients' cells, may identify potential targets for therapy., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The GGA family of clathrin adaptor proteins mediates the intracellular trafficking of transmembrane proteins by interacting with DXXLL-type sorting signals on the latter. These signals were originally identified at the carboxy-termini of the transmembrane cargo proteins. Subsequent studies, however, showed that internal DXXLL sorting motifs occur within the N- or C-terminal cytoplasmic domains of cargo molecules. The GGAs themselves also contain internal DXXLL motifs that serve to auto-regulate GGA function. A recent study challenged the notion that internal DXXLL signals are competent for binding to GGAs. Since the question of whether GGA adaptors interact with internal DXXLL motifs is fundamental to the identification of bona fide GGA cargo, and to an accurate understanding of GGA regulation within cells, we have extended our previous findings. We now present additional evidence confirming that GGAs do interact with internal DXXLL motifs. We also summarize the recent reports from other laboratories documenting internal GGA binding motifs., (© 2012 John Wiley & Sons A/S.)

The N-terminal domain (TD) of the clathrin heavy chain is folded into a seven-bladed β-propeller that projects inward from the polyhedral outer clathrin coat. As the most membrane-proximal portion of assembled clathrin, the TD is a major protein-protein interaction node. Contact with the TD β-propeller occurs through short peptide sequences typically located within intrinsically disordered segments of coat components that usually are elements of the membrane-apposed, inner 'adaptor' coat layer. A huge variation in TD-binding motifs is known and now four spatially discrete interaction surfaces upon the β-propeller have been delineated. An important operational feature of the TD interaction sites in vivo is functional redundancy. The recent discovery that 'pitstop' chemical inhibitors apparently occupy only one of the four TD interaction surfaces, but potently block clathrin-mediated endocytosis, warrants careful consideration of the underlying molecular basis for this inhibition., (© 2012 John Wiley & Sons A/S.)

Biogenesis of lysosome-related organelle complex-1 (BLOC-1) is one of the four multi-subunit complexes implicated in sorting cargo to lysosome-related organelles, as loss of function of any of these complexes causes Hermansky-Pudlak syndrome. Eight subunits of BLOC-1 interact with each other and with many other proteins. Identifying new interactors of BLOC-1 will increase understanding of its mechanism of action, and studies in model organisms are useful for finding such interactors. PSI-BLAST searches identify homologues in diverse model organisms, but there are significant gaps for BLOC-1, with none of its eight subunits found in Saccharomyces cerevisiae. Here we use more sensitive searches to identify distant homologues for three BLOC-1 subunits in S. cerevisiae: Blos1, snapin and cappuccino (cno). Published data on protein interactions show that in yeast these are likely to form a complex with three other proteins. One of these is the yeast homologue of the previously uncharacterized KxDL protein, which also interacts with Blos1 and cno in higher eukaryotes, suggesting that KxDL proteins are key interactors with BLOC-1., (© 2011 John Wiley & Sons A/S.)

The cytosolic adaptors GGA1-3 mediate sorting of transmembrane proteins displaying a C-terminal acidic dileucine motif (DXXLL) in their cytosolic domain. GGA1 and GGA3 contain similar but intrinsic motifs that are believed to serve as autoinhibitory sites activated by the phosphorylation of a serine positioned three residues upstream of the DXXLL motif. In the present study, we have subjected the widely acknowledged concept of GGA1 autoinhibition to a thorough structural and functional examination. We find that (i) the intrinsic motif of GGA1 is inactive, (ii) only C-terminal DXXLL motifs constitute active GGA binding sites, (iii) while aspartates and phosphorylated serines one or two positions upstream of the DXXLL motif increase GGA1 binding, phosphoserines further upstream have little or no influence and (iv) phosphorylation of GGA1 does not affect its conformation or binding to Sortilin and SorLA. Taken together, our findings seem to refute the functional significance of GGA autoinhibition in particular and of intrinsic GGA binding motifs in general.

Clathrin-coated vesicles (CCVs) sort proteins at the plasma membrane, endosomes and trans Golgi network for multiple membrane traffic pathways. Clathrin recruitment to membranes and its self-assembly into a polyhedral coat depends on adaptor molecules, which interact with membrane-associated vesicle cargo. To determine how adaptors induce clathrin recruitment and assembly, we mapped novel interaction sites between these coat components. A site in the ankle domain of the clathrin triskelion leg was identified that binds a common site on the appendages of tetrameric [AP1 and AP2] and monomeric (GGA1) adaptors. Mutagenesis and modeling studies suggested that the clathrin-GGA1 appendage interface is nonlinear, unlike other peptide-appendage interactions, but overlaps with a sandwich domain binding site for accessory protein peptides, allowing for competitive regulation of coated vesicle formation. A novel clathrin box in the GGA1 hinge region was also identified and shown to mediate membrane recruitment of clathrin, while disruption of the clathrin-GGA1 appendage interaction did not affect recruitment. Thus, the distinct sites for clathrin-adaptor interactions perform distinct functions, revealing new aspects to regulation of CCV formation.

Epsin 1 engages several core components of the endocytic clathrin coat, yet the precise mode of operation of the protein remains controversial. The occurrence of tandem ubiquitin-interacting motifs (UIMs) suggests that epsin could recognize a ubiquitin internalization tag, but the association of epsin with clathrin-coat components or monoubiquitin is reported to be mutually exclusive. Here, we show that endogenous epsin 1 is clearly an integral component of clathrin coats forming at the cell surface and is essentially absent from caveolin-1-containing structures under normal conditions. The UIM region of epsin 1 associates directly with polyubiquitin chains but has extremely poor affinity for monoubiquitin. Polyubiquitin binding is retained when epsin synchronously associates with phosphoinositides, the AP-2 adaptor complex and clathrin. The enrichment of epsin within clathrin-coated vesicles purified from different tissue sources varies and correlates with sorting of multiubiquitinated cargo, and in cultured cells, polyubiquitin, rather than non-conjugable monoubiquitin, promotes rapid internalization. As epsin interacts with eps15, which also contains a UIM region that binds to polyubiquitin, epsin and eps15 appear to be central components of the vertebrate poly/multiubiquitin-sorting endocytic clathrin machinery.

BACE (beta-site amyloid precursor protein cleaving enzyme, beta-secretase) is a type-I membrane protein which functions as an aspartic protease in the production of beta-amyloid peptide, a causative agent of Alzheimer's disease. Its cytoplasmic tail has a characteristic acidic-cluster dileucine motif recognized by the VHS domain of adaptor proteins, GGAs (Golgi-localizing, gamma-adaptin ear homology domain, ARF-interacting). Here we show that BACE is colocalized with GGAs in the trans-Golgi network and peripheral structures, and phosphorylation of a serine residue in the cytoplasmic tail enhances interaction with the VHS domain of GGA1 by about threefold. The X-ray crystal structure of the complex between the GGA1-VHS domain and the BACE C-terminal peptide illustrates a similar recognition mechanism as mannose 6-phosphate receptors except that a glutamine residue closes in to fill the gap created by the shorter BACE peptide. The serine and lysine of the BACE peptide point their side chains towards the solvent. However, phosphorylation of the serine affects the lysine side chain and the peptide backbone, resulting in one additional hydrogen bond and a stronger electrostatic interaction with the VHS domain, hence the reversible increase in affinity.

The cytoplasmic tail of the H,K-ATPase beta-subunit contains a putative tyrosine-based motif that directs the beta-subunit's basolateral sorting when it is expressed in Madin-Darby Canine Kidney (MDCK) cells. When expressed in LLC-PK(1) cells, however, the beta-subunit is localized to the apical membrane. Several proteins that contain tyrosine-based motifs, including the low-density lipoprotein and transferrin receptors, show a similar sorting 'defect' when expressed in LLC-PK(1) cells. For low-density lipoprotein and transferrin receptors, this behavior is due to the differential expression of the mu 1B subunit of the AP-1B clathrin adaptor complex. mu 1B is expressed by MDCK cells, but not LLC-PK(1) cells, and transfection of mu 1B into LLC-PK(1) cells restores basolateral localization of low-density lipoprotein and transferrin receptors. For the beta-subunit, however, mu B expression in LLC-PK(1) cells does not induce its basolateral expression. We found that the beta-subunit interacts with both mu 1B and mu 1A in vitro and in vivo. The capacity to participate in a mu 1B interaction therefore is not sufficient to program the beta-subunit's basolateral localization in MDCK cells. Our data suggest that the H,K-ATPase beta-subunit's basolateral sorting signal is either masked in certain epithelial cells, or requires an interaction with sorting machinery other than AP-1B for delivery to the basolateral plasma membrane.

The trans-Golgi network is the major sorting compartment of the secretory pathway for protein, lipid and membrane traffic. There is a constant flow of membrane and cargo to and from this compartment. Evidence is emerging that the trans-Golgi network has multiple biochemically and functionally distinct subdomains, each of which contributes to the combined sorting and transport requirements of this dynamic compartment. The recruitment of distinct arrays of protein complexes to trans-Golgi network membranes is likely to produce the diversity of structure and biochemistry observed amongst subdomains that serve to generate different carriers or maintain resident trans-Golgi network components. This review discusses how these subdomains may be formed and examines the molecular players involved, including G proteins, clathrin adaptors and golgin tethers. Diversity within these protein families is highlighted and shown to be critical for the functionality of the trans-Golgi network, as a mediator of protein sorting and membrane transport, and for the maintenance of Golgi structure.