The Cytosolic Adaptor AP‐1A Is Essential for the Trafficking and Function of Niemann‐Pick Type C Proteins

Niemann‐Pick type C (NPC) disease is a fatal neurodegenerative disorder characterized by over‐accumulation of low‐density lipoprotein‐derived cholesterol and glycosphingolipids in late endosomes/lysosomes (LE/L) throughout the body. Human mutations in either NPC1 or NPC2 genes have been directly associated with impaired cholesterol efflux from LE/L. Independent from its role in cholesterol homeostasis and its NPC2 partner, NPC1 was unexpectedly identified as a critical player controlling intracellular entry of filoviruses such as Ebola. In this study, a yeast three‐hybrid system revealed that the NPC1 cytoplasmic tail directly interacts with the clathrin adaptor protein AP‐1 via its acidic/di‐leucine motif. Consequently, a nonfunctional AP‐1A cytosolic complex resulted in a typical NPC‐like phenotype mainly due to a direct impairment of NPC1 trafficking to LE/L and a partial secretion of NPC2. Furthermore, the mislocalization of NPC1 was not due to cholesterol accumulation in LE/L, as it was not rescued upon treatment with Mβ‐cyclodextrin, which almost completely eliminated intracellular free cholesterol. Our cumulative data demonstrate that the cytosolic clathrin adaptor AP‐1A is essential for the lysosomal targeting and function of NPC1 and NPC2.     

The kinesin KIF16B mediates apical transcytosis of transferrin receptor in AP‐1B‐deficient epithelia

Polarized epithelial cells take up nutrients from the blood through receptors that are endocytosed and recycle back to the basolateral plasma membrane (PM) utilizing the epithelial‐specific clathrin adaptor AP‐1B. Some native epithelia lack AP‐1B and therefore recycle cognate basolateral receptors to the apical PM, where they carry out important functions for the host organ. Here, we report a novel transcytotic pathway employed by AP‐1B‐deficient epithelia to relocate AP‐1B cargo, such as transferrin receptor (TfR), to the apical PM. Lack of AP‐1B inhibited basolateral recycling of TfR from common recycling endosomes (CRE), the site of function of AP‐1B, and promoted its transfer to apical recycling endosomes (ARE) mediated by the plus‐end kinesin KIF16B and non‐centrosomal microtubules, and its delivery to the apical membrane mediated by the small GTPase rab11a. Hence, our experiments suggest that the apical recycling pathway of epithelial cells is functionally equivalent to the rab11a‐dependent TfR recycling pathway of non‐polarized cells. They define a transcytotic pathway important for the physiology of native AP‐1B‐deficient epithelia and report the first microtubule motor involved in transcytosis.     

Identification of annexin A1 as a novel substrate for E6AP‐mediated ubiquitylation

E6‐associated protein (E6AP) is a cellular ubiquitin protein ligase that mediates ubiquitylation and degradation of p53 in conjunction with the high‐risk human papillomavirus E6 proteins. However, the physiological functions of E6AP are poorly understood. To identify a novel biological function of E6AP, we screened for binding partners of E6AP using GST pull‐down and mass spectrometry. Here we identified annexin A1, a member of the annexin superfamily, as an E6AP‐binding protein. Ectopic expression of E6AP enhanced the degradation of annexin A1 in vivo. RNAi‐mediated downregulation of endogenous E6AP increased the levels of endogenous annexin A1 protein. E6AP interacted with annexin A1 and induced its ubiquitylation in a Ca²⁺‐dependent manner. GST pull‐down assay revealed that the annexin repeat domain III of annexin A1 is important for the E6AP binding. Taken together, our data suggest that annexin A1 is a novel substrate for E6AP‐mediated ubiquitylation. Our findings raise the possibility that E6AP may play a role in controlling the diverse functions of annexin A1 through the ubiquitin‐proteasome pathway. J. Cell. Biochem. 106: 1123–1135, 2009. © 2009 Wiley‐Liss, Inc.     

Conformational regulation of AP1 and AP2 clathrin adaptor complexes

Heterotetrameric clathrin adaptor protein complexes (APs) orchestrate the formation of coated vesicles for transport among organelles of the cell periphery. AP1 binds membranes enriched for phosphatidylinositol 4‐phosphate, such as the trans Golgi network, while AP2 associates with phosphatidylinositol 4,5‐bisphosphate of the plasma membrane. At their respective membranes, AP1 and AP2 bind the cytoplasmic tails of transmembrane protein cargo and clathrin triskelions, thereby coupling cargo recruitment to coat polymerization. Structural, biochemical and genetic studies have revealed that APs undergo conformational rearrangements and reversible phosphorylation to cycle between different activity states. While membrane, cargo and clathrin have been demonstrated to promote AP activation, growing evidence supports that membrane‐associated proteins such as Arf1 and FCHo also stimulate this transition. APs may be returned to the inactive state via a regulated process involving phosphorylation and a protein called NECAP. Finally, because antiviral mechanisms often rely on appropriate trafficking of membrane proteins, viruses have evolved novel strategies to evade host defenses by influencing the conformation of APs. This review will cover recent advances in our understanding of the molecular inputs that stimulate AP1 and AP2 to adopt structurally and functionally distinct configurations.     

TgDrpC,an atypical dynamin‐related protein in Toxoplasma gondii,is associated with vesicular transport factors and parasite division

Dynamin‐related proteins (Drps) are involved in diverse processes such as organelle division and vesicle trafficking. The intracellular parasite Toxoplasma gondii possesses three distinct Drps. TgDrpC, whose function remains unresolved, is unusual in that it lacks a conserved GTPase Effector Domain, which is typically required for function. Here, we show that TgDrpC localizes to cytoplasmic puncta; however, in dividing parasites, TgDrpC redistributes to the growing edge of the daughter cells. By conditional knockdown, we determined that loss of TgDrpC stalls division and leads to rapid deterioration of multiple organelles and the IMC. We also show that TgDrpC interacts with proteins that exhibit homology to those involved in vesicle transport, including members of the adaptor complex 2. Two of these proteins, a homolog of the adaptor protein 2 (AP‐2) complex subunit alpha‐1 and a homolog of the ezrin–radixin–moesin (ERM) family proteins, localize to puncta and associate with the daughter cells. Consistent with the association with vesicle transport proteins, re‐distribution of TgDrpC to the IMC during division is dependent on post‐Golgi trafficking. Together, these results support that TgDrpC contributes to vesicle trafficking and is critical for stability of parasite organelles and division.     

The Sybtraps: Control of Synaptobrevin Traffic by Synaptophysin, α‐Synuclein and AP‐180

Synaptobrevin II (sybII) is a key fusogenic molecule on synaptic vesicles (SVs) therefore the active maintenance of both its conformation and location in sufficient numbers on this organelle is critical in both mediating and sustaining neurotransmitter release. Recently three proteins have been identified having key roles in the presentation, trafficking and retrieval of sybII during the fusion and endocytosis of SVs. The nerve terminal protein α‐synuclein catalyses sybII entry into SNARE complexes, whereas the monomeric adaptor protein AP‐180 is required for sybII retrieval during SV endocytosis. Overarching these events is the tetraspan SV protein synaptophysin, which is a major sybII interaction partner on the SV. This review will evaluate recent studies to propose working models for the control of sybII traffic by synaptophysin and other Sybtraps (syb II tra fficking p artners ) and suggest how dysfunction in sybII traffic may contribute to human disease.      

During Human Melanoma Progression AP‐1 Binding Pairs are Altered with Loss of c‐Jun In Vitro

We demonstrated previously that c‐Jun, JunB and c‐Fos RNA were dysregulated in metastatic melanoma cells compared with normal human melanocytes. The purpose of this study was to evaluate the distribution in composition of AP‐1 dimers in human melanoma pathogenesis. We investigated AP‐1 dimer pairing in radial growth phase‐like (RGP) (w3211) and vertical growth phase‐like (VGP) (w1205) human melanoma cells and metastatic cell lines (cloned from patients, c83‐2c, c81‐46A, A375, respectively) compared with melanocytes using electrophoretic mobility shift assay (EMSA), Western blot and transfection analyses. There are progressive variations in AP‐1 composition in different melanoma cell lines compared with normal melanocytes, in which c‐Jun, JunD and FosB were involved in AP‐1 complexes. In w3211, c‐Jun, JunD and Fra‐1 were involved in AP‐1 binding, while in w1205, overall AP‐1 binding activity was decreased significantly and supershift binding was detected only with JunD antibodies. In metastatic c81‐46A and A375 cells, only JunD was involved in AP‐1 binding activity, but in a third (c83‐2c) c‐Jun, JunD and Fra‐1 were present. Western blot evaluation detected c‐Jun in melanocytes and w3211, but this component was decreased significantly or was not detectable in w1205, c81‐46A and A375 cells. In contrast, JunD protein was elevated in c81‐46A and c83‐2c cells compared with melanocytes and RGP and VGP cell lines. Normal melanocytes and c83‐2c cells (which have c‐Jun involved in AP‐1 binding), transfected with c‐Jun antisense and treated with cisplatin, showed higher viability compared with untransfected cells, while in c81‐46A cells (in which only JunD is detectable) no change in cell viability was observed following treatment with cisplatin and c‐jun antisense transfection. A dominant‐negative c‐Jun mutant (TAM67) significantly increased the soft agar colony formation of w3211 and c83‐2c cells. These results suggest that components of AP‐1, especially c‐Jun, may offer a new target for the prevention or treatment of human melanoma progression.     

Exit of GPI‐Anchored Proteins from the ER Differs in Yeast and Mammalian Cells

Previous studies have shown that yeast glycosylphosphatidylinositol‐anchored proteins (GPI‐APs) and other secretory proteins are preferentially incorporated into distinct coat protein II (COPII) vesicle populations for their transport from the endoplasmic reticulum (ER) to the Golgi apparatus, and that incorporation of yeast GPI‐APs into COPII vesicles requires specific lipid interactions. We compared the ER exit mechanism and segregation of GPI‐APs from other secretory proteins in mammalian and yeast cells. We find that, unlike yeast, ER‐to‐Golgi transport of GPI‐APs in mammalian cells does not depend on sphingolipid synthesis. Whereas ER exit of GPI‐APs is tightly dependent on Sar1 in mammalian cells, it is much less so in yeast. Furthermore, in mammalian cells, GPI‐APs and other secretory proteins are not segregated upon COPII vesicle formation, in contrast to the remarkable segregation seen in yeast. These findings suggest that GPI‐APs use different mechanisms to concentrate in COPII vesicles in the two organisms, and the difference might explain their propensity to segregate from other secretory proteins upon ER exit.     

Clathrin functions in the absence of heterotetrameric adaptors and AP180-related proteins in yeast.

The major coat proteins of clathrin-coated vesicles are the clathrin triskelion and heterotetrameric associated protein (AP) complexes. The APs are thought to be involved in cargo capture and recruitment of clathrin to the membrane during endocytosis and sorting in the trans-Golgi network/endosomal system. AP180 is an abundant coat protein in brain clathrin-coated vesicles, and it has potent clathrin assembly activity. In Saccharomyces cerevisiae, there are 13 genes encoding homologs of heterotetrameric AP subunits and two genes encoding AP180-related proteins. To test the model that clathrin function is dependent on the heterotetrameric APs and/or AP180 homologs, yeast strains containing multiple disruptions in AP subunit genes, as well as in the two YAP180 genes, were constructed. Surprisingly, the AP deletion strains did not display the phenotypes associated with clathrin deficiency, including slowed growth and endocytosis, defective late Golgi protein retention and impaired cytosol to vacuole/autophagy function. Clathrin-coated vesicles isolated from multiple AP deletion mutants were morphologically indistinguishable from those from wild-type cells. These results indicate that clathrin function and recruitment onto membranes are not dependent upon heterotetrameric adaptors or AP180 homologs in yeast. Therefore, alternative mechanisms for clathrin assembly and coated vesicle formation, as well as the role of AP complexes and AP180-related proteins in these processes, must be considered.     

Differential Roles of Grb2 and AP‐2 in p38 MAPK‐ and EGF‐Induced EGFR Internalization

The epidermal growth factor receptor ( EGFR ) is an important regulator of normal growth and differentiation, and it is involved in the pathogenesis of many cancers. Endocytic downregulation is central in terminating EGFR signaling after ligand stimulation. It has been shown that p38 MAPK activation also can induce EGFR endocytosis. This endocytosis lacks many of the characteristics of ligand‐induced EGFR endocytosis. We compared the two types of endocytosis with regard to the requirements for proteins in the internalization machinery. Both types of endocytosis require clathrin, but while epidermal growth factor (EGF) ‐induced EGFR internalization also required Grb 2 , p38 MAPK ‐induced internalization did not. Interestingly , AP ‐2 knock down blocked p38 MAPK ‐induced EGFR internalization, but only mildly affected EGF ‐induced internalization. In line with this, simultaneously mutating two AP ‐2 interaction sites in EGFR affected p38 MAPK ‐induced internalization much more than EGF ‐induced EGFR internalization. Thus, it seems that EGFR in the two situations uses different sets of internalization mechanisms.     

AP‐1A Controls Secretory Granule Biogenesis and Trafficking of Membrane Secretory Granule Proteins

The adaptor protein 1A complex (AP‐1A) transports cargo between the trans‐Golgi network (TGN) and endosomes. In professional secretory cells, AP‐1A also retrieves material from immature secretory granules (SGs). The role of AP‐1A in SG biogenesis was explored using AtT‐20 corticotrope tumor cells expressing reduced levels of the AP‐1A μ1A subunit. A twofold reduction in μ1A resulted in a decrease in TGN cisternae and immature SGs and the appearance of regulated secretory pathway components in non‐condensing SGs. Although basal secretion of endogenous SG proteins was unaffected, secretagogue‐stimulated release was halved. The reduced μ1A levels interfered with the normal trafficking of carboxypeptidase D (CPD) and peptidylglycine α‐amidating monooxygenase‐1 (PAM‐1), integral membrane enzymes that enter immature SGs. The non‐condensing SGs contained POMC products and PAM‐1, but not CPD. Based on metabolic labeling and secretion experiments, the cleavage of newly synthesized PAM‐1 into PHM was unaltered, but PHM basal secretion was increased in sh‐μ1A PAM‐1 cells. Despite lacking a canonical AP‐1A binding motif, yeast two‐hybrid studies demonstrated an interaction between the PAM‐1 cytosolic domain and AP‐1A. Coimmunoprecipitation experiments with PAM‐1 mutants revealed an influence of the luminal domains of PAM‐1 on this interaction. Thus, AP‐1A is crucial for normal SG biogenesis, function and composition.      

A dominant‐negative form of Arabidopsis AP‐3 β‐adaptin improves intracellular pH homeostasis

Intracellular pH (pH_i) is a crucial parameter in cellular physiology but its mechanisms of homeostasis are only partially understood. To uncover novel roles and participants of the pH_i regulatory system, we have screened an Arabidopsis mutant collection for resistance of seed germination to intracellular acidification induced by weak organic acids (acetic, propionic, sorbic). The phenotypes of one identified mutant, weak acid‐tolerant 1‐1D (wat1‐1D) are due to the expression of a truncated form of AP‐3 β‐adaptin (encoded by the PAT2 gene) that behaves as a as dominant‐negative. During acetic acid treatment the root epidermal cells of the mutant maintain a higher pH_i and a more depolarized plasma membrane electrical potential than wild‐type cells. Additional phenotypes of wat1‐1D roots include increased rates of acetate efflux, K⁺ uptake and H⁺ efflux, the latter reflecting the in vivo activity of the plasma membrane H⁺‐ATPase. The in vitro activity of the enzyme was not increased but, as the H⁺‐ATPase is electrogenic, the increased ion permeability would allow a higher rate of H⁺ efflux. The AP‐3 adaptor complex is involved in traffic from Golgi to vacuoles but its function in plants is not much known. The phenotypes of the wat1‐1D mutant can be explained if loss of function of the AP‐3 β‐adaptin causes activation of channels or transporters for organic anions (acetate) and for K⁺ at the plasma membrane, perhaps through miss‐localization of tonoplast proteins. This suggests a role of this adaptin in trafficking of ion channels or transporters to the tonoplast.     

AGAP1/AP‐3‐dependent endocytic recycling of M5 muscarinic receptors promotes dopamine release

Of the five mammalian muscarinic acetylcholine (ACh) receptors, M₅ is the only subtype expressed in midbrain dopaminergic neurons, where it functions to potentiate dopamine release. We have identified a direct physical interaction between M₅ and the AP‐3 adaptor complex regulator AGAP1. This interaction was specific with regard to muscarinic receptor (MR) and AGAP subtypes, and mediated the binding of AP‐3 to M₅. Interaction with AGAP1 and activity of AP‐3 were required for the endocytic recycling of M₅ in neurons, the lack of which resulted in the downregulation of cell surface receptor density after sustained receptor stimulation. The elimination of AP‐3 or abrogation of AGAP1–M₅ interaction in vivo decreased the magnitude of presynaptic M₅‐mediated dopamine release potentiation in the striatum. Our study argues for the presence of a previously unknown receptor‐recycling pathway that may underlie mechanisms of G‐protein‐coupled receptor (GPCR) homeostasis. These results also suggest a novel therapeutic target for the treatment of dopaminergic dysfunction.     

AP180 Couples Protein Retrieval to Clathrin‐Mediated Endocytosis of Synaptic Vesicles

How clathrin‐mediated endocytosis (CME) retrieves vesicle proteins into newly formed synaptic vesicles (SVs) remains a major puzzle. Besides its roles in stimulating clathrin‐coated vesicle formation and regulating SV size, the clathrin assembly protein AP180 has been identified as a key player in retrieving SV proteins. The mechanisms by which AP180 recruits SV proteins are not fully understood. Here, we show that following acute inactivation of AP180 in Drosophila, SV recycling is severely impaired at the larval neuromuscular synapse based on analyses of FM 1‐43 uptake and synaptic ultrastructure. More dramatically, AP180 activity is important to maintain the integrity of SV protein complexes at the plasma membrane during endocytosis. These observations suggest that AP180 normally clusters SV proteins together during recycling. Consistent with this notion, SV protein composition and distribution are altered in AP180 mutant flies. Finally, AP180 co‐immunoprecipitates with SV proteins, including the vesicular glutamate transporter and neuronal synaptobrevin. These results reveal a new mode by which AP180 couples protein retrieval to CME of SVs. AP180 is also genetically linked to Alzheimer's disease. Hence, the findings of this study may provide new mechanistic insight into the role of AP180 dysfunction in Alzheimer's disease.      

Clathrin and AP1 are required for apical sorting of glycosyl phosphatidyl inositol‐anchored proteins in biosynthetic and recycling routes in Madin‐Darby canine kidney cells

Recently, studies in animal models demonstrate potential roles for clathrin and AP1 in apical protein sorting in epithelial tissue. However, the precise functions of these proteins in apical protein transport remain unclear. Here, we reveal mistargeting of endogenous glycosyl phosphatidyl inositol‐anchored proteins (GPI‐APs) and soluble secretory proteins in Madin‐Darby canine kidney (MDCK) cells upon clathrin heavy chain or AP1 subunit knockdown (KD). Using a novel directional endocytosis and recycling assay, we found that these KD cells are not only affected for apical sorting of GPI‐APs in biosynthetic pathway but also for their apical recycling and basal‐to‐apical transcytosis routes. The apical distribution of the t‐SNARE syntaxin 3, which is known to be responsible for selective targeting of various apical‐destined cargo proteins in both biosynthetic and endocytic routes, is compromised suggesting a molecular explanation for the phenotype in KD cells. Our results demonstrate the importance of biosynthetic and endocytic routes for establishment and maintenance of apical localization of GPI‐APs in polarized MDCK cells.      

Characterization of a novel murine preadipocyte line,AP‐18, isolated from subcutaneous tissue: Analysis of adipocyte‐related gene expressions

Adipocyte lines are a useful tool for adipocyte research. Recently, a new preadipocyte line designated AP‐18 was established from subcutaneous tissue of the C3H/He mouse. In this study, we further characterized AP‐18 cells. Adipocyte differentiation was assessed by accumulation of fat droplets stained by Oil Red O. The expression of the preadipocyte‐ or adipocyte‐specific genes and adipocytokine genes was analysed qualitatively by RT‐PCR and quantitatively by real‐time PCR in comparison with the LM cell, a murine fibroblast line, and the 3T3‐L1 cell, respectively. AP‐18 cells were fibroblastoid in maintenance culture. After the confluence, fat droplets were accumulated in 50–60% of the cells cultured in the medium alone and in 70–90% of the cells cultured with insulin within 2 to 3 weeks. The fat accumulation was not promoted by the addition of dexamethazone, IBMX (3‐isobutyl‐1‐methylxanthine) or troglitazone in combination with insulin, which were obligatory for differentiation of the 3T3‐L1 cell, a murine preadipocyte line. Throughout the differentiation, AP‐18 cells expressed Pref‐1, LPL, C/EBPβ, C/EBPδ, RXRα, C/EBPα, PPARγ, RXRγ, aP2, GLUT4, SCD1, UCP2, UCP3, TNFα, resistin, leptin, adiponectin and PAI‐1 genes, but not the UCP1 gene, indicating that the cell is derived from WAT (white adipose tissue). The time course of these gene expressions was similar to that of 3T3‐L1 cells, although the expressions were slower and lower in AP‐18 cells. These data indicate that AP‐18 cells are preadipocytes originated from WAT and differentiate into adipocytes under more physiological conditions than 3T3‐L1 cells. AP‐18 may be useful in adipocyte research.