We have recently shown that two proteins related to two of the adaptor subunits of clathrincoated vesicles, p47 (mu3) and beta-NAP (beta3B), are part of an adaptor-like complex not associated with clathrin (Simpson, F., N.A. Bright, M.A. West, L.S. Newman, R.B. Darnell, and M.S. Robinson, 1996. J. Cell Biol. 133:749-760). In the present study we have searched the EST database and have identified, cloned, and sequenced a ubiquitously expressed homologue of beta-NAP, beta3A, as well as homologues of the alpha/gamma and sigma adaptor subunits, delta and sigma3, which are also ubiquitously expressed. Antibodies raised against recombinant delta and sigma3 show that they are the other two subunits of the adaptor-like complex. We are calling this complex AP-3, a name that has also been used for the neuronalspecific phosphoprotein AP180, but we feel that it is a more appropriate designation for an adaptor-related heterotetramer. Immunofluorescence using anti-delta antibodies reveals that the AP-3 complex is associated with the Golgi region of the cell as well as with more peripheral structures. These peripheral structures show only limited colocalization with endosomal markers and may correspond to a postTGN biosynthetic compartment. The delta subunit is closely related to the protein product of the Drosophila garnet gene, which when mutated results in reduced pigmentation of the eyes and other tissues. Because pigment granules are believed to be similar to lysosomes, this suggests either that the AP-3 complex may be directly involved in trafficking to lysosomes or alternatively that it may be involved in another pathway, but that missorting in that pathway may indirectly lead to defects in pigment granules.

Eukaryotic cells internalize transmembrane receptors via clathrin-mediated endocytosis, but it remains unclear how the machinery underpinning this process is regulated. We recently discovered that membrane-associated muniscin proteins such as FCHo and SGIP initiate endocytosis by converting the AP2 clathrin adaptor complex to an open, active conformation that is then phosphorylated (Hollopeter et al., 2014). Here we report that loss of ncap-1, the sole C. elegans gene encoding an adaptiN Ear-binding Coat-Associated Protein (NECAP), bypasses the requirement for FCHO-1. Biochemical analyses reveal AP2 accumulates in an open, phosphorylated state in ncap-1 mutant worms, suggesting NECAPs promote the closed, inactive conformation of AP2. Consistent with this model, NECAPs preferentially bind open and phosphorylated forms of AP2 in vitro and localize with constitutively open AP2 mutants in vivo. NECAPs do not associate with phosphorylation-defective AP2 mutants, implying that phosphorylation precedes NECAP recruitment. We propose NECAPs function late in endocytosis to inactivate AP2.

LZTFL1 participates in immune synapse formation, ciliogenesis, and the localization of ciliary proteins, and knockout of LZTFL1 induces abnormal distribution of heterotetrameric adaptor protein complex-1 (AP-1) in the Lztfl1-knockout mouse photoreceptor cells, suggesting that LZTFL1 is involved in intracellular transport. Here, we demonstrate that in vitro LZTFL1 directly binds to AP-1 and AP-2 and coimmunoprecipitates AP-1 and AP-2 from cell lysates. DxxFxxLxxxR motif of LZTFL1 is essential for these bindings, suggesting LZTFL1 has roles in AP-1 and AP-2-mediated protein trafficking. Since AP-1 and AP-2 are known to be involved in transferrin receptor 1 (TfR1) trafficking, the effect of LZTFL1 on TfR1 recycling was analyzed. TfR1, AP-1 and LZTFL1 from cell lysates could be coimmunoprecipitated. However, pull-down results indicate there is no direct interaction between TfR1 and LZTFL1, suggesting that LZTFL1 interaction with TfR1 is indirect through AP-1. We report the colocalization of LZTFL1 and AP-1, AP-1 and TfR1 as well as LZTFL1 and TfR1 in the perinuclear region (PNR) and the cytoplasm, suggesting a potential complex between LZTFL1, AP-1 and TfR1. The results from the disruption of adaptin recruitment with brefeldin A treatment suggested ADP-ribosylation factor-dependent localization of LZFL1 and AP-1 in the PNR. Knockdown of AP-1 reduces the level of LZTFL1 in the PNR, suggesting that AP-1 plays a role in LZTFL1 trafficking. Knockout of LZTFL1 reduces the cell surface level and the rate of internalization of TfR1, leading to a decrease of transferrin uptake, efflux, and internalization. However, knockout of LZTFL1 did not affect the cell surface levels of epidermal growth factor receptor and cation-independent mannose 6-phosphate receptor, indicating that LZTFL1 specifically regulates the cell surface level of TfR1. These data support a novel role of LZTFL1 in regulating the cell surface TfR1 level by interacting with AP-1 and AP-2.

Multimeric adaptors are broadly involved in vesicle-mediated membrane trafficking. AP2 adaptor, in particular, plays a central role in clathrin-mediated endocytosis (CME) by recruiting cargo and clathrin to endocytic sites. It is generally thought that trafficking adaptors such as AP2 adaptor assemble spontaneously. In this work, however, we discovered that AP2 adaptor assembly is an ordered process controlled by alpha and gamma adaptin binding protein (AAGAB), an uncharacterized factor identified in our genome-wide genetic screen of CME. AAGAB guides the sequential association of AP2 subunits and stabilizes assembly intermediates. Without the assistance of AAGAB, AP2 subunits fail to form the adaptor complex, leading to their degradation. The function of AAGAB is abrogated by a mutation that causes punctate palmoplantar keratoderma type 1 (PPKP1), a human skin disease. Since other multimeric trafficking adaptors operate in an analogous manner to AP2 adaptor, their assembly likely involves a similar regulatory mechanism.

Members of the conserved SNF1/AMP-activated protein kinase (AMPK) family regulate cellular responses to environmental and nutritional stress in eukaryotes. Yeast SNF1 and animal AMPKs form a complex with regulatory SNF4/AMPKgamma and SIP1/SIP2/GAL83/AMPKbeta subunits. The beta-subunits function as target selective adaptors that anchor the catalytic kinase and regulator SNF4/gamma-subunits to their kinase association (KIS) and association with the SNF1 complex (ASC) domains. Here we demonstrate that plant SNF1-related protein kinases (SnRKs) interact with an adaptor-regulator protein, AKINbetagamma, in which an N-terminal KIS domain characteristic of beta-subunits is fused with a C-terminal region related to the SNF4/AMPKgamma proteins. AKINbetagamma is constitutively expressed in plants, suppresses the yeast delta snf4 mutation, and shows glucose-regulated interaction with the Arabidopsis SnRK, AKIN11. Our results suggest that evolution of AKINbetagamma reflects a unique function of SNF1-related protein kinases in plant glucose and stress signalling.

The AP-2 complex is a heterotetrameric endocytic cargo-binding adaptor that facilitates uptake of membrane proteins during mammalian clathrin-mediated endocytosis. While budding yeast has clear homologues of all four AP-2 subunits which form a complex and localize to endocytic sites in vivo, the function of yeast AP-2 has remained enigmatic. Here, we demonstrate that AP-2 is required for hyphal growth in Candida albicans and polarized cell responses in Saccharomyces cerevisiae. Deletion of APM4, the cargo-binding mu subunit of AP-2, causes defects in pseudohyphal growth, generation of a mating projection and the cell wall damage response. In an apm4 null mutant, the cell wall stress sensor Mid2 is unable to relocalize to the tip of a mating projection following pheromone addition, or to the mother bud neck in response to cell wall damage. A direct binding interaction between Mid2 and the mu homology domain of Apm4 further supports a model in which AP-2 binds Mid2 to facilitate its internalization and relocalization in response to specific signals. Thus, Mid2 is the first cargo for AP-2 identified in yeast. We propose that endocytic recycling of Mid2 and other components is required for polarized cell responses ensuring cell wall deposition and is tightly monitored during cell growth.

A Drosophila cDNA (BAD1) encoding a structural and assembly-competent homologue of the mammalian coated pit beta-adaptins (beta and beta') has been cloned and sequenced. In its amino-terminal region (residues 1-575), the BAD1 sequence appears intermediate between that of the mammalian beta-adaptin and a predicted sequence, from cDNA 105a, which appears to code for a version of beta'-adaptin. To test its functional characteristics, a 'myc'-tagged version of BAD1 was expressed in Cos cells. The BAD1 protein was detected most clearly in plasma membrane coated pits, where it colocalized with alpha-adaptin, although other coated pits were noted which apparently did not contain alpha-adaptin. However, these are probably gamma-adaptin containing pits, as BAD1 was also found colocalized with gamma-adaptin in Golgi coated pits in which, typically, alpha-adaptin is absent. Immunoprecipitation experiments confirmed that the BAD1 protein was present in both types of adaptor complex, unlike beta-adaptin which complexes with alpha-adaptin and beta'-adaptin which partners gamma-adaptin exclusively. In spite of this, BAD1 expression does not appear to mix alpha-adaptin and gamma-adaptin distribution amongst all the coated pits: thus the location of these adaptor complexes in mammalian cells does not depend on the differences between beta subunits but rather on membrane-specific interactions of other adaptor polypeptides. The differential interaction of beta with alpha-adaptin and beta' with gamma-adaptin in mammalian cells is likely to depend on the few non-conservative differences between their respective sequences and BAD1. Four of these (one with respect to beta and three versus 105a) are clustered in a particular region (residues 155 to 305), which may therefore represent a domain that influences the choice of partner adaptin.

The heterotetrameric AP2 adaptor (alpha, beta 2, mu 2 and sigma 2 subunits) plays a central role in clathrin-mediated endocytosis. We present the protein recruitment function and 1.7 A resolution structure of its beta 2-appendage domain to complement those previously determined for the mu 2 subunit and alpha appendage. Using structure-directed mutagenesis, we demonstrate the ability of the beta 2 appendage alone to bind directly to clathrin and the accessory proteins AP180, epsin and eps15 at the same site. Clathrin polymerization is promoted by binding of clathrin simultaneously to the beta 2-appendage site and to a second site on the adjacent beta 2 hinge. This results in the displacement of the other ligands from the beta 2 appendage. Thus clathrin binding to an AP2-accessory protein complex would cause the controlled release of accessory proteins at sites of vesicle formation.

Integrins are heterodimeric cell-surface receptors with key functions in cell-cell and cell-matrix adhesion. Integrin α and β subunits are present throughout the metazoans, but it is unclear whether the subunits predate the origin of multicellular organisms. Several component domains have been detected in bacteria, one of which, a specific 7-bladed β-propeller domain, is a unique feature of the integrin α subunits. Here, we describe a structure-derived motif, which incorporates key features of each blade from the X-ray structures of human αIIbβ3 and αVβ3, includes elements of the FG-GAP/Cage and Ca(2+)-binding motifs, and is specific only for the metazoan integrin domains. Separately, we searched for the metazoan integrin type β-propeller domains among all available sequences from bacteria and unicellular eukaryotic organisms, which must incorporate seven repeats, corresponding to the seven blades of the β-propeller domain, and so that the newly found structure-derived motif would exist in every repeat. As the result, among 47 available genomes of unicellular eukaryotes we could not find a single instance of seven repeats with the motif. Several sequences contained three repeats, a predicted transmembrane segment, and a short cytoplasmic motif associated with some integrins, but otherwise differ from the metazoan integrin α subunits. Among the available bacterial sequences, we found five examples containing seven sequential metazoan integrin-specific motifs within the seven repeats. The motifs differ in having one Ca(2+)-binding site per repeat, whereas metazoan integrins have three or four sites. The bacterial sequences are more conserved in terms of motif conservation and loop length, suggesting that the structure is more regular and compact than those example structures from human integrins. Although the bacterial examples are not full-length integrins, the full-length metazoan-type 7-bladed β-propeller domains are present, and sometimes two tandem copies are found.

Kinetoplastid RNA (k-RNA) editing is a complex process in the mitochondria of kinetoplastid protozoa, including Trypanosoma brucei, that involves the guide RNA-directed insertion and deletion of uridines from precursor-mRNAs to produce mature, translatable mRNAs. k-RNA editing is performed by multiprotein complexes called editosomes. Additional non-editosome components termed k-RNA-editing accessory factors affect the extent of editing of specific RNAs or classes of RNAs. The T. brucei p22 protein was identified as one such accessory factor. Here we show that p22 contributes to cell growth in the procyclic form of T. brucei and functions as a cytochrome oxidase subunit II-specific k-RNA-editing accessory factor. To gain insight into its functions, we solved the crystal structure of the T. brucei p22 protein to 2.0-A resolution. The p22 structure consists of a six-stranded, antiparallel beta-sheet flanked by five alpha-helices. Three p22 subunits combine to form a tight trimer that is primarily stabilized by interactions between helical residues. One side of the trimer is strikingly acidic, while the opposite face is more neutral. Database searches show p22 is structurally similar to human p32, which has a number of functions, including regulation of RNA splicing. p32 interacts with a number of target proteins via its alpha1 N-terminal helix, which is among the most conserved regions between p22 and p32. Co-immunoprecipitation studies showed that p22 interacts with the editosome and the k-RNA accessory protein, TbRGG2, and alpha1 of p22 was shown to be important for the p22-TbRGG2 interaction. Thus, these combined studies suggest that p22 mediates its role in k-RNA editing by acting as an adaptor protein.

TMEM39A, encoding an ER-localized transmembrane protein, is a susceptibility locus for multiple autoimmune diseases. The molecular function of TMEM39A remains completely unknown. Here we demonstrated that TMEM39A, also called SUSR2, modulates autophagy activity by regulating the spatial distribution and levels of PtdIns(4)P. Depletion of SUSR2 elevates late endosomal/lysosomal PtdIns(4)P levels, facilitating recruitment of the HOPS complex to promote assembly of the SNARE complex for autophagosome maturation. SUSR2 knockdown also increases the degradative capability of lysosomes. Mechanistically, SUSR2 interacts with the ER-localized PtdIns(4)P phosphatase SAC1 and also the COPII SEC23/SEC24 subunits to promote the ER-to-Golgi transport of SAC1. Retention of SAC1 on the ER in SUSR2 knockdown cells increases the level of PtdIns(3)P produced by the VPS34 complex, promoting autophagosome formation. Our study reveals that TMEM39A/SUSR2 acts as an adaptor protein for efficient export of SAC1 from the ER and provides insights into the pathogenesis of diseases associated with TMEM39A mutations.