Endocytic Functions Research Articles

Real-Time Sensing of Enteropathogenic E. coli-Induced Effects on Epithelial Host Cell Height, Cell-Substrate Interactions, and Endocytic Processes by Infrared Surface Plasmon Spectroscopy

Enteropathogenic Escherichia coli (EPEC) is an important, generally non-invasive, bacterial pathogen that causes diarrhea in humans. The microbe infects mainly the enterocytes of the small intestine. Here we have applied our newly developed infrared surface plasmon resonance (IR-SPR) spectroscopy approach to study how EPEC infection affects epithelial host cells. The IR-SPR experiments showed that EPEC infection results in a robust reduction in the refractive index of the infected cells. Assisted by confocal and total internal reflection microscopy, we discovered that the microbe dilates the intercellular gaps and induces the appearance of fluid-phase-filled pinocytic vesicles in the lower basolateral regions of the host epithelial cells. Partial cell detachment from the underlying substratum was also observed. Finally, the waveguide mode observed by our IR-SPR analyses showed that EPEC infection decreases the host cell's height to some extent. Together, these observations reveal novel impacts of the pathogen on the host cell architecture and endocytic functions. We suggest that these changes may induce the infiltration of a watery environment into the host cell, and potentially lead to failure of the epithelium barrier functions. Our findings also indicate the great potential of the label-free IR-SPR approach to study the dynamics of host-pathogen interactions with high spatiotemporal sensitivity.

LRP-1: A Checkpoint for the Extracellular Matrix Proteolysis

Low-density lipoprotein receptor-related protein-(LRP-1) is a large endocytic receptor that binds more than 35 ligands and exhibits signaling properties. Proteinases capable of degrading extracellular matrix (ECM), called matrix proteinases in this paper, are mainly serine proteinases: the activators of plasminogen into plasmin, tissue-type (tPA) and urokinase-type (uPA) plasminogen activators, and the members of the matrix metalloproteinase (MMP) family. LRP-1 is responsible for clearing matrix proteinases, complexed or not with inhibitors. This paper attempts to summarize some aspects on the cellular and molecular bases of endocytic and signaling functions of LRP-1 that modulate extra- and pericellular levels of matrix proteinases.

Multifunctional protein APPL2 contributes to survival of human glioma cells

Some endocytic proteins have recently been shown to play a role in tumorigenesis. In this study, we demonstrate that APPL2, an adapter protein with known endocytic functions, is upregulated in 40% cases of glioblastoma multiforme, the most common and aggressive cancer of the central nervous system. The silencing of APPL2 expression by small interfering RNAs (siRNAs) in glioma cells markedly reduces cell survival under conditions of low growth factor availability and enhances apoptosis (measured by executor caspase activity). Long-term depletion of APPL2 by short hairpin RNAs (shRNAs), under regular growth factor availability, suppresses the cell transformation abilities, assessed by inhibited colony formation in soft agar and by reduced xenograft tumor growth in vivo. At the molecular level, the negative effect of APPL2 knockdown on cell survival is not due to the alterations in AKT or GSK3β activities which were reported to be modulated by APPL proteins. Instead, we attribute the reduced cell survival upon APPL2 depletion to the changes in gene expression, in particular to the upregulation of apoptosis-related genes, such as UNC5B (a proapoptotic dependence receptor) and HRK (harakiri, an activator of apoptosis, which antagonizes anti-apoptotic function of Bcl2). In support of this notion, the loss of glioma cell survival upon APPL2 knockdown can be rescued either by an excess of netrin-1, the prosurvival ligand of UNC5B or by simultaneous silencing of HRK. Consistently, APPL2 overexpression reduces expression of HRK and caspase activation in cells treated with apoptosis inducers, resulting in the enhancement of cell viability. This prosurvival activity of APPL2 is independent of its endosomal localization. Cumulatively, our data indicate that a high level of APPL2 protein might enhance glioblastoma growth by maintaining low expression level of genes responsible for cell death induction.

Epigenetic Modulation of the Biophysical Properties of Drug-Resistant Cell Lipids to Restore Drug Transport and Endocytic Functions

In our recent studies exploring the biophysical characteristics of resistant cell lipids, and the role they play in drug transport, we demonstrated the difference of drug-resistant breast cancer cells from drug-sensitive cells in lipid composition and biophysical properties, suggesting that cancer cells acquire a drug-resistant phenotype through the alteration of lipid synthesis to inhibit intracellular drug transport to protect from cytotoxic effect. In cancer cells, epigenetic changes (e.g., DNA hypermethylation) are essential to maintain this drug-resistant phenotype. Thus, altered lipid synthesis may be linked to epigenetic mechanisms of drug resistance. We hypothesize that reversing DNA hypermethylation in resistant cells with an epigenetic drug could alter lipid synthesis, changing the cell membrane's biophysical properties to facilitate drug delivery to overcome drug resistance. Herein we show that treating drug-resistant breast cancer cells (MCF-7/ADR) with the epigenetic drug 5-aza-2'-deoxycytidine (decitabine) significantly alters cell lipid composition and biophysical properties, causing the resistant cells to acquire biophysical characteristics similar to those of sensitive cell (MCF-7) lipids. Following decitabine treatment, resistant cells demonstrated increased sphingomyelinase activity, resulting in a decreased sphingomyelin level that influenced lipid domain structures, increased membrane fluidity, and reduced P-glycoprotein expression. Changes in the biophysical characteristics of resistant cell lipids facilitated doxorubicin transport and restored endocytic function for drug delivery with a lipid-encapsulated form of doxorubicin, enhancing the drug efficacy. In conclusion, we have established a new mechanism for efficacy of an epigenetic drug, mediated through changes in lipid composition and biophysical properties, in reversing cancer drug resistance.

Pancreatic Stellate Cells Do Not Exhibit Features of Antigen-Presenting Cells

Major histocompatibility complex (MHC) class II molecules are expressed on professional antigen-presenting cells (APCs), and pancreatic stellate cells (PSCs) have endocytic and phagocytic functions and play a role in the immune responses of the pancreas. The aim of the present study was to investigate whether PSCs exhibit features of APCs. Rat and human PSCs were cultured with interferon-γ (IFN-γ) or an exogenous antigen, ovalbumin (OVA), and they were analyzed for expression of MHC II molecules by flow cytometry and reverse transcription-polymerase chain reaction. The cells simulated with IFN-γ expressed very little or no MHC class II molecules or human leukocyte antigen (HLA)-DR at the transcriptional level. Stimulation with IFN-γ failed to induce expression of MHC class II molecules and HLA-DR molecules according to the results of flow cytometry. Dual-color flow cytometric analysis showed that approximately 95% of the PSCs took up OVA; however, none of the cells that took up OVA expressed MHC class II molecules or HLA-DR molecules. Pancreatic stellate cells do not seem to be responsible for the MHC class II-dependent pathway of antigen presentation, suggesting that PSCs do not play a role in adaptive immunity as APCs.

Different endocytic functions of AGEF-1 in C. elegans coelomocytes

BackgroundADP-ribosylation factors (ARFs) are a family of small GTP-binding proteins that play roles in membrane dynamics and vesicle trafficking. AGEF-1, which is thought to act as a guanine nucleotide exchange factor of class I ARFs, is required for caveolin-1 body formation and receptor-mediated endocytosis in oocytes of Caenorhabditis elegans. This study explores additional roles of AGEF-1 in endocytic transport. Methodsagef-1 expression was knocked down by using RNAi in C. elegans. Markers that allow analysis of endocytic transport in scavenger cells were investigated for studying the effect of AGEF-1 on different steps of membrane transport. ResultsKnockdown of AGEF-1 levels results in two apparent trafficking defects in coelomocytes of C. elegans. First, there is a delay in the uptake of solutes from the extracellular medium. Second, there is a dramatic enlargement of the sizes of lysosomes, even though lysosomal acidification is normal and degradation still occurs. ConclusionOur results suggest that AGEF-1 regulates endosome/lysosome fusion or fission events, in addition to earlier steps in endocytic transport. General significanceAGEF-1 is the first identified GTPase regulator that functions at the lysosome fusion or fission stage of the endocytic pathway. Our study provides insight into lysosome dynamics in C. elegans.

Asrij Maintains the Stem Cell Niche and Controls Differentiation during Drosophila Lymph Gland Hematopoiesis

Several signaling pathways control blood cell (hemocyte) development in the Drosophila lymph gland. Mechanisms that modulate and integrate these signals are poorly understood. Here we report that mutation in a conserved endocytic protein Asrij affects signal transmission and causes aberrant lymph gland hematopoiesis. Mammalian Asrij (Ociad1) is expressed in stem cells of the blood vascular system and is implicated in several cancers. We found that Drosophila Asrij is a pan-hemocyte marker and localizes to a subset of endocytic vesicles. Loss of asrij causes hyperproliferation of lymph gland lobes coupled with increased hemocyte differentiation, thereby depleting the pool of quiescent hemocyte precursors. This co-relates with fewer Col+ cells in the hematopoietic stem cell niche of asrij mutants. Asrij null mutants also show excess specification of crystal cells that express the RUNX factor Lozenge (Lz), a target of Notch signaling. Asrij mutant lymph glands show increased N in sorting endosomes suggesting aberrant trafficking. In vitro assays also show impaired traffic of fluorescent probes in asrij null hemocytes. Taken together our data suggest a role for Asrij in causing increased Notch signaling thereby affecting hemocyte differentiation. Thus, conserved endocytic functions may control blood cell progenitor quiescence and differentiation.

<em>In vivo</em> Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (˜100 μm) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2. SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7. With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11. In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

<em>In vivo</em> Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (˜100 μm) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2.SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7.With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11.In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

Determinants of endocytic membrane geometry, stability, and scission

During endocytic vesicle formation, distinct subdomains along the membrane invagination are specified by different proteins, which bend the membrane and drive scission. Bin-Amphiphysin-Rvs (BAR) and Fer-CIP4 homology-BAR (F-BAR) proteins can induce membrane curvature and have been suggested to facilitate membrane invagination and scission. Two F-BAR proteins, Syp1 and Bzz1, are found at budding yeast endocytic sites. Syp1 arrives early but departs from the endocytic site before formation of deep membrane invaginations and scission. Using genetic, spatiotemporal, and ultrastructural analyses, we demonstrate that Bzz1, the heterodimeric BAR domain protein Rvs161/167, actin polymerization, and the lipid phosphatase Sjl2 cooperate, each through a distinct mechanism, to induce membrane scission in yeast. Additionally, actin assembly and Rvs161/167 cooperate to drive formation of deep invaginations. Finally, we find that Bzz1, acting at the invagination base, stabilizes endocytic sites and functions with Rvs161/167, localized along the tubule, to achieve proper endocytic membrane geometry necessary for efficient scission. Together, our results reveal that dynamic interplay between a lipid phosphatase, actin assembly, and membrane-sculpting proteins leads to proper membrane shaping, tubule stabilization, and scission.

Monocyte- and Endothelial-Derived Microparticles Induce an Inflammatory Phenotype in Human Podocytes

Background/Aims: Proteinuria is associated with cardiovascular and chronic kidney disease. Microparticles (MPs) are bioactive vesicles shed from activated cells and also linked to cardiovascular disease. MP-like structures have been identified in the glomerular basement membrane, urinary space and between the glomerular basement membrane and the podocyte. We hypothesised that circulating MPs may provide a link between vascular injury and kidney diseases by inducing podocyte phenotypic alterations, thus propagating glomerular dysfunction and proteinuria. Methods:Human umbilical vein endothelial cells and U937 monocytes were stimulated with TNF-α to produce MPs. These MPs were confirmed by electron microscopy, and added to differentiated podocyte monolayers to determine effects on podocyte albumin endocytosis and the production of soluble mediators. Results:Monocyte and endothelial MPs upregulated podocyte production of pro-inflammatory mediators monocyte chemoattractant protein-1 (p < 0.001) and interleukin-6 (p < 0.001). Only monocyte MPs upregulated podocyte secretion of VEGF (p < 0.001), known to regulate glomerular permeability. Endothelial MPs decreased podocyte albumin endocytosis by 13% compared to control cells (p < 0.01). Conclusion:MPs alter endocytic functions of podocytes and induce secretion of pro-inflammatory cytokines, potentially leading to glomerular inflammation in vivo and the development of proteinuria. This study identifies a potential pathophysiological role for circulating MPs in the kidney through effects on the podocyte.

Pattern Recognition Scavenger Receptor CD204 Attenuates Toll-like Receptor 4-induced NF-κB Activation by Directly Inhibiting Ubiquitination of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6

The collaboration and cross-talk between different classes of innate pattern recognition receptors are crucial for a well coordinated inflammatory response and host defense. Here we report a previously unrecognized role of scavenger receptor A (SRA; also known as CD204) as a signaling regulator in the context of Toll-like receptor 4 (TLR4) activation. We show that SRA/CD204 deficiency leads to greater sensitivity to LPS-induced endotoxic shock. SRA/CD204 down-regulates inflammatory gene expression in dendritic cells by suppressing TLR4-induced activation of the transcription factor NF-κB. For the first time, we demonstrate that SRA/CD204 executes its regulatory functions by directly interacting with the TRAF-C domain of TNF receptor-associated factor 6 (TRAF6), resulting in inhibition of TRAF6 dimerization and ubiquitination. The attenuation of NF-κB activity by SRA/CD204 is independent of its ligand-binding domain, indicating that the signaling-regulatory feature of SRA/CD204 can be uncoupled from its conventional endocytic functions. Collectively, we have identified the molecular linkage between SRA/CD204 and the TLR4 signaling pathways, and our results reveal a novel mechanism by which a non-TLR pattern recognition receptor restricts TLR4 activation and consequent inflammatory response.

Interaction of activated Rab5 with actin-bundling proteins, L- and T-plastin and its relevance to endocytic functions in mammalian cells

Rab5 is a GTP-binding protein that is crucial for endocytic machinery functions. We previously identified L-plastin as a binding protein for Rab5, using an affinity column with constitutively active Rab5. L- and T-plastin are isoforms of a plastin protein family belonging to actin-bundling proteins that are implicated in the regulation of cell morphology, lamellipodium protrusion, bacterial invasion and tumor progression. However, the physiological relevance of Rab5 binding to plastin has remained unclear. Here, we show that L- and T-plastin interacted only with activated Rab5 and that they co-localized with Rab5 on the plasma membrane and endosome. Rab5 activity was also higher in both L- and T-plastin over-expressing Cos-1 cells. Furthermore, expression of L- and T-plastin increased the rate of fluid-phase endocytosis. These findings imply that the Rab5 is either activated or the activity is sustained by interaction with plastin, and that this interaction influences endocytic activity.

Post-translational proteolytic events influence LRP-1 functions

The low-density lipoprotein receptor-related protein (LRP-1) is a membrane receptor displaying both endocytic scavenging and signaling functions. In this review, we briefly present post-translational proteolytic processes targeting this receptor and speculate on their possible influence on LRP-1 biological functions.

A Novel GGA-Binding Site Is Required for Intracellular Sorting Mediated by Stabilin-1

Stabilin-1 is a unique scavenger receptor that combines endocytic and intracellular sorting functions in macrophages. Stabilin-1 mediates the endocytosis of acetylated low-density lipoprotein (acLDL), SPARC, and growth hormone family member placental lactogen (PL). At the same time, stabilin-1 is involved in trans-Golgi network-to-endosome routing of the endogenous chitinase-like protein SI-CLP (stabilin-interacting chitinase-like protein). A DDSLL motif in the cytoplasmic tail of stabilin-1 interacts with GGA adaptors; however, the deletion of DDSLL reduces but does not abrogate this interaction. Here, we identified a novel GGA-binding site, EDDADDD, in the cytoplasmic tail of stabilin-1. The deletion of EDDADDD impaired and the deletion of both the DDSLL and EDDADDD sites abrogated the interaction of stabilin-1 with GGAs. The surface exposure of stabilin-1 and stabilin-1-mediated endocytosis of acLDL, SPARC, and PL were not affected by the deletion either of DDSLL or EDDADDD or both. At the same time, both GGA-binding sites were necessary for the intracellular sorting of SI-CLP performed by stabilin-1. Our data indicate that the novel GGA-binding site EDDADDD is essential for stabilin-1-mediated intracellular sorting but is not required for endocytosis.

A Contiguous Compartment Functions as Endoplasmic Reticulum and Endosome/Lysosome in Giardia lamblia

The dynamic evolution of organelle compartmentalization in eukaryotes and how strictly compartmentalization is maintained are matters of ongoing debate. While the endoplasmic reticulum (ER) is classically envisioned as the site of protein cotranslational translocation, it has recently been proposed to have pluripotent functions. Using transfected reporter constructs, organelle-specific markers, and functional enzyme assays, we now show that in an early-diverging protozoan, Giardia lamblia, endocytosis and subsequent degradation of exogenous proteins occur in the ER or in an adjacent and communicating compartment. The Giardia endomembrane system is simple compared to those of typical eukaryotes. It lacks peroxisomes, a classical Golgi apparatus, and canonical lysosomes. Giardia orthologues of mammalian lysosomal proteases function within an ER-like tubulovesicular compartment, which itself can dynamically communicate with clathrin-containing vacuoles at the periphery of the cell to receive endocytosed proteins. These primitive characteristics support Giardia's proposed early branching and could serve as a model to study the compartmentalization of endocytic and lysosomal functions into organelles distinct from the ER. This system also may have functional similarity to the retrograde transport of toxins and major histocompatibility complex class I function in the ER of mammals.

Nuclear Trafficking and Functions of Endocytic Proteins Implicated in Oncogenesis

A subset of proteins predominantly associated with early endosomes or implicated in clathrin-mediated endocytosis can shuttle between the cytoplasm and the nucleus. Although the endocytic functions of these proteins have been extensively studied, much less effort has been expended in exploring their nuclear roles. Membrane trafficking proteins can affect signalling and proliferation and this can be achieved either at a nuclear or endocytic level. Furthermore, some proteins, such as Huntingtin interacting protein 1, are known as cancer biomarkers. This review will highlight the limits of our understanding of their nuclear functions and the relevance of this to signalling and oncogenesis.

PKC induces internalization and retention of the EAAC1 glutamate transporter in recycling endosomes of MDCK cells

Here we show that stimulation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) treatment induces a time-dependent decrease in glutamate transport activity due to relocalization of the excitatory amino acid carrier 1 (EAAC1) glutamate transporter from the apical surface of polarized epithelial Madin-Darby canine kidney (MDCK) cells to intracellular compartments. The PKC-induced internalization of EAAC1 is negatively regulated by the calcineurin inhibitor cyclosporine A and by the expression of a dominant-negative mutant of the endocytic protein dynamin 1, a well-known target of the phosphatase activity of calcineurin. Using 32P-metabolic labeling experiments, we found unchanged levels of phosphorylated EAAC1, indicating that EAAC1 relocalization does not depend on PKC and calcineurin modification of the transporter, while we found that a target of these modifications was the serine778 residue of dynamin, a calcineurin substrate that in its dephosphorylated form activates the endocytic functions of dynamin. These data suggest that PMA stimulates endogenous dynamin and that this activation is required to mediate internalization of EAAC1 in MDCK cells. By immunofluorescence experiments with endosomal markers we demonstrated that internalized EAAC1 accumulates in endosomes also containing the basolateral betaine-GABA transporter BGT1 and activated PKCalpha. The sustained activation of PKC was required to maintain the transporters in the endosomal compartment, while a posttreatment with a PKC-specific inhibitor induced the recycling of the transporters to their appropriate surfaces. Taken together, our data indicate that PKC activity regulates EAAC1 surface density in MDCK cells by inducing its internalization and retention in PKCalpha-labeled recycling endosomes common to apical and basolateral proteins.

A PH domain within OCRL bridges clathrin-mediated membrane trafficking to phosphoinositide metabolism

OCRL, whose mutations are responsible for Lowe syndrome and Dent disease, and INPP5B are two similar proteins comprising a central inositol 5-phosphatase domain followed by an ASH and a RhoGAP-like domain. Their divergent NH2-terminal portions remain uncharacterized. We show that the NH2-terminal region of OCRL, but not of INPP5B, binds clathrin heavy chain. OCRL, which in contrast to INPP5B visits late stage endocytic clathrin-coated pits, was earlier shown to contain another binding site for clathrin in its COOH-terminal region. NMR structure determination further reveals that despite their primary sequence dissimilarity, the NH2-terminal portions of both OCRL and INPP5B contain a PH domain. The novel clathrin-binding site in OCRL maps to an unusual clathrin-box motif located in a loop of the PH domain, whose mutations reduce recruitment efficiency of OCRL to coated pits. These findings suggest an evolutionary pressure for a specialized function of OCRL in bridging phosphoinositide metabolism to clathrin-dependent membrane trafficking.

The Single ENTH‐Domain Protein of Trypanosomes; Endocytic Functions and Evolutionary Relationship with Epsin

Epsin N-terminal homology (ENTH) domains occur in proteins of either the epsin or epsin-related (epsinR) form. They principally function in clathrin-mediated trafficking and membrane deformation. Both epsin and epsinR possess clathrin-binding motifs, but only epsin incorporates a ubiquitin-interaction motif (UIM). To better understand the origins of ENTH-domain proteins and their functions, we performed detailed comparative genomics and phylogenetics on the epsin family. The epsin ENTH-UIM configuration is an architecture restricted to yeast and animals. Further, we undertook functional analysis in Trypanosoma brucei (T. brucei), a divergent organism possessing a single ENTH-domain protein (TbEpsinR). TbEpsinR has a cellular location similar to both epsin and epsinR at plasma membrane clathrin budding sites and endosomal compartments, and associates with clathrin, as demonstrated by coimmunoprecipitation. Knockdown of TbEpsinR leads to a significant decrease in the intracellular pools of multiple surface antigens, without affecting bulk membrane internalization. Therefore, despite lacking the UIM, TbEpsinR maintains a similar role to metazoan epsin in endocytosis and participates as a clathrin-associated adaptor. We suggest that recruitment of a UIM to the ENTH-domain proteins was not essential for participation in endocytosis of ubiquitylated molecules, and is presumably a specific innovation restricted to higher eukaryotes.