Temporal Ordering in Endocytic Clathrin-Coated Vesicle Formation via AP2 Phosphorylation.

Clathrin-mediated endocytosis (CME) is key to maintaining the transmembrane protein composition of cells' limiting membranes. During mammalian CME, a reversible phosphorylation event occurs on Thr156 of the µ2 subunit of the main endocytic clathrin adaptor, AP2. We show that this phosphorylation event starts during clathrin-coated pit (CCP) initiation and increases throughout CCP lifetime. µ2Thr156 phosphorylation favors a new, cargo-bound conformation of AP2 and simultaneously creates a binding platform for the endocytic NECAP proteins but without significantly altering AP2's cargo affinity in vitro. We describe the structural bases of both. NECAP arrival at CCPs parallels that of clathrin and increases with µ2Thr156 phosphorylation. In turn, NECAP recruits drivers of late stages of CCP formation, including SNX9, via a site distinct from where NECAP binds AP2. Disruption of the different modules of this phosphorylation-based temporal regulatory system results in CCP maturation being delayed and/or stalled, hence impairing global rates of CME.

Epsin deficiency impairs endocytosis by stalling the actin-dependent invagination of endocytic clathrin-coated pits.

Epsin is an evolutionarily conserved endocytic clathrin adaptor whose most critical function(s) in clathrin coat dynamics remain(s) elusive. To elucidate such function(s), we generated embryonic fibroblasts from conditional epsin triple KO mice. Triple KO cells displayed a dramatic cell division defect. Additionally, a robust impairment in clathrin-mediated endocytosis was observed, with an accumulation of early and U-shaped pits. This defect correlated with a perturbation of the coupling between the clathrin coat and the actin cytoskeleton, which we confirmed in a cell-free assay of endocytosis. Our results indicate that a key evolutionary conserved function of epsin, in addition to other roles that include, as we show here, a low affinity interaction with SNAREs, is to help generate the force that leads to invagination and then fission of clathrin-coated pits.

Clathrin pit-mediated endocytosis of neutrophil elastase and cathepsin G by cancer cells.

Neutrophil elastase (NE) is a neutrophil-derived serine proteinase with broad substrate specificity. We have recently demonstrated that NE is capable of entering tumor cell endosomes and processing novel intracellular substrates. In the current study, we sought to determine the mechanism by which NE enters tumor cells. Our results show that NE enters into early endosomal antigen-1(+) endosomes in a dynamin- and clathrin-dependent but flotillin-1- and caveolin-1-independent fashion. Cathepsin G (but not proteinase-3) also enters tumor endosomes via the same mechanism. We utilized (125)I-labeled NE to demonstrate that NE binds to the surface of cancer cells. Incubation of radiolabeled NE with lung cancer cells displays a dissociation constant (K(d)) of 284 nm. Because NE is known to bind to heparan sulfate- and chondroitin sulfate-containing proteoglycans, we treated cells with glycanases to remove these confounding factors, which did not significantly diminish cell surface binding or endosomal entry. Thus, NE and CG bind to the surface of cancer cells, presumably to a cell surface receptor, and subsequently undergo clathrin pit-mediated endocytosis.

Sphingomyelin synthase 1-generated sphingomyelin plays an important role in transferrin trafficking and cell proliferation.

Transferrin (Tf) endocytosis and recycling are essential for iron uptake and the regulation of cell proliferation. Tf and Tf receptor (TfR) complexes are internalized via clathrin-coated pits composed of a variety of proteins and lipids and pass through early endosomes to recycling endosomes. We investigated the role of sphingomyelin (SM) synthases (SMS1 and SMS2) in clathrin-dependent trafficking of Tf and cell proliferation. We employed SM-deficient lymphoma cells that lacked SMSs and that failed to proliferate in response to Tf. Transfection of SMS1, but not SMS2, enabled these cells to incorporate SM into the plasma membrane, restoring Tf-mediated proliferation. SM-deficient cells showed a significant reduction in clathrin-dependent Tf uptake compared with the parental SM-producing cells. Both SMS1 gene transfection and exogenous short-chain SM treatment increased clathrin-dependent Tf uptake in SM-deficient cells, with the Tf being subsequently sorted to Rab11-positive recycling endosomes. We observed trafficking of the internalized Tf to late/endolysosomal compartments, and this was not dependent on the clathrin pathway in SM-deficient cells. Thus, SMS1-mediated SM synthesis directs Tf-TfR to undergo clathrin-dependent endocytosis and recycling, promoting the proliferation of lymphoma cells.

Solitary and repetitive binding motifs for the AP2 complex alpha-appendage in amphiphysin and other accessory proteins.

Adaptor protein (AP) complexes bind to transmembrane proteins destined for internalization and to membrane lipids, so linking cargo to the accessory internalization machinery. This machinery interacts with the appendage domains of APs, which have platform and beta-sandwich subdomains, forming the binding surfaces for interacting proteins. Proteins that interact with the subdomains do so via short motifs, usually found in regions of low structural complexity of the interacting proteins. So far, up to four motifs have been identified that bind to and partially compete for at least two sites on each of the appendage domains of the AP2 complex. Motifs in individual accessory proteins, their sequential arrangement into motif domains, and partial competition for binding sites on the appendage domains coordinate the formation of endocytic complexes in a temporal and spatial manner. In this work, we examine the dominant interaction sequence in amphiphysin, a synapse-enriched accessory protein, which generates membrane curvature and recruits the scission protein dynamin to the necks of coated pits, for the platform subdomain of the alpha-appendage. The motif domain of amphiphysin1 contains one copy of each of a DX(F/W) and FXDXF motif. We find that the FXDXF motif is the main determinant for the high affinity interaction with the alpha-adaptin appendage. We describe the optimal sequence of the FXDXF motif using thermodynamic and structural data and show how sequence variation controls the affinities of these motifs for the alpha-appendage.

Loss of the Atp2c1 secretory pathway Ca(2+)-ATPase (SPCA1) in mice causes Golgi stress, apoptosis, and midgestational death in homozygous embryos and squamous cell tumors in adult heterozygotes.

Loss of one copy of the human ATP2C1 gene, encoding SPCA1 (secretory pathway Ca(2+)-ATPase isoform 1), causes Hailey-Hailey disease, a skin disorder. We performed targeted mutagenesis of the Atp2c1 gene in mice to analyze the functions of this Golgi membrane Ca(2+) pump. Breeding of heterozygous mutants yielded a normal Mendelian ratio among embryos on gestation day 9.5; however, null mutant (Spca1(-/-)) embryos exhibited growth retardation and did not survive beyond gestation day 10.5. Spca1(-/-) embryos had an open rostral neural tube, but hematopoiesis and cardiovascular development were ostensibly normal. Golgi membranes of Spca1(-/-) embryos were dilated, had fewer stacked leaflets, and were expanded in amount, consistent with increased Golgi biogenesis. The number of Golgi-associated vesicles was also increased, and rough endoplasmic reticulum had fewer ribosomes. Coated pits, junctional complexes, desmosomes, and basement membranes appeared normal in mutant embryos, indicating that processing and trafficking of proteins in the secretory pathway was not massively impaired. However, apoptosis was increased, possibly the result of secretory pathway stress, and a large increase in cytoplasmic lipid was observed in mutant embryos, consistent with impaired handling of lipid by the Golgi. Adult heterozygous mice appeared normal and exhibited no evidence of Hailey-Hailey disease; however, aged heterozygotes had an increased incidence of squamous cell tumors of keratinized epithelial cells of the skin and esophagus. These data show that loss of the Golgi Ca(2+) pump causes Golgi stress, expansion of the Golgi, increased apoptosis, and embryonic lethality and demonstrates that SPCA1 haploinsufficiency causes a genetic predisposition to cancer.

In vivo, dendritic cells can cross-present virus-like particles using an endosome-to-cytosol pathway.

Recombinant parvovirus-like particles (PPV-VLPs) are particulate exogenous Ags that induce strong CTL response in the absence of adjuvant. In the present report to decipher the mechanisms responsible for CTL activation by such exogenous Ag, we analyzed ex vivo and in vitro the mechanisms of capture and processing of PPV-VLPs by dendritic cells (DCs). In vivo, PPV-VLPs are very efficiently captured by CD8alpha- and CD8alpha+ DCs and then localize in late endosomes of DCs. Macropinocytosis and lipid rafts participate in PPV-VLPs capture. Processing of PPV-VLPs does not depend upon recycling of MHC class I molecules, but requires vacuolar acidification as well as proteasome activity, TAP translocation, and neosynthesis of MHC class I molecules. This study therefore shows that in vivo DCs can cross-present PPV-VLPs using an endosome-to-cytosol processing pathway.

A rugged energy landscape mechanism for trapping of transmembrane receptors during endocytosis.

Efficient clathrin-mediated endocytosis of transmembrane receptors requires that clathrin-coated pits retain the receptors long enough to allow vesicle formation and internalization. In many cases, however, the receptors can exhibit mean lifetimes in coated pits much shorter than the lifetime of the pit at the plasma membrane. A rugged energy landscape for binding, which produces a broad distribution of residence times, ensures a significant probability of times much greater than the mean and would allow efficient trapping of proteins in these cases. We used fluorescence correlation spectroscopy and total internal reflection microscopy to measure the kinetics of movement of a C5a receptor-yellow fluorescent protein fusion in living cells. These experiments demonstrate that clusters of trapped receptors exhibit fluctuations in fluorescence intensity that vary in time scale over 2 orders of magnitude. Most of the variation in intensity is likely due to the motion of the receptors in the plane of the plasma membrane, although it is not possible to rule out a small contribution from motion orthogonal to the plane of the membrane. The broad time scale distribution of the intensity fluctuations is consistent with a rugged energy landscape mechanism for trapping of the receptors. This mechanism, which allows efficient trapping to coexist with rapid exchange, may also be relevant to other biological processes involving binding in heterogeneous chemical environments.

Molecular mechanisms of angiotensin II (AT1A) receptor endocytosis.

1. Angiotensin II (AngII) initiates a variety of cellular responses through activation of type 1 (AT1; with subtypes AT1A and AT1B) and type 2 (AT2) cell surface angiotensin receptors. Both AT1 and AT2 receptors couple to heterotrimeric guanyl nucleotide binding proteins (G-proteins) and generate intracellular signals following recognition of extracellular AngII, but only AT1 is targeted for the rapid ligand-stimulated endocytosis (internalization) typical of many plasma membrane receptors. 2. AT1 endocytosis proceeds through clathrin-coated pits and is independent of G-protein coupling which predicts that the AngII-AT1 receptor complex attains a conformation necessary for interaction with the endocytotic machinery, but separate from receptor signalling activation. 3. The function of AT1 endocytosis and the reason for the disparity between AT1 and AT2 endocytosis is not fully appreciated, but the latter probably reflects differences in the primary amino acid sequence of these two receptor types. 4. For many receptors that undergo internalization, it has been established that internalization motifs (2-6 amino acids, often incorporating crucial tyrosine and hydrophobic amino acids) within the cytoplasmic regions of the receptor mediate the selective recruitment of activated receptors into clathrin-coated pits and vesicles. 5. Mutagenesis studies on the AT1A receptor, aimed at identifying such motifs, reveal that sites within the third cytoplasmic loop and the cytoplasmic carboxyl terminal region are important for AngII-stimulated AT1A receptor endocytosis.