Adaptor heat shock protein complex formation regulates trafficking of the asialoglycoprotein receptor.
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In the asialoglycoprotein receptor (ASGPR) endocytic pathway, internalized receptors pass through early, recycling, and sorting endosomal compartments before returning to the cell surface. Sorting motifs in the cytoplasmic domain (CD) and protein interactions with these sequences presumably direct receptor trafficking. Previous studies have shown that association of a potential sorting heat shock protein (HSP) heterocomplex with the ASGPR-CD was regulated by casein kinase 2 (CK2)-mediated phosphorylation. Mass spectrometry and immunoblot analyses identified five of these ASGPR-CD-associated proteins as the molecular chaperones glycoprotein 96, HSP70, HSP90, cyclophilin A, and FK 506 binding protein. The present study was undertaken to determine whether any of the adaptor protein complexes (AP1, AP2, or AP3) were selectivity associated with the ASGPR-CD. In conjunction with molecular chaperones, AP2 and AP1 were recovered from a CK2 phosphorylated agarose-GSH-GST-ASGPR-CD matrix. Binding of AP3 was independent of the phosphorylation status of the CD matrix. Inhibition of CK2-mediated phosphorylation with tetrabromobenzotriazole prevented AP recovery within an immunoadsorbed ASGPR complex. Rapamycin, which dissociates the HSP heterocomplex from ASGPR-CD, thereby altering receptor trafficking also, inhibited AP association. Similar results were obtained with an inhibitor of HSP90 heterocomplex formation, geldanmycin. The data presented provide evidence that recruitment of AP1 and AP2, which is necessary for appropriate receptor trafficking, is mediated by the interaction of AP with the ASGPR-CD-bound HSP complex. (+info)
HIV-1 Nef stabilizes AP-1 on membranes without inducing ARF1-independent de novo attachment.
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HIV-1 Nef affects the trafficking of numerous cellular proteins to optimize viral replication and evade host defenses. The adaptor protein (AP) complexes, which form part of the cytoplasmic coat of endosomal vesicles, are key cellular co-factors for Nef. Nef binds these complexes and alters their physiologic cycle of attachment and release from membranes. Specifically, while AP-1 normally becomes cytosolic when attachment events are blocked by inhibition of the GTPase cycle of ADP-ribosylation factor-1 (ARF1), the complex remains membrane-associated in Nef-expressing cells. To investigate the mechanism of this effect, we used a permeabilized cell system to detect the de novo attachment of exogenous AP-1 to endosomal membranes. Nef did not mediate de novo attachment independently of ARF1, despite its ability to maintain the association of AP-1 with endosomal membranes when the activity of ARF1 was blocked. We conclude that Nef stabilizes AP complexes on endosomal membranes after ARF1-dependent attachment. This stabilization may facilitate coat formation and stimulate the trafficking of multiple cellular proteins. (+info)
Anti-inflammatory mechanisms of phenanthroindolizidine alkaloids.
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The molecular mechanisms for the anti-inflammatory activity of phenanthroindolizidine alkaloids were examined in an in vitro system mimicking acute inflammation by studying the suppression of lipopolysaccharide (LPS)/interferon-gamma (IFNgamma)-induced nitric oxide production in RAW264.7 cells. Two of the phenanthroindolizidine alkaloids, NSTP0G01 (tylophorine) and NSTP0G07 (ficuseptine-A), exhibited potent suppression of nitric oxide production and did not show significant cytotoxicity to the LPS/IFNgamma-stimulated RAW264.7 cells, in contrast to their respective cytotoxic effects on cancer cells. Tylophorine was studied further to investigate the responsible mechanisms. It was found to inhibit the induced protein levels of tumor necrosis factor-alpha, inducible nitric-oxide synthase (iNOS), and cyclooxygenase (COX)-II. It also inhibited the activation of murine iNOS and COX-II promoter activity. However, of the two common responsive elements of iNOS and COX-II promoters, nuclear factor-kappaB (NF-kappaB) and adaptor protein (AP)1, only AP1 activation was inhibited by tylophorine in the LPS/IFNgamma-stimulated RAW264.7 cells. Further studies showed that the tylophorine enhanced the phosphorylation of Akt and thus decreased the expression and phosphorylation levels of c-Jun protein, thereby causing the subsequent inhibition of AP1 activity. Furthermore, the tylophorine was able to block mitogen-activated protein/extracellular signal-regulated kinase kinase 1 activity and its downstream signaling activation of NF-kappaB and AP1. Thus, NSTP0G01 exerts its anti-inflammatory effects by inhibiting expression of the proinflammatory factors and related signaling pathways. (+info)
Modulation of cellular protein trafficking by human immunodeficiency virus type 1 Nef: role of the acidic residue in the ExxxLL motif.
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The nef gene contributes to the replication of primate lentiviruses by altering the trafficking of cellular proteins involved in adaptive immunity (class I and II major histocompatibility complex [MHC]) and viral transmission (CD4 and DC-SIGN). A conserved acidic leucine-based sequence (E(160)xxxLL) within human immunodeficiency virus type 1 (HIV-1) Nef binds to the cellular adaptor protein (AP) complexes, which mediate protein sorting into endosomal vesicles. The leucine residues in this motif are required for the down-regulation of CD4 and for the up-regulation of DC-SIGN and the invariant chain of MHC class II, but the role of the acidic residue is unclear. Here, substitution of E160 with uncharged residues impaired the ability of Nef to up-regulate the expression of the invariant chain and DC-SIGN at the cell surface, whereas substitution with a basic residue was required for a similar effect on the down-regulation of CD4. All substitutions of E160 relieved the Nef-mediated block to transferrin uptake. E160 was required for the efficient interaction of Nef with AP-1 and AP-3 and for the stabilization of these complexes on endosomal membranes in living cells. Systematic mutation of the ExxxLL sequence together with correlation of binding and functional data leads to the hypotheses that AP-1 and AP-3 are major cofactors for the effect of Nef on the trafficking of transferrin, are less important but contribute to the modulation of the invariant chain and DC-SIGN, and are least critical for the modulation of CD4. The data suggest that the E160 residue plays a differential role in the modulation of leucine-dependent Nef-targets and support a model in which distinct AP complexes are used by Nef to modulate different cellular proteins. (+info)
Sorting of Pmel17 to melanosomes through the plasma membrane by AP1 and AP2: evidence for the polarized nature of melanocytes.
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Adaptor proteins (AP) play important roles in the sorting of proteins from the trans-Golgi network, but how they function in the sorting of various melanosome-specific proteins such as Pmel17, an essential structural component of melanosomes, in melanocytes is unknown. We characterized the processing and trafficking of Pmel17 via adaptor protein complexes within melanocytic cells. Proteomics analysis detected Pmel17, AP1 and AP2, but not AP3 or AP4 in early melanosomes. Real-time PCR, immunolabeling and tissue in-situ hybridization confirmed the coexpression of AP1 isoforms mu1A and mu1B (expressed only in polarized cells) in melanocytes and keratinocytes, but expression of mu1B is missing in some melanoma cell lines. Transfection with AP1 isoforms (mu1A or mu1B) showed two distinct distribution patterns that involved Pmel17, and only mu1B was able to restore the sorting of Pmel17 to the plasma membrane in cells lacking mu1B expression. Finally, we established that expression of mu1B is regulated physiologically in melanocytes by UV radiation or DKK1. These results show that Pmel17 is sorted to melanosomes by various intracellular routes, directly or indirectly through the plasma membrane, and the presence of basolateral elements in melanocytes suggests their polarized nature. (+info)
Laa1p, a conserved AP-1 accessory protein important for AP-1 localization in yeast.
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AP-1 and Gga adaptors participate in clathrin-mediated protein transport between the trans-Golgi network and endosomes. Both adaptors contain homologous domains that act to recruit accessory proteins involved in clathrin-coated vesicle formation, but the spectrum of known adaptor-binding partners is limited. This study describes an evolutionarily conserved protein of Saccharomyces cerevisiae, Laa1p (Yjl207cp), that interacts and functions specifically with AP-1. Deletion of LAA1, when combined with a conditional mutation in clathrin heavy chain or deletion of GGA genes, accentuated growth defects and increased disruption of clathrin-dependent alpha-factor maturation and transport of carboxypeptidase Y to the vacuole. In contrast, such genetic interactions were not observed between deletions of LAA1 and AP-1 subunit genes. Laa1p preferentially interacted with AP-1 compared with Gga proteins by glutathione S-transferase-fusion affinity binding and coimmunoprecipitations. Localization of AP-1 and Laa1p, but not Gga proteins, was highly sensitive to brefeldin A, an inhibitor of ADP-ribosylation factor (Arf) activation. Importantly, deletion of LAA1 caused mislocalization of AP-1, especially in cells at high density (postdiauxic shift), but it did not affect Gga protein distribution. Our results identify Laa1p as a new determinant of AP-1 localization, suggesting a model in which Laa1p and Arf cooperate to direct stable association of AP-1 with appropriate intracellular membranes. (+info)
The clathrin adaptor complex 1 directly binds to a sorting signal in Ste13p to reduce the rate of its trafficking to the late endosome of yeast.
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Yeast trans-Golgi network (TGN) membrane proteins maintain steady-state localization by constantly cycling to and from endosomes. In this study, we examined the trafficking itinerary and molecular requirements for delivery of a model TGN protein A(F-->A)-alkaline phosphatase (ALP) to the prevacuolar/endosomal compartment (PVC). A(F-->A)-ALP was found to reach the PVC via early endosomes (EEs) with a half-time of approximately 60 min. Delivery of A(F-->A)-ALP to the PVC was not dependent on either the GGA or adaptor protein 1 (AP-1) type of clathrin adaptors, which are thought to function in TGN to PVC and TGN to EE transport, respectively. Surprisingly, in cells lacking the function of both GGA and AP-1 adaptors, A(F-->A)-ALP transport to the PVC was dramatically accelerated. A 12-residue cytosolic domain motif of A(F-->A)-ALP was found to mediate direct binding to AP-1 and was sufficient to slow TGN-->EE-->PVC trafficking. These results suggest a model in which this novel sorting signal targets A(F-->A)-ALP into clathrin/AP-1 vesicles at the EE for retrieval back to the TGN. (+info)
Distinct roles for TGN/endosome epsin-like adaptors Ent3p and Ent5p.
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Clathrin adaptors are key factors in clathrin-coated vesicle formation, coupling clathrin to cargo and/or the lipid bilayer. A physically interacting network of three classes of adaptors participate in clathrin-mediated traffic between the trans-Golgi network (TGN) and endosomes: AP-1, Gga proteins, and epsin-like proteins. Here we investigate functional relationships within this network through transport assays and protein localization analysis in living yeast cells. We observed that epsin-like protein Ent3p preferentially localized with Gga2p, whereas Ent5p distributed equally between AP-1 and Gga2p. Ent3p was mislocalized in Gga-deficient but not in AP-1-deficient cells. In contrast, Ent5p retained localization in cells lacking either or both AP-1 and Gga proteins. The Ent proteins were dispensable for AP-1 or Gga localization. Synthetic genetic growth and alpha-factor maturation defects were observed when ent5Delta but not ent3Delta was introduced together with deletions of the GGA genes. In AP-1-deficient cells, ent3Delta and to a lesser extent ent5Delta caused minor alpha-factor maturation defects, but together resulted in a near-lethal phenotype. Deletions of ENT3 and ENT5 also displayed synthetic defects similar to, but less severe than, synthetic effects of AP-1 and Gga inactivation. These results differentiate Ent3p and Ent5p function in vivo, suggesting that Ent3p acts primarily with Gga proteins, whereas Ent5p acts with both AP-1 and Gga proteins but is more critical for AP-1-mediated transport. The data also support a model in which the Ent adaptors provide important accessory functions to AP-1 and Gga proteins in TGN/endosome traffic. (+info)