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PubMed2006

General and cell-type specific mechanisms target TRPP2/PKD-2 to cilia.

Ciliary localization of the transient receptor potential polycystin 2 channel (TRPP2/PKD-2) is evolutionarily conserved, but how TRPP2 is targeted to cilia is not known. In this study, we characterize the motility and localization of PKD-2, a TRPP2 homolog, in C. elegans sensory neurons. We demonstrate that GFP-tagged PKD-2 moves bidirectionally in the dendritic compartment. Furthermore, we show a requirement for different molecules in regulating the ciliary localization of PKD-2. PKD-2 is directed to moving dendritic particles by the UNC-101/adaptor protein 1 (AP-1) complex. When expressed in non-native neurons, PKD-2 remains in cell bodies and is not observed in dendrites or cilia, indicating that cell-type specific factors are required for directing PKD-2 to the dendrite. PKD-2 stabilization in cilia and cell bodies requires LOV-1, a functional partner and a TRPP1 homolog. In lov-1 mutants, PKD-2 is greatly reduced in cilia and forms abnormal aggregates in neuronal cell bodies. Intraflagellar transport (IFT) is not essential for PKD-2 dendritic motility or access to the cilium, but may regulate PKD-2 ciliary abundance. We propose that both general and cell-type-specific factors govern TRPP2/PKD-2 subcellular distribution by forming at least two steps involving somatodendritic and ciliary sorting decisions.

Adaptor Protein Complex 1↗
PubMed2005

Ubiquitously expressed secretory carrier membrane proteins (SCAMPs) 1-4 mark different pathways and exhibit limited constitutive trafficking to and from the cell surface.

Secretory carrier membrane proteins (SCAMPs) 1-4 are ubiquitously expressed and are major components of the eukaryotic cell surface recycling system. We investigated whether different SCAMPs function along distinct pathways and whether they behave like itinerant cargoes or less mobile trafficking machinery. In NRK cells, we show by immunofluorescence microscopy that different SCAMPs are concentrated mostly adjacent to one another in the trans-Golgi network and endosomal recycling compartment. By immunoelectron microscopy, they were shown to be close neighbors on individual transferrin-containing endosomal elements and on the plasma membrane. Within the internal endosomal network, SCAMPs are located distal to rab5-containing endosomes, and the individual isoforms appear to mark pathways that diverge from the constitutive recycling route and that may be distinguished by different adaptors, especially AP-1 and AP-3. Based on comparisons of SCAMP localization with endocytosed transferrin as well as live imaging of GFP-SCAMP1, we show that SCAMPs are concentrated within the motile population of early and recycling endosomes; however, they are not detected in newly formed transferrin-containing endocytic vesicles or in vesicles recycling transferrin to the surface. Also, they are not detected in constitutive secretory carriers marked by VSV-G. Their minimal recycling to the surface is reflected by their inability to relocate to the plasma membrane upon inhibition of endocytosis. Thus SCAMPs exhibit limited exchange between the cell surface and internal recycling systems, but within each of these sites, they form a mosaic with individual isoforms marking distinct pathways and potentially functioning as trafficking machinery at sites of vesicle formation and fusion. A corollary of these findings is that early endosomes exist as a distinct SCAMP-containing compartment and are not formed de novo by fusion of endocytic vesicles.

Adaptor Protein Complex 1↗
PubMed2004

The cytoplasmic tail of the cation-independent mannose 6-phosphate receptor contains four binding sites for AP-1.

The trafficking of the cation-independent mannose 6-phosphate receptor between the trans-Golgi network and endosomes requires binding of sorting determinants in the cytoplasmic tail of the receptor to adaptor protein complex-1 (AP-1). Using a GST pull-down binding assay, four binding motifs were identified in the cytoplasmic tail: a tyrosine-based motif ((26)YSKV(29)), an internal dileucine-based motif ((39)ETEWLM(44)), and two casein kinase 2 sites ((84)DSEDE(88) and (154)DDSDED(159)). The YSKV motif mediated the strongest interaction with AP-1 and the two CK2 motifs bound AP-1 only when they were phosphorylated. The COOH-terminal dileucines were not required for interaction with AP-1.

Adaptor Protein Complex 1↗
PubMed2003

Phosphatidylinositol 4 phosphate regulates targeting of clathrin adaptor AP-1 complexes to the Golgi.

Phosphatidylinositol 4 phosphate [PI(4)P] is essential for secretion in yeast, but its role in mammalian cells is unclear. Current paradigms propose that PI(4)P acts primarily as a precursor to phosphatidylinositol 4,5 bisphosphate (PIP2), an important plasma membrane regulator. We found that PI(4)P is enriched in the mammalian Golgi, and used RNA interference (RNAi) of PI4KIIalpha, a Golgi resident phosphatidylinositol 4 kinase, to determine whether PI(4)P directly regulates the Golgi. PI4KIIalpha RNAi decreases Golgi PI(4)P, blocks the recruitment of clathrin adaptor AP-1 complexes to the Golgi, and inhibits AP-1-dependent functions. This AP-1 binding defect is rescued by adding back PI(4)P. In addition, purified AP-1 binds PI(4)P, and anti-PI(4)P inhibits the in vitro recruitment of cytosolic AP-1 to normal cellular membranes. We propose that PI4KIIalpha establishes the Golgi's unique lipid-defined organelle identity by generating PI(4)P-rich domains that specify the docking of the AP-1 coat machinery.

Adaptor Protein Complex 1↗
PubMed2002

Drs2p-dependent formation of exocytic clathrin-coated vesicles in vivo.

The small GTP binding protein ARF has been implicated in budding clathrin-coated vesicles (CCVs) from Golgi and endosomal membranes. An arf1 synthetic lethal screen identified DRS2/SWA3 along with a clathrin heavy-chain conditional allele (chc1-5/swa5-1) and SWA2, encoding the yeast auxilin-like protein involved in uncoating CCVs. Drs2p/Swa3p is a P-type ATPase and a potential aminophospholipid translocase that localizes to the trans-Golgi network (TGN) in yeast. Genetic and phenotypic analyses of drs2Delta mutants suggested that Drs2p was required for clathrin function. To address a potential role for Drs2p in CCV formation from the TGN in vivo, we have performed epistasis analyses between drs2 and mutations that cause accumulation of distinct populations of post-Golgi vesicles. We find that Drs2p is required to form a specific class of secretory vesicles that accumulate when the actin cytoskeleton is disrupted. Accumulation of these vesicles also requires clathrin and is perturbed by mutation of AP-1, but not AP-2, AP-3, or GGA adaptins. Most of the accumulated vesicles are uncoated; however, clathrin coats can be partially stabilized on these vesicles by deletion of SWA2. These data provide the first in vivo evidence for an integral membrane protein requirement in forming CCVs.

Adaptor Protein Complex 1↗
PubMed2002

The endocytic apparatus of the kinetoplastida. Part II: machinery and components of the system.

Endocytic systems within eukaryotic cells are a diverse set of intracellular transport pathways responsible for uptake, recycling, interaction with the exocytic system and degradation of molecules. Each of these pathways requires the interaction of distinct protein components that function in macromolecule sorting, control of transport rates and in membrane biogenesis. In the second of two articles on kinetoplastida endocytosis, the endocytic system in Trypanosoma brucei is considered as a model, and the molecules that control this system and the protein components of the endocytic pathway are discussed. We also consider novel mechanisms for sorting that have been proposed to operate in trypanosomes.

Adaptor Protein Complex 1↗
PubMed2002

AP-1 in Toxoplasma gondii mediates biogenesis of the rhoptry secretory organelle from a post-Golgi compartment.

We have previously demonstrated that Toxoplasma gondii has a tyrosine-based sorting system, which mediates protein targeting to the lysosome-like rhoptry secretory organelle. We now show that rhoptry protein targeting is also dependent on a dileucine motif and occurs from a post-Golgi endocytic organelle to mature rhoptries in an adaptin-dependent fashion. The T. gondii AP-1 adaptin complex is implicated in this transport because the micro1 chain of T. gondii AP-1 (a) was localized to multivesicular endosomes and the limiting and luminal membranes of the rhoptries; (b) bound to endocytic tyrosine motifs in rhoptry proteins, but not in proteins from dense granule secretory organelles; (c) when mutated in predicted tyrosine-binding motifs, led to accumulation of the rhoptry protein ROP2 in a post-Golgi multivesicular compartment; and (d) when depleted via antisense mRNA, resulted in accumulation of multivesicular endosomes and immature rhoptries. These are the first results to implicate AP-1 in transport from a post-Golgi compartment to a mature secretory organelle and substantially expand the role for AP-1 in anterograde protein transport.

Adaptor Protein Complex 1↗
PubMed2003

AP-1 binding to sorting signals and release from clathrin-coated vesicles is regulated by phosphorylation.

The adaptor protein complex-1 (AP-1) sorts and packages membrane proteins into clathrin-coated vesicles (CCVs) at the TGN and endosomes. Here we show that this process is highly regulated by phosphorylation of AP-1 subunits. Cell fractionation studies revealed that membrane-associated AP-1 differs from cytosolic AP-1 in the phosphorylation status of its beta1 and mu1 subunits. AP-1 recruitment onto the membrane is associated with protein phosphatase 2A (PP2A)-mediated dephosphorylation of its beta1 subunit, which enables clathrin assembly. This Golgi-associated isoform of PP2A exhibits specificity for phosphorylated beta1 compared with phosphorylated mu1. Once on the membrane, the mu1 subunit undergoes phosphorylation, which results in a conformation change, as revealed by increased sensitivity to trypsin. This conformational change is associated with increased binding to sorting signals on the cytoplasmic tails of cargo molecules. Dephosphorylation of mu1 (and mu2) by another PP2A-like phosphatase reversed the effect and resulted in adaptor release from CCVs. Immunodepletion and okadaic acid inhibition studies demonstrate that PP2A is the cytosolic cofactor for Hsc-70-mediated adaptor uncoating. A model is proposed where cyclical phosphorylation/dephosphorylation of the subunits of AP-1 regulate its function from membrane recruitment until its release into cytosol.

Adaptor Protein Complex 1↗
PubMed2004

Recycling endosomes can serve as intermediates during transport from the Golgi to the plasma membrane of MDCK cells.

The AP-1B clathrin adaptor complex is responsible for the polarized transport of many basolateral membrane proteins in epithelial cells. Localization of AP-1B to recycling endosomes (REs) along with other components (exocyst subunits and Rab8) involved in AP-1B-dependent transport suggested that RE might be an intermediate between the Golgi and the plasma membrane. Although the involvement of endosomes in the secretory pathway has long been suspected, we now present direct evidence using four independent methods that REs play a role in basolateral transport in MDCK cells. Newly synthesized AP-1B-dependent cargo, vesicular stomatitis virus glycoprotein G (VSV-G), was found by video microscopy, immunoelectron microscopy, and cell fractionation to enter transferrin-positive REs within a few minutes after exit from the trans-Golgi network. Although transient, RE entry appears essential because enzymatic inactivation of REs blocked VSV-G delivery to the cell surface. Because an apically targeted VSV-G mutant behaved similarly, these results suggest that REs not only serve as an intermediate but also as a common site for polarized sorting on the endocytic and secretory pathways.

Adaptor Protein Complex 1↗
PubMed2005

An AP-1/clathrin coat plays a novel and essential role in forming the Weibel-Palade bodies of endothelial cells.

Clathrin provides an external scaffold to form small 50-100-nm transport vesicles. In contrast, formation of much larger dense-cored secretory granules is driven by selective aggregation of internal cargo at the trans-Golgi network; the only known role of clathrin in dense-cored secretory granules formation is to remove missorted proteins by small, coated vesicles during maturation of these spherical organelles. The formation of Weibel-Palade bodies (WPBs) is also cargo driven, but these are cigar-shaped organelles up to 5 mum long. We hypothesized that a cytoplasmic coat might be required to make these very different structures, and we found that new and forming WPBs are extensively, sometimes completely, coated. Overexpression of an AP-180 truncation mutant that prevents clathrin coat formation or reduced AP-1 expression by small interfering RNA both block WPB formation. We propose that, in contrast to other secretory granules, cargo aggregation alone is not sufficient to form immature WPBs and that an external scaffold that contains AP-1 and clathrin is essential.

Adaptor Protein Complex 1↗
PubMed2005

Transcytosis of NgCAM in epithelial cells reflects differential signal recognition on the endocytic and secretory pathways.

NgCAM is a cell adhesion molecule that is largely axonal in neurons and apical in epithelia. In Madin-Darby canine kidney cells, NgCAM is targeted to the apical surface by transcytosis, being first inserted into the basolateral domain from which it is internalized and transported to the apical domain. Initial basolateral transport is mediated by a sequence motif (Y(33)RSL) decoded by the AP-1B clathrin adaptor complex. This motif is a substrate in vitro for tyrosine phosphorylation by p60src, a modification that disrupts NgCAM's ability to interact with clathrin adaptors. Based on the behavior of various NgCAM mutants, it appears that after arrival at the basolateral surface, the AP-1B interaction site is silenced by phosphorylation of Tyr(33). This slows endocytosis and inhibits basolateral recycling from endosomes, resulting in NgCAM transcytosis due to a cryptic apical targeting signal in its extracellular domain. Thus, transcytosis of NgCAM and perhaps other membrane proteins may reflect the spatial regulation of recognition by adaptors such as AP-1B.

Adaptor Protein Complex 1↗
PubMed2003

Visualization of TGN to endosome trafficking through fluorescently labeled MPR and AP-1 in living cells.

We have stably expressed in HeLa cells a chimeric protein made of the green fluorescent protein (GFP) fused to the transmembrane and cytoplasmic domains of the mannose 6-phosphate/insulin like growth factor II receptor in order to study its dynamics in living cells. At steady state, the bulk of this chimeric protein (GFP-CI-MPR) localizes to the trans-Golgi network (TGN), but significant amounts are also detected in peripheral, tubulo-vesicular structures and early endosomes as well as at the plasma membrane. Time-lapse videomicroscopy shows that the GFP-CI-MPR is ubiquitously detected in tubular elements that detach from the TGN and move toward the cell periphery, sometimes breaking into smaller tubular fragments. The formation of the TGN-derived tubules is temperature dependent, requires the presence of intact microtubule and actin networks, and is regulated by the ARF-1 GTPase. The TGN-derived tubules fuse with peripheral, tubulo-vesicular structures also containing the GFP-CI-MPR. These structures are highly dynamic, fusing with each other as well as with early endosomes. Time-lapse videomicroscopy performed on HeLa cells coexpressing the CFP-CI-MPR and the AP-1 complex whose gamma-subunit was fused to YFP shows that AP-1 is present not only on the TGN and peripheral CFP-CI-MPR containing structures but also on TGN-derived tubules containing the CFP-CI-MPR. The data support the notion that tubular elements can mediate MPR transport from the TGN to a peripheral, tubulo-vesicular network dynamically connected with the endocytic pathway and that the AP-1 coat may facilitate MPR sorting in the TGN and endosomes.

Adaptor Protein Complex 1↗
PubMed2003

The AP-1 clathrin-adaptor is required for lysosomal enzymes sorting and biogenesis of the contractile vacuole complex in Dictyostelium cells.

Adaptor protein complexes (AP) are major components of the cytoplasmic coat found on clathrin-coated vesicles. Here, we report the molecular and functional characterization of Dictyostelium clathrin-associated AP-1 complex, which in mammalian cells, participates mainly in budding of clathrin-coated vesicles from the trans-Golgi network (TGN). The gamma-adaptin AP-1 subunit was cloned and shown to belong to a Golgi-localized 300-kDa protein complex. Time-lapse analysis of cells expressing gamma-adaptin tagged with the green-fluorescent protein demonstrates the dynamics of AP-1-coated structures leaving the Golgi apparatus and rarely moving toward the TGN. Targeted disruption of the AP-1 medium chain results in viable cells displaying a severe growth defect and a delayed developmental cycle compared with parental cells. Lysosomal enzymes are constitutively secreted as precursors, suggesting that protein transport between the TGN and lysosomes is defective. Although endocytic protein markers are correctly localized to endosomal compartments, morphological and ultrastructural studies reveal the absence of large endosomal vacuoles and an increased number of small vacuoles. In addition, the function of the contractile vacuole complex (CV), an osmoregulatory organelle is impaired and some CV components are not correctly targeted.

Adaptor Protein Complex 1↗
PubMed2005

The aftiphilin/p200/gamma-synergin complex.

Aftiphilin is a protein that was recently identified in database searches for proteins with motifs that interact with AP-1 and clathrin, but its function is unknown. Here we demonstrate that aftiphilin has a second, atypical clathrin binding site, YQW, that colocalizes with AP-1 by immunofluorescence, and that is enriched in clathrin-coated vesicles (CCVs), confirming that it is a bona fide component of the CCV machinery. By gel filtration, aftiphilin coelutes with two other AP-1 binding partners, p200a and gamma-synergin. Antibodies against any one of these three proteins immunoprecipitate the other two, and knocking down any of the three proteins by siRNA causes a reduction in the levels of the other two, indicating that they form a stable complex. Like AP-1-depleted cells, aftiphilin-depleted cells missort a CD8-furin chimera and the lysosomal enzyme cathepsin D. However, whereas AP-1-depleted cells recycle endocytosed transferrin more slowly than untreated cells, aftiphilin-depleted cells accumulate endocytosed transferrin in a peripheral compartment and recycle it more rapidly. These observations show that in general, the aftiphilin/p200/gamma-synergin complex facilitates AP-1 function, but the complex may have additional functions as well, because of the opposing effects of the two knockdowns on transferrin recycling.

Adaptor Protein Complex 1↗
PubMed2003

Modulation of endotoxin-induced endothelial activity by microtubule depolymerization.

BACKGROUND: Endotoxin not only activates the Toll-mediated signaling pathway within endothelial cells that leads to neutrophil migration but also causes the polymerization of microtubules. The potential role of this polymerization event, however, is unknown. METHODS: Human umbilical vein endothelial cells stimulated with endotoxin were pretreated with or without the microtubule depolymerizing agent colchicine. Toll-mediated signaling events and protein production were in turn investigated by Western blot, gel shift, and enzyme-linked immunosorbent assay. Finally, neutrophil adhesion was assayed fluorometrically under the various conditions. RESULTS: Endotoxin led to activation of the various Toll-mediated pathways, production of intercellular adhesion molecule-1 and interleukin-8, and subsequent neutrophil adhesion. Pretreatment with colchicine led to selective inhibition of anti-dual phosphorylated extracellular signal-regulated kinase-1/2, anti-dual phosphorylated c-jun N-terminal kinase, and adaptor protein-1; selective enhancement of p38; and no effect on nuclear factor-kappaB. This selective modulation of intracellular signaling resulted in attenuated intercellular adhesion molecule-1, interleukin-8 and prostaglandin E2 production, but enhanced cyclooxygenase-2 expression. As a result, microtubule disruption led to a significant reduction in neutrophil adhesion. CONCLUSION: Microtubule formation is essential to optimal endotoxin-induced intracellular signaling through anti-dual phosphorylated extracellular signal-regulated kinase-1/2, anti-dual phosphorylated c-jun N-terminal kinase, and adaptor protein-1. Failure of these signaling events is associated with a marked reduction in the formation of a proadhesive phenotype that may prove to be beneficial in modulating neutrophil recruitment during sepsis.

Adaptor Protein Complex 1↗
PubMed2005

Regulation of matrix metalloproteinase 9 expression by Epstein-Barr virus nuclear antigen 3C and the suppressor of metastasis Nm23-H1.

Epstein-Barr virus latent protein EBNA3C has been shown to bind Nm23-H1, a known suppresser of cell migration and metastasis and a regulator of the guanine exchange factor Tiam-1. This interaction results in cellular translocation of Nm23-H1 to the nucleus and suppression of the antimigratory effect in vitro. Furthermore, these proteins can synergistically increase transcription of a basal promoter when targeted to DNA by fusion to a Gal4 DNA binding domain. In this report, we show that EBNA3C and Nm23-H1 can cooperate to upregulate expression of MMP-9, known to be expressed in aggressive forms of lymphomas. This upregulation resulted in increased levels of MMP-9 mRNA, as well as a detectable increase in MMP-9 gelatinolytic activity. Specific mutations in the MMP-9 promoter showed that the Ap1 and NFkappaB binding sites are important for upregulation by the proteins. Additionally, it was shown for the first time that EBNA3C and Nm23-H1 can bind subunits of these transcription factors. This suggests that the ability of EBNA3C to reverse the antimigratory effects of Nm23-H1 is likely to be in part through the synergistic upregulation of MMP-9, mediated through interactions with the AP1 and NFkappaB transcription factors.

Adaptor Protein Complex 1↗