Phosphorylation of ADAPTOR PROTEIN-2 µ-adaptin by ADAPTOR-ASSOCIATED KINASE1 regulates the tropic growth of Arabidopsis roots.

ADAPTOR-ASSOCIATED PROTEIN KINASE1 (AAK1) is a known regulator of clathrin-mediated endocytosis in mammals. Human AAK1 phosphorylates the µ2 subunit of the ADAPTOR PROTEIN-2 (AP-2) complex (AP2M) and plays important roles in cell differentiation and development. Previous interactome studies discovered the association of AAK1 with AP-2 in Arabidopsis (Arabidopsis thaliana), but its function was unclear. Here, genetic analysis revealed that the Arabidopsis aak1 and ap2m mutants both displayed altered root tropic growth, including impaired touch- and gravity-sensing responses. In Arabidopsis, AAK1-phosphorylated AP2M on Thr-163, and expression of the phospho-null version of AP2M in the ap2m mutant led to an aak1-like phenotype, whereas the phospho-mimic forms of AP2M rescued the aak1 mutant. In addition, we found that the AAK1-dependent phosphorylation state of AP2M modulates the frequency distribution of endocytosis. Our data indicate that the phosphorylation of AP2M on Thr-163 by AAK1 fine-tunes endocytosis in the Arabidopsis root to control its tropic growth.

Cellular uptake of chitosan and its role in antifungal action against Penicillium expansum.

Chitosan has wide-spectrum antimicrobial activity but knowledge of its antifungal mechanism is still incomplete. In this study, transcriptome of Penicillium expansum upon chitosan treatment was analyzed by RNA-Seq. KEGG enrichment analysis revealed that endocytosis as well as other physiological pathways was regulated by chitosan treatment. Clathrin adaptor protein mu-subunit (PeCAM) gene, which encodes a protein associated with clathrin-dependent endocytosis, was up-regulated after chitosan treatment. Deletion of PeCAM resulted in changes of conidial, hyphal and colonial morphology. Confocal microscopy images of the distribution of fluorescein isothiocyanate-labeled chitosan confirmed cellular internalization of chitosan. However, deletion of PeCAM almost completely blocked uptake of chitosan into fungal cells and ΔPeCAM mutant exhibited less sensitivity to chitosan compared with wild type, suggesting that chitosan uptake is mediated by clathrin-dependent endocytosis and internalized chitosan also plays an important role in its antifungal activity. Collectively, our results provide a new insight into the antifungal mechanism of chitosan.

Unconventional endocytosis and trafficking of transferrin receptor induced by iron.

The posttranslational regulation of transferrin receptor (TfR1) is largely unknown. We investigated whether iron availability affects TfR1 endocytic cycle and protein stability in HepG2 hepatoma cells exposed to ferric ammonium citrate (FAC). NH4Cl and bafilomycin A1, but not the proteasomal inhibitor MG132, prevented the FAC-mediated decrease in TfR1 protein levels, thus indicating lysosomal involvement. Knockdown experiments showed that TfR1 lysosomal degradation is independent of 1) endocytosis mediated by the clathrin adaptor AP2; 2) Tf, which was suggested to facilitate TfR1 internalization; 3) H-ferritin; and 4) MARCH8, previously implicated in TfR1 degradation. Notably, FAC decreased the number of TfR1 molecules at the cell surface and increased the Tf endocytic rate. Colocalization experiments confirmed that, upon FAC treatment, TfR1 was endocytosed in an AP2- and Tf-independent pathway and trafficked to the lysosome for degradation. This unconventional endocytic regulatory mechanism aimed at reducing surface TfR1 may represent an additional posttranslational control to prevent iron overload. Our results show that iron is a key regulator of the trafficking of TfR1, which has been widely used to study endocytosis, often not considering its function in iron homeostasis.

The MUDENG Augmentation: A Genesis in Anti-Cancer Therapy?

Despite multitudes of reports on cancer remedies available, we are far from being able to declare that we have arrived at that defining anti-cancer therapy. In recent decades, researchers have been looking into the possibility of enhancing cell death-related signaling pathways in cancer cells using pro-apoptotic proteins. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and Mu-2/AP1M2 domain containing, death-inducing (MUDENG, MuD) have been established for their ability to bring about cell death specifically in cancer cells. Targeted cell death is a very attractive term when it comes to cancer, since most therapies also affect normal cells. In this direction TRAIL has made noteworthy progress. This review briefly sums up what has been done using TRAIL in cancer therapeutics. The importance of MuD and what has been achieved thus far through MuD and the need to widen and concentrate on applicational aspects of MuD has been highlighted. This has been suggested as the future perspective of MuD towards prospective progress in cancer research.

CDK5 inhibits the clathrin-dependent internalization of TRPV1 by phosphorylating the clathrin adaptor protein AP2µ2.

Transient receptor potential vanilloid 1 (TRPV1), a nonselective, ligand-gated cation channel, responds to multiple noxious stimuli and is targeted by many kinases that influence its trafficking and activity. Studies on the internalization of TRPV1 have mainly focused on that induced by capsaicin or other agonists. Here, we report that constitutive internalization of TRPV1 occurred in a manner dependent on clathrin, dynamin, and adaptor protein complex 2 (AP2). The µ2 subunit of AP2 (AP2µ2) interacted directly with TRPV1 and was required for its constitutive internalization. Cyclin-dependent kinase 5 (CDK5) phosphorylated AP2µ2 at Ser45, which reduced the interaction between TRPV1 and AP2µ2, leading to decreased TRPV1 internalization. Intrathecal delivery of a cell-penetrating fusion peptide corresponding to the Cdk5 phosphorylation site in AP2µ2, which competed with AP2µ2 for phosphorylation by Cdk5, increased the abundance of TRPV1 on the surface of dorsal root ganglion neurons and reduced complete Freund's adjuvant (CFA)-induced inflammatory thermal hyperalgesia in rats. In addition to describing a mechanism of TRPV1 constitutive internalization and its inhibition by CDK5, these findings demonstrate that CDK5 promotes inflammatory thermal hyperalgesia by reducing TRPV1 internalization, providing previously unidentified insights into the search for drug targets to treat pain.

A Recurrent Missense Variant in AP2M1 Impairs Clathrin-Mediated Endocytosis and Causes Developmental and Epileptic Encephalopathy.

The developmental and epileptic encephalopathies (DEEs) are heterogeneous disorders with a strong genetic contribution, but the underlying genetic etiology remains unknown in a significant proportion of individuals. To explore whether statistical support for genetic etiologies can be generated on the basis of phenotypic features, we analyzed whole-exome sequencing data and phenotypic similarities by using Human Phenotype Ontology (HPO) in 314 individuals with DEEs. We identified a de novo c.508C>T (p.Arg170Trp) variant in AP2M1 in two individuals with a phenotypic similarity that was higher than expected by chance (p = 0.003) and a phenotype related to epilepsy with myoclonic-atonic seizures. We subsequently found the same de novo variant in two individuals with neurodevelopmental disorders and generalized epilepsy in a cohort of 2,310 individuals who underwent diagnostic whole-exome sequencing. AP2M1 encodes the µ-subunit of the adaptor protein complex 2 (AP-2), which is involved in clathrin-mediated endocytosis (CME) and synaptic vesicle recycling. Modeling of protein dynamics indicated that the p.Arg170Trp variant impairs the conformational activation and thermodynamic entropy of the AP-2 complex. Functional complementation of both the µ-subunit carrying the p.Arg170Trp variant in human cells and astrocytes derived from AP-2µ conditional knockout mice revealed a significant impairment of CME of transferrin. In contrast, stability, expression levels, membrane recruitment, and localization were not impaired, suggesting a functional alteration of the AP-2 complex as the underlying disease mechanism. We establish a recurrent pathogenic variant in AP2M1 as a cause of DEEs with distinct phenotypic features, and we implicate dysfunction of the early steps of endocytosis as a disease mechanism in epilepsy.

SGIP1α functions as a selective endocytic adaptor for the internalization of synaptotagmin 1 at synapses.

Proper sorting of exocytosed synaptic vesicle (SV) proteins into individual SVs during endocytosis is of the utmost importance for the fidelity of subsequent neurotransmission. Recent studies suggest that each SV protein is sorted into individual SVs by its own dedicated adaptors as well as by association between SV proteins. The SH3-containing GRB2-like protein 3-interacting protein 1 (SGIP1), an ortholog of Fer/Cip4 homology domain-only (FCHo) proteins, contains a µ-homology domain (µHD) and binds AP-2 and Eps15, thus functioning as an endocytic regulator of clathrin-mediated endocytosis (CME). Its longest isoform SGIP1α is predominantly expressed in the brain but the functional significance of SGIP1 in SV recycling remains unknown. Here, we found that SGIP1α, a brain-specific long isoform of SGIP1 binds synaptotagmin1 (Syt1) via its µHD and promotes the internalization of Syt1 on the neuronal surface. The small hairpin RNA (shRNA)-mediated knockdown (KD) of SGIP1α caused selective impairment of Syt1 internalization at hippocampal synapses and it was fully rescued by coexpression of the shRNA-resistant form of SGIP1α in KD neurons. We further found that the µHD of SGIP1α is structurally similar to those of AP-2 and stonin2, and mutations at Trp771 and Lys781, which correspond to Syt1-recognition motifs of AP-2 and stonin2, to Ala bound less efficiently to Syt1 and failed to rescue the endocytic defect of Syt1 caused by KD. Our results indicate that SGIP1α is an endocytic adaptor dedicated to the retrieval of surface-stranded Syt1. Since endocytic sorting of Syt1 is also mediated by the overlapping activities of synaptic vesicle glycoprotein 2A/B (SV2A/B) and stonin2, our results suggest that complementary fail-safe mechanism by these proteins ensures high fidelity of Syt1 retrieval.

The AP2M1 gene expression is a promising biomarker for predicting survival of patients with hepatocellular carcinoma.

There is a growing need for the discovery of new prognostic factors for cases where the scoring and staging system of hepatocellular carcinoma (HCC) does not result in a clear definition. We analyzed whether AP-2 complex subunit mu (AP2M1) expression could be a new prognostic marker for HCC based on the roles of AP2M1 in influencing hepatocyte growth factor (HGF) promoter regulation and hepatitis C virus (HCV) assembly. Patient data were extracted from cohorts of the Gene Expression Omnibus (GSE10186), International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). Differential expression value between matched cancer and normal liver was identified using ICGC cohort. Subsequently, we compared AP2M1 expression as a prognostic gene with other well-known prognostic genes for HCC, using the time-dependent area under the curve (AUC) of the Uno's C-index, the AUC value of the receiver operating characteristics at 5 years, Kaplan-Meier survival curve, and multivariate analysis. Particularly, TCGA and GSE10186 patients were divided into subgroups based on alcohol intake, hepatitis B, and C viral infections, and analyzed in the same methods. The AP2M1 expression values in patients with cancer were much higher than matched normal liver. The AP2M1 level showed excellent prognosis predictions in comparison with existing markers in the three independent cohorts (n = 647). In particular, it was more predictive of prognosis than other markers in alcohol intake and HCV infections. In conclusion, we were confident that AP2M1 provides sufficient value as a new prognostic marker for HCC especially patients with HCV infection and/or alcohol intake.

Varicella-Zoster Virus ORF9p Binding to Cellular Adaptor Protein Complex 1 Is Important for Viral Infectivity.

ORF9p (homologous to herpes simplex virus 1 [HSV-1] VP22) is a varicella-zoster virus (VZV) tegument protein essential for viral replication. Even though its precise functions are far from being fully described, a role in the secondary envelopment of the virus has long been suggested. We performed a yeast two-hybrid screen to identify cellular proteins interacting with ORF9p that might be important for this function. We found 31 ORF9p interaction partners, among which was AP1M1, the µ subunit of the adaptor protein complex 1 (AP-1). AP-1 is a heterotetramer involved in intracellular vesicle-mediated transport and regulates the shuttling of cargo proteins between endosomes and the trans-Golgi network via clathrin-coated vesicles. We confirmed that AP-1 interacts with ORF9p in infected cells and mapped potential interaction motifs within ORF9p. We generated VZV mutants in which each of these motifs was individually impaired and identified leucine 231 in ORF9p to be critical for the interaction with AP-1. Disrupting ORF9p binding to AP-1 by mutating leucine 231 to alanine in ORF9p strongly impaired viral growth, most likely by preventing efficient secondary envelopment of the virus. Leucine 231 is part of a dileucine motif conserved among alphaherpesviruses, and we showed that VP22 of Marek's disease virus and HSV-2 also interacts with AP-1. This indicates that the function of this interaction in secondary envelopment might be conserved as well.IMPORTANCE Herpesviruses are responsible for infections that, especially in immunocompromised patients, can lead to severe complications, including neurological symptoms and strokes. The constant emergence of viral strains resistant to classical antivirals (mainly acyclovir and its derivatives) pleads for the identification of new targets for future antiviral treatments. Cellular adaptor protein (AP) complexes have been implicated in the correct addressing of herpesvirus glycoproteins in infected cells, and the discovery that a major constituent of the varicella-zoster virus tegument interacts with AP-1 reveals a previously unsuspected role of this tegument protein. Unraveling the complex mechanisms leading to virion production will certainly be an important step in the discovery of future therapeutic targets.

Apical secretion of Wnt1 in polarized epithelial cells is regulated by exocyst-mediated trafficking.

Wnts are glycosylated proteins secreted from various cell types including mesenchymal, hematopoietic and epithelial cells. Directional secretion of Wnts in polarized epithelial cells is unique; Wnt11 is secreted apically, whereas Wnt5a and Wnt3a are secreted basolaterally. Here, we found that Wnt1 is equivalently secreted both apically and basolaterally in MDCK cells. Wnt1 was modified with a complex- or hybrid-type glycan at Asn29 and Asn359 and the high-mannose- or hybrid-type glycan at Asn316. Although glycosylation of Wnt11 at the N-terminal site was shown to be essential for its apical secretion, glycosylation of Asn29 of Wnt1 was not required. Instead, the apical secretion of Wnt1 was inhibited by knockdown of Sec6 and Sec8, suggesting that Wnt1 is secreted apically via exocyst-mediated transport. Basolateral secretion of Wnt1 was mediated by clathrin and AP-1, in mechanism similar to that used by Wnt5a and Wnt3a. Although Wingless was reported to be transcytosed to the basolateral region in the Drosophila wing disc, transcytosis was not involved in the basolateral secretion of Wnt1. Thus, the polarized secretion of Wnt1 is regulated by different mechanisms than other Wnts.

Contribution of the clathrin adaptor AP-1 subunit µ1 to acidic cluster protein sorting.

Acidic clusters act as sorting signals for packaging cargo into clathrin-coated vesicles (CCVs), and also facilitate down-regulation of MHC-I by HIV-1 Nef. To find acidic cluster sorting machinery, we performed a gene-trap screen and identified the medium subunit (µ1) of the clathrin adaptor AP-1 as a top hit. In µ1 knockout cells, intracellular CCVs still form, but acidic cluster proteins are depleted, although several other CCV components were either unaffected or increased, indicating that cells can compensate for long-term loss of AP-1. In vitro experiments showed that the basic patch on µ1 that interacts with the Nef acidic cluster also contributes to the binding of endogenous acidic cluster proteins. Surprisingly, µ1 mutant proteins lacking the basic patch and/or the tyrosine-based motif binding pocket could rescue the µ1 knockout phenotype completely. In contrast, these mutants failed to rescue Nef-induced down-regulation of MHC class I, suggesting a possible mechanism for attacking the virus while sparing the host cell.

µ2-Dependent endocytosis of N-cadherin is regulated by ß-catenin to facilitate neurite outgrowth.

Circuit formation in the brain requires neurite outgrowth throughout development to establish synaptic contacts with target cells. Active endocytosis of several adhesion molecules facilitates the dynamic exchange of these molecules at the surface and promotes neurite outgrowth in developing neurons. The endocytosis of N-cadherin, a calcium-dependent adhesion molecule, has been implicated in the regulation of neurite outgrowth, but the mechanism remains unclear. Here, we identified that a fraction of N-cadherin internalizes through clathrin-mediated endocytosis (CME). Two tyrosine-based motifs in the cytoplasmic domain of N-cadherin recognized by the µ2 subunit of the AP-2 adaptor complex are responsible for CME of N-cadherin. Moreover, ß-catenin, a core component of the N-cadherin adhesion complex, inhibits N-cadherin endocytosis by masking the 2 tyrosine-based motifs. Removal of ß-catenin facilitates µ2 binding to N-cadherin, thereby increasing clathrin-mediated N-cadherin endocytosis and neurite outgrowth without affecting the steady-state level of surface N-cadherin. These results identify and characterize the mechanism controlling N-cadherin endocytosis through ß-catenin-regulated µ2 binding to modulate neurite outgrowth.

The cytoplasmic tail of L-selectin interacts with the adaptor-protein complex AP-1 subunit µ1A via a novel basic binding motif.

L-selectin regulates leukocyte adhesion and rolling along the endothelium. Proteins binding to the cytoplasmic tail of L-selectin regulate L-selectin functions. We used L-selectin cytoplasmic tail peptide pulldown assays combined with high sensitivity liquid chromatography/mass spectrometry to identify novel L-selectin tail-binding proteins. Incubation of the L-selectin tail with cell extracts from phorbol 12-myristate 13-acetate-stimulated Raw 264.7 macrophages resulted in the binding of µ1A of the clathrin-coated vesicle AP-1 complex. Furthermore, full-length GST-µ1A and the GST-µ1A C-terminal domain, but not the GST-µ1A N-terminal domain, bind to L-selectin tail peptide, and the intracellular pool of L-selectin colocalizes with AP-1 at the trans-Golgi network. We identified a novel basic protein motif consisting of a cluster of three dibasic residues (356RR357, 359KK360, and 362KK363) in the membrane-proximal domain of the L-selectin tail as well as a doublet of aspartic acid residues (369DD370) in the membrane-distal end of the L-selectin tail involved in µ1A binding. Stimulation of Raw 264.7 macrophages with PMA augmented the amount of µ1A associated with anti-L-selectin immunoprecipitates. However, full-length GST-µ1A did not bind to the phospho-L-selectin tail or phospho-mimetic S364D L-selectin tail. Accordingly, we propose that phosphorylation of µ1A is required for interaction with the L-selectin tail and that L-selectin tail phosphorylation may regulate this interaction in vivo Molecular docking of the L-selectin tail to µ1A was used to identify the µ1A surface domain binding the L-selectin tail and to explain how phosphorylation of the L-selectin tail abrogates µ1A interaction. Our findings indicate that L-selectin is transported constitutively by the AP-1 complex, leading to the formation of a trans-Golgi network reserve pool and that phosphorylation of the L-selectin tail blocks AP-1-dependent retrograde transport of L-selectin.

Regulation of clathrin-mediated endocytosis by hierarchical allosteric activation of AP2.

The critical initiation phase of clathrin-mediated endocytosis (CME) determines where and when endocytosis occurs. Heterotetrameric adaptor protein 2 (AP2) complexes, which initiate clathrin-coated pit (CCP) assembly, are activated by conformational changes in response to phosphatidylinositol-4,5-bisphosphate (PIP2) and cargo binding at multiple sites. However, the functional hierarchy of interactions and how these conformational changes relate to distinct steps in CCP formation in living cells remains unknown. We used quantitative live-cell analyses to measure discrete early stages of CME and show how sequential, allosterically regulated conformational changes activate AP2 to drive both nucleation and subsequent stabilization of nascent CCPs. Our data establish that cargoes containing Yxxφ motif, but not dileucine motif, play a critical role in the earliest stages of AP2 activation and CCP nucleation. Interestingly, these cargo and PIP2 interactions are not conserved in yeast. Thus, we speculate that AP2 has evolved as a key regulatory node to coordinate CCP formation and cargo sorting and ensure high spatial and temporal regulation of CME.

Sorting signals for PIN1 trafficking and localization.

PIN-FORMED (PIN) family proteins direct polar auxin transport based on their asymmetric (polar) localization at the plasma membrane. In the case of PIN1, it mainly localizes to the basal (rootward) plasma membrane domain of stele cells in root meristems. Vesicular trafficking events, such as clathrin-dependent PIN1 endocytosis and polar recycling, are probably the main determinants for PIN1 polar localization. However, very little is known about the signals which may be involved in binding the µ-adaptin subunit of clathrin adaptor complexes (APs) for sorting of PIN1 within clathrin-coated vesicles, which can determine its trafficking and localization. We have performed a systematic mutagenesis analysis to investigate putative sorting motifs in the hydrophilic loop of PIN1. We have found that a non-canonical motif, based in a phenylalanine residue, through the binding of µA(µ2)- and µD(µ3)-adaptin, is important for PIN1 endocytosis and for PIN1 traffcking along the secretory pathway, respectively. In addition, tyrosine-based motifs, which also bind different µ-adaptins, could also contribute to PIN1 trafficking and localization.

Upregulation of µ3A Drives Homeostatic Plasticity by Rerouting AMPAR into the Recycling Endosomal Pathway.

Synaptic scaling is a form of homeostatic plasticity driven by transcription-dependent changes in AMPA-type glutamate receptor (AMPAR) trafficking. To uncover the pathways involved, we performed a cell-type-specific screen for transcripts persistently altered during scaling, which identified the µ subunit (µ3A) of the adaptor protein complex AP-3A. Synaptic scaling increased µ3A (but not other AP-3 subunits) in pyramidal neurons and redistributed dendritic µ3A and AMPAR to recycling endosomes (REs). Knockdown of µ3A prevented synaptic scaling and this redistribution, while overexpression (OE) of full-length µ3A or a truncated µ3A that cannot interact with the AP-3A complex was sufficient to drive AMPAR to REs. Finally, OE of µ3A acted synergistically with GRIP1 to recruit AMPAR to the dendritic membrane. These data suggest that excess µ3A acts independently of the AP-3A complex to reroute AMPAR to RE, generating a reservoir of receptors essential for the regulated recruitment to the synaptic membrane during scaling up.

Sorting Motifs Involved in the Trafficking and Localization of the PIN1 Auxin Efflux Carrier.

In contrast with the wealth of recent reports about the function of µ-adaptins and clathrin adaptor protein (AP) complexes, there is very little information about the motifs that determine the sorting of membrane proteins within clathrin-coated vesicles in plants. Here, we investigated putative sorting signals in the large cytosolic loop of the Arabidopsis (Arabidopsis thaliana) PIN-FORMED1 (PIN1) auxin transporter, which are involved in binding µ-adaptins and thus in PIN1 trafficking and localization. We found that Phe-165 and Tyr-280, Tyr-328, and Tyr-394 are involved in the binding of different µ-adaptins in vitro. However, only Phe-165, which binds µA(µ2)- and µD(µ3)-adaptin, was found to be essential for PIN1 trafficking and localization in vivo. The PIN1:GFP-F165A mutant showed reduced endocytosis but also localized to intracellular structures containing several layers of membranes and endoplasmic reticulum (ER) markers, suggesting that they correspond to ER or ER-derived membranes. While PIN1:GFP localized normally in a µA (µ2)-adaptin mutant, it accumulated in big intracellular structures containing LysoTracker in a µD (µ3)-adaptin mutant, consistent with previous results obtained with mutants of other subunits of the AP-3 complex. Our data suggest that Phe-165, through the binding of µA (µ2)- and µD (µ3)-adaptin, is important for PIN1 endocytosis and for PIN1 trafficking along the secretory pathway, respectively.

Genome-wide association analysis of self-reported events in 6135 individuals and 252 827 controls identifies 8 loci associated with thrombosis.

Thrombotic diseases are among the leading causes of morbidity and mortality in the world. To add insights into the genetic regulation of thrombotic disease, we conducted a genome-wide association study (GWAS) of 6135 self-reported blood clots events and 252 827 controls of European ancestry belonging to the 23andMe cohort of research participants. Eight loci exceeded genome-wide significance. Among the genome-wide significant results, our study replicated previously known venous thromboembolism (VTE) loci near the F5, FGA-FGG, F11, F2, PROCR and ABO genes, and the more recently discovered locus near SLC44A2 In addition, our study reports for the first time a genome-wide significant association between rs114209171, located upstream of the F8 structural gene, and thrombosis risk. Analyses of expression profiles and expression quantitative trait loci across different tissues suggested SLC44A2, ILF3 and AP1M2 as the three most plausible candidate genes for the chromosome 19 locus, our only genome-wide significant thrombosis-related locus that does not harbor likely coagulation-related genes. In addition, we present data showing that this locus also acts as a novel risk factor for stroke and coronary artery disease (CAD). In conclusion, our study reveals novel common genetic risk factors for VTE, stroke and CAD and provides evidence that self-reported data on blood clots used in a GWAS yield results that are comparable with those obtained using clinically diagnosed VTE. This observation opens up the potential for larger meta-analyses, which will enable elucidation of the genetics of thrombotic diseases, and serves as an example for the genetic study of other diseases.

The alternate AP-1 adaptor subunit Apm2 interacts with the Mil1 regulatory protein and confers differential cargo sorting.

Heterotetrameric adaptor protein complexes are important mediators of cargo protein sorting in clathrin-coated vesicles. The cell type-specific expression of alternate µ 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 µ 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 µ chain. Here we show that the variant µ 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 γ/σ 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.

Basolateral sorting of the Mg²âº transporter CNNM4 requires interaction with AP-1A and AP-1B.

Ancient conserved domain protein/cyclin M (CNNM) 4 is an evolutionarily conserved Mg(2+) transporter that localizes at the basolateral membrane of the intestinal epithelia. Here, we show the complementary importance of clathrin adaptor protein (AP) complexes AP-1A and AP-1B in basolateral sorting of CNNM4. We first confirmed the basolateral localization of both endogenous and ectopically expressed CNNM4 in Madin-Darby Canine Kidney cells, which form highly polarized epithelia in culture. Single knockdown of µ1B, a cargo-recognition subunit of AP-1B, did not affect basolateral localization, but simultaneous knockdown of the µ1A subunit of AP-1A abrogated localization. Mutational analyses showed the importance of three conserved dileucine motifs in CNNM4 for both basolateral sorting and interaction with µ1A and µ1B. These results imply that CNNM4 is sorted to the basolateral membrane by the complementary function of AP-1A and AP-1B.