MEX3C interacts with adaptor-related protein complex 2 and involves in miR-451a exosomal sorting.
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ABSTRACT:
SUBMITTER: Lu P
PROVIDER: S-EPMC5628917 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Adaptor protein complexes (APs) function as vesicle coat components in different membrane traffic pathways; however, there are a number of pathways for which there is still no candidate coat. To find novel coat components related to AP complexes, we have searched the expressed sequence tag database and have identified, cloned, and sequenced a new member of each of the four AP subunit families. We have shown by a combination of coimmunoprecipitation and yeast two-hybrid analysis that these four proteins (epsilon, beta4, mu4, and sigma4) are components of a novel adaptor-like heterotetrameric complex, which we are calling AP-4. Immunofluorescence reveals that AP-4 is localized to approximately 10-20 discrete dots in the perinuclear region of the cell. This pattern is disrupted by treating the cells with brefeldin A, indicating that, like other coat proteins, the association of AP-4 with membranes is regulated by the small GTPase ARF. Immunogold electron microscopy indicates that AP-4 is associated with nonclathrin-coated vesicles in the region of the trans-Golgi network. The mu4 subunit of the complex specifically interacts with a tyrosine-based sorting signal, indicating that, like the other three AP complexes, AP-4 is involved in the recognition and sorting of cargo proteins with tyrosine-based motifs. AP-4 is of relatively low abundance, but it is expressed ubiquitously, suggesting that it participates in a specialized trafficking pathway but one that is required in all cell types.
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Project description:Heterotetrameric adaptor protein complexes are important mediators of cargo protein sorting in clathrin-coated vesicles. The cell type-specific expression of alternate ? chains creates distinct forms of AP-1 with altered cargo sorting, but how these subunits confer differential function is unclear. Whereas some studies suggest the ? subunits specify localization to different cellular compartments, others find that the two forms of AP-1 are present in the same vesicle but recognize different cargo. Yeast have two forms of AP-1, which differ only in the ? chain. Here we show that the variant ? chain Apm2 confers distinct cargo-sorting functions. Loss of Apm2, but not of Apm1, increases cell surface levels of the v-SNARE Snc1. However, Apm2 is unable to replace Apm1 in sorting Chs3, which requires a dileucine motif recognized by the ?/? subunits common to both complexes. Apm2 and Apm1 colocalize at Golgi/early endosomes, suggesting that they do not associate with distinct compartments. We identified a novel, conserved regulatory protein that is required for Apm2-dependent sorting events. Mil1 is a predicted lipase that binds Apm2 but not Apm1 and contributes to its membrane recruitment. Interactions with specific regulatory factors may provide a general mechanism to diversify the functional repertoire of clathrin adaptor complexes.
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Project description: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.
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Project description:Exosomes are emerging mediators of intercellular communication; whether the release of exosomes has an effect on the exosome donor cells in addition to the recipient cells has not been investigated to any extent. Here, we examine different exosomal miRNA expression profiles in primary mouse colon tumour, liver metastasis of colon cancer and naive colon tissues. In more advanced disease, higher levels of tumour suppressor miRNAs are encapsulated in the exosomes. miR-193a interacts with major vault protein (MVP). Knockout of MVP leads to miR-193a accumulation in the exosomal donor cells instead of exosomes, inhibiting tumour progression. Furthermore, miR-193a causes cell cycle G1 arrest and cell proliferation repression through targeting of Caprin1, which upregulates Ccnd2 and c-Myc. Human colon cancer patients with more advanced disease show higher levels of circulating exosomal miR-193a. In summary, our data demonstrate that MVP-mediated selective sorting of tumour suppressor miRNA into exosomes promotes tumour progression.
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Project description:For the outcome of a hepatitis B virus (HBV) infection, the viral L envelope protein with its pre-S domain performs pivotal functions by mediating attachment of HBV to liver cells, envelopment of viral capsids, release of (sub)viral particles, regulation of supercoiled DNA amplification, and transcriptional transactivation. To assess its multiple functions and host-protein assistance involved, we initiated a two-hybrid screen using the L-specific pre-S1 domain as bait. With this approach, we have identified gamma2-adaptin, a putative member of the clathrin adaptor proteins responsible for protein sorting and trafficking, as a specific binding partner of L protein. Evidence for a physical interaction between L protein and gamma2-adaptin was also demonstrated by affinity chromatography and coimmunoprecipitation, and the binding sites were mapped to the L-specific pre-S1 domain and the gamma2-adaptin-specific ear domain. The specificity of the interaction was further sustained by the failure of gamma1-adaptin, a closely related gamma2-adaptin homologue, to associate with L protein. Analysis of an L mutant protein indicates that the L-gamma2-adaptin interaction strictly depends on the pre-S1 domain of transmembrane L protein oriented to the cytosol and thus appears to occur in the cytosolic environment. Interestingly, coexpression of the two interacting partners in transfected cells resulted in recruitment of gamma2-adaptin by L protein onto cis-Golgi-like structures, strongly indicating that the association is physiologically relevant. Together, the results suggest a role for gamma2-adaptin in L-mediated processes of viral biogenesis and/or pathogenesis, such as facilitating and guiding HBV assembly.
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Project description:We constructed a novel chicken (Gallus gallus) lung cDNA library fused inside yeast acting domain vector (pGADT7). Using yeast two-hybrid screening with highly pathogenic avian influenza (HPAI) nucleoprotein (NP) from the strain (A/chicken/Malaysia/5858/2004(H5N1)) as bait, and the Gallus gallus lung cDNA library as prey, a novel interaction between the Gallus gallus cellular RNA export adaptor protein Aly/REF and the viral NP was identified. This interaction was confirmed and validated with mammalian two hybrid studies and co-immunoprecipitation assay. Cellular localization studies using confocal microscopy showed that NP and Aly/REF co-localize primarily in the nucleus. Further investigations by mammalian two hybrid studies into the binding of NP of other subtypes of influenza virus such as the swine A/New Jersey/1976/H1N1 and pandemic A/Malaysia/854/2009(H1N1) to human Aly/REF, also showed that the NP of these viruses interacts with human Aly/REF. Our findings are also supported by docking studies which showed tight and favorable binding between H5N1 NP and human Aly/REF, using crystal structures from Protein Data Bank. siRNA knockdown of Aly/REF had little effect on the export of HPAI NP and other viral RNA as it showed no significant reduction in virus titer. However, UAP56, another component of the TREX complex, which recruits Aly/REF to mRNA was found to interact even better with H5N1 NP through molecular docking studies. Both these proteins also co-localizes in the nucleus at early infection similar to Aly/REF. Intriguingly, knockdown of UAP56 in A549 infected cells shows significant reduction in viral titer (close to 10 fold reduction). Conclusively, our study have opened new avenues for research of other cellular RNA export adaptors crucial in aiding viral RNA export such as the SRSF3, 9G8 and ASF/SF2 that may play role in influenza virus RNA nucleocytoplasmic transport.
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Project description:Fused Toes (FTS) is a member of a small group of inactive variant E2 ubiquitin-conjugating enzyme domain-containing proteins of unknown function. Through proteomic analysis of FTS complexes purified from human embryonic kidney 293T cells, we identified a new multiprotein complex, the FHF complex, containing FTS, members of the microtubule-binding Hook family of coiled-coil proteins (Hook1, Hook2, and Hook3), and a previously uncharacterized 107-kDa protein, FTS and Hook Interacting Protein (FHIP). FTS associated with a conserved C-terminal motif in Hook proteins in the yeast two-hybrid system and in tissue culture cells, and Hook proteins were found to form homo- and heterodimers. The approximately 500-kDa FHF complex contained all three Hook proteins, and small interfering RNA depletion experiments suggest that Hook proteins can interact interchangeably within this complex. Hook proteins as well as FTS interact with members of both the class B and class C components of the homotypic vesicular protein sorting (HOPS) complex. Depletion of FTS by RNA interference affects both the trafficking of epidermal growth factor from early-to-late endosome/lysosomes and the efficiency by which overexpression of the HOPS component Vps18 promotes clustering of lysosomal-associated membrane protein 1-positive endosome/lysosomes. These data suggest that the FTS/Hook/FHIP complex functions to promote vesicle trafficking and/or fusion via the HOPS complex.
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Project description:Adaptor protein (AP) complexes sort cargo into vesicles for transport from one membrane compartment of the cell to another. Four distinct AP complexes have been identified, which are present in most eukaryotes. We report the existence of a fifth AP complex, AP-5. Tagged AP-5 localises to a late endosomal compartment in HeLa cells. AP-5 does not associate with clathrin and is insensitive to brefeldin A. Knocking down AP-5 subunits interferes with the trafficking of the cation-independent mannose 6-phosphate receptor and causes the cell to form swollen endosomal structures with emanating tubules. AP-5 subunits can be found in all five eukaryotic supergroups, but they have been co-ordinately lost in many organisms. Concatenated phylogenetic analysis provides robust resolution, for the first time, into the evolutionary order of emergence of the adaptor subunit families, showing AP-3 as the basal complex, followed by AP-5, AP-4, and AP-1 and AP-2. Thus, AP-5 is an evolutionarily ancient complex, which is involved in endosomal sorting, and which has links with hereditary spastic paraplegia.
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Project description:Myosin Va (MyoVa) is an actin-based molecular motor that plays key roles in the final stages of secretory pathways, including neurotransmitter release. Several studies have addressed how MyoVa coordinates the trafficking of secretory vesicles, but why this molecular motor is found in exosomes is still unclear. In this work, using a yeast two-hybrid screening system, we identified the direct interaction between the globular tail domain (GTD) of MyoVa and four protein components of exosomes: the WD repeat-containing protein 48 (WDR48), the cold shock domain-containing protein E1 (CSDE1), the tandem C2 domain-containing protein 1 (TC2N), and the enzyme spermine synthase (SMS). The interaction between the GTD of MyoVa and SMS was further validated in vitro and displayed a K d in the low micromolar range (3.5 ± 0.5 µM). SMS localized together with MyoVa in cytoplasmic vesicles of breast cancer MCF-7 and neuroblastoma SH-SY5Y cell lines, known to produce exosomes. Moreover, MYO5A knockdown decreased the expression of SMS gene and rendered the distribution of SMS protein diffuse, supporting a role for MyoVa in SMS expression and targeting.
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Project description:Multifunctional adapter and chaperone protein Daxx participates in the regulation of a number of mainly transcription-related processes. Most notably in a complex with chromatin-remodelling ATPase ATRX, Daxx serves as a histone H3.3 chaperone at telomeric regions and certain genes. In this report we document that Daxx interacts with another chromatin-remodelling, ATPase Brg1. We confirm the Daxx-Brg1 association both in vitro and in cells and show that Daxx interacts with Brg1 in high-molecular-weight complexes. Ectopic co-expression of Daxx with Brg1 and PML could shift disperse nuclear localisation of Brg1 into PML bodies. Mapping the Daxx-Brg1 interaction revealed that Daxx preferentially binds the region between Brg1 N-terminal QLQ and HSA domains, but also weakly interacts with its C-terminal part. Brg1 interacted with both the central and N-terminal parts of Daxx. SiRNA-mediated down-regulation of Daxx in SW13 adrenal carcinoma cells markedly enhanced expression of Brg1-activated genes CD44 or SCEL, suggesting that Daxx either directly through Brg1 and/or indirectly via other factors is a negative regulator of their transcription. Our findings point to Brg1 as another chromatin-remodelling protein that might similarly, as ATRX, target Daxx to specific chromatin regions where it can carry out its chromatin- and transcription-regulating functions.
| S-EPMC5600331 | biostudies-literature