Multiple routes of protein transport from endosomes to the trans Golgi network

Proteins use multiple routes for transport from endosomes to the Golgi complex. Shiga and cholera toxins and TGN38/46 are routed from early and recycling endosomes, while mannose 6-phosphate receptors are routed from late endosomes. The identification of distinct molecular requirements for each of these pathways makes it clear that mammalian cells have evolved more complex targeting mechanisms and routes than previously anticipated.     

Delivery of the Cu-transporting ATPase ATP7B to the plasma membrane in Xenopus oocytes

Cu-transporting ATPase ATP7B (Wilson disease protein) is essential for the maintenance of intracellular copper concentration. In hepatocytes, ATP7B is required for copper excretion, which is thought to occur via a transient delivery of the ATP7B- and copper-containing vesicles to the apical membrane. The currently available experimental systems do not allow analysis of ATP7B at the cell surface. Using epitope insertion, we identified an extracellular loop into which the HA-epitope can be introduced without inhibiting ATP7B activity. The HA-tagged ATP7B was expressed in Xenopus oocytes and the presence of ATP7B at the plasma membrane was demonstrated by electron microscopy, freeze-fracture experiments, and surface luminescence measurements in intact cells. Neither the deletion of the entire N-terminal copper-binding domain nor the inactivating mutation of catalytic Asp1027 affected delivery to the plasma membrane of oocytes. In contrast, surface targeting was decreased for the ATP7B variants with mutations in the ATP-binding site or the intra-membrane copper-binding site, suggesting that ligand-stabilized conformation(s) are important for ATP7B trafficking. The developed system provides significant advantages for studies that require access to both sides of ATP7B in the membrane.     

GGA1 overexpression attenuates amyloidogenic processing of the amyloid precursor protein in Niemann-Pick type C cells

Alzheimer’s disease (AD) and a rare inherited disorder of cholesterol transport, Niemann-Pick type C (NPC) share several similarities including aberrant APP processing and increased Aβ production. Previously, we have shown that the AD-like phenotype in NPC model cells involves cholesterol-dependent enhanced APP cleavage by β-secretase and accumulation of both APP and BACE1 within endocytic compartments. Since retrograde transport of BACE1 from endocytic compartments to the trans-Golgi network (TGN) is regulated by the Golgi-localized γ-ear containing ADP ribosylation factor-binding protein 1 (GGA1), we analyzed in this work a potential role of GGA1 in the AD-like phenotype of NPC1-null cells. Overexpression of GGA1 caused a shift in APP processing towards the non-amyloidogenic pathway by increasing the localization of APP at the cell surface. However, the observed effect appear to be independent on the subcellular localization and phosphorylation state of BACE1. These findings show that the AD-like phenotype of NPC model cells can be partly reverted by promoting a non-amyloidogenic processing of APP through the upregulation of GGA1 supporting its preventive role against AD.     

JM4 is a four-transmembrane protein binding to the CCR5 receptor

The CC chemokine receptor 5 (CCR5) is a major co-receptor for human immunodeficiency virus (HIV) and CCR5 mutants lacking the carboxy (C)-terminus interfere with HIV infection. Therefore, we analysed the C-terminus of CCR5 and here describe Jena-Muenchen 4 (JM4), a novel CCR5-interacting protein. JM4 is membrane-associated, co-precipitates with CCR5, and is ubiquitously expressed. It shares about 62% sequence similarity with JWA and glutamate transporter-associated protein 3-18 (GTRAP3-18), a regulator of an amino acid transporter. JWA, like JM4, is a four-transmembrane protein, which binds to the CCR5 receptor. Furthermore, JM4, JWA, and GTRAP3-18 co-localise and heterodimerise indicating a functional relationship. JM4 co-localises with calnexin in the endoplasmic reticulum and with the mannose 6-phosphate receptor in the Golgi. JM4 and GTRAP3-18 harbor a Rab-acceptor motif, indicating a function in vesicle formation at the Golgi complex. In conclusion, we describe a CCR5-interacting protein, which is suggested to function in trafficking and membrane localisation of the receptor, possibly also other receptors or amino acid transporters.     

Entamoeba histolytica: FYVE-finger domains, phosphatidylinositol 3-phosphate biosensors, associate with phagosomes but not fluid filled endosomes

Endocytosis is an important virulence function for Entamoeba histolytica, the causative agent of amoebic dysentery. Although a number of E. histolytica proteins that regulate this process have been identified, less is known about the role of lipids. In other systems, phosphatidylinositol 3-phosphate (PI3P), a product of phosphatidylinositol 3-kinase (PI 3-kinase), has been shown to be required for endocytosis. FYVE-finger domains are protein motifs that bind specifically to PI3P. Using a PI3P biosensor consisting of glutathione-S-transferase (GST) fused to two tandem FYVE-finger domains, we have localized PI3P to phagosomes but not fluid-phase pinosomes in E. histolytica, suggesting a role for PI3P in phagocytosis. Treatment of cells with PI 3-kinase inhibitors impaired GST-2 x FYVE-phagosome association supporting the authenticity of the biosensor staining. However, treatment with PI 3-kinase inhibitors did not inhibit E. histolytica-particle interaction, indicating that PI3P is not required for the initial step, but is required for subsequent steps of phagocytosis.