GTPases and lipid kinases regulate membrane traffic along the endocytic pathway by mechanisms that are not completely understood. Fusion between early endosomes requires phosphatidylinositol-3-OH kinase (PI(3)K) activity as well as the small GTPase Rab5. Excess Rab5-GTP complex restores endosome fusion when PI(3)K is inhibited. Here we identify the early-endosomal autoantigen EEA1 which binds the PI(3)K product phosphatidylinositol-3-phosphate, as a new Rab5 effector that is required for endosome fusion. The association of EEA1 with the endosomal membrane requires Rab5-GTP and PI(3)K activity, and excess Rab5-GTP stabilizes the membrane association of EEA1 even when PI(3)K is inhibited. The identification of EEA1 as a direct Rab5 effector provides a molecular link between PI(3)K and Rab5, and its restricted distribution to early endosomes indicates that EEA1 may confer directionality to Rab5-dependent endocytic transport.

Vitamin B (B12; also known as cobalamin) is a B vitamin that has an important role in cellular metabolism, especially in DNA synthesis, methylation and mitochondrial metabolism. Clinical B12 deficiency with classic haematological and neurological manifestations is relatively uncommon. However, subclinical deficiency affects between 2.5% and 26% of the general population depending on the definition used, although the clinical relevance is unclear. B12 deficiency can affect individuals at all ages, but most particularly elderly individuals. Infants, children, adolescents and women of reproductive age are also at high risk of deficiency in populations where dietary intake of B12-containing animal-derived foods is restricted. Deficiency is caused by either inadequate intake, inadequate bioavailability or malabsorption. Disruption of B12 transport in the blood, or impaired cellular uptake or metabolism causes an intracellular deficiency. Diagnostic biomarkers for B12 status include decreased levels of circulating total B12 and transcobalamin-bound B12, and abnormally increased levels of homocysteine and methylmalonic acid. However, the exact cut-offs to classify clinical and subclinical deficiency remain debated. Management depends on B12 supplementation, either via high-dose oral routes or via parenteral administration. This Primer describes the current knowledge surrounding B12 deficiency, and highlights improvements in diagnostic methods as well as shifting concepts about the prevalence, causes and manifestations of B12 deficiency.

EEA1, a 162-kDa autoantigen associated with subacute cutaneous systemic lupus erythematosus, is a coiled-coil protein localized to early endosomes and cytosol. At its C terminus, the protein contains a cysteinerich motif, which is shared with Vps27, Fab1, and Vac1, yeast proteins implicated in membrane traffic (Mu, F. T., Callaghan, J. M., Steele-Mortimer, O., Stenmark, H., Parton, R. G., Campbell, P. L., McCluskey, J., Yeo, J. P., Tock, E. P., and Toh, B. H. (1995) J. Biol. Chem. 270, 13503-13511). Here we show that this motif constitutes a genuine zinc binding domain, which we term the FYVE finger (based on the first letters of four proteins containing this motif). Profile-based data base searches identified the FYVE finger in 11 distinct proteins. The FYVE finger-containing C terminus of EEA1 was found to bind 2 mol equivalents of Zn 2؉. Mutations of conserved histidine and cysteine residues in the FYVE motif independently reduced zinc binding to 1 mol equivalent. Confocal immunofluorescence microscopy of transfected HEp2 cells revealed that the C-terminal part (residues 1277-1411) of EEA1 colocalizes extensively with a GTPasedeficient mutant of the early endosomal GTPase Rab5, while deletion of the FYVE finger or mutations that interfere with zinc binding cause a cytosolic localization. These results implicate the FYVE finger in the specific localization of EEA1 to endosomes.Endocytosis involves the cellular uptake of extracellular compounds by the invagination and pinching off of defined areas of the plasma membrane. The so formed endocytic vesicles fuse with early endosomes, from where the endocytosed material can be relocated to a number of alternative destinations (1). The sorting function of the early endosome compartment has been extensively studied (2), but still our knowledge about the molecular basis for endocytosis and endosome sorting is fragmentary. Only few molecules have so far been found specifically associated with the early endosome compartment. One of them is Rab5, a GTPase regulating homotypic fusion between early endosomes (3), and, presumably, the heterotypic fusion between endocytic vesicles and early endosomes (4). Recently, autoimmune sera from some patients with subacute systemic lupus erythematosus were found to react with a 162-kDa peripheral membrane protein specifically localized to early endosomes (5). This autoantigen, called early endosome antigen 1 (EEA1) 1 , comprises extensive coiled-coil regions, and at its N and C termini it contains sequence motifs reminiscent of zinc fingers, protein structures originally found in nucleic acid binding proteins (6, 7). The C-terminal zinc-finger-like domain is particularly interesting, as it is conserved among several non-nuclear proteins, some of which are involved in intracellular trafficking (5,8). In this report we have focused on this domain, which we have now found in 11 different proteins. We show that it binds two Zn 2ϩ ions and plays a major role in the intracellular localization of EEA1. EXPERIMENTAL PROCEDURES Materials-Hydrox...

Early endosomes are cellular compartments receiving endocytosed material and sorting them for vesicular transport to late endosomes and lysosomes or for recycling to the plasma membrane. We have cloned a human cDNA encoding an evolutionarily conserved 180-kDa protein on early endosomes named EEA1 (Early Endosome Antigen1). EEA1 is associated with early endosomes since it co-localizes by immunofluorescence with the transferrin receptor and with Rab5 but not with Rab7. Immunoelectron microscopy shows that it is associated with tubulovesicular early endosomes containing internalized bovine serum albumin-gold. EEA1 is a hydrophilic peripheral membrane protein present in cytosol and membrane fractions. It partitions in the aqueous phase after Triton X-114 solubilization and is extracted from membranes by 0.3 M NaCl. It is a predominantly alpha-helical protein sharing 17-20% sequence identity with the myosins and contains a calmodulin-binding IQ motif. It is flanked by metal-binding, cysteine "finger" motifs. The COOH-terminal fingers, Cys-X2-Cys-X12-Cys-X2-Cys and Cys-X2-Cys-X16-Cys-X2-Cys, are present within a region that is strikingly homologous with Saccharomyces cerevisiae FAB1 protein required for endocytosis and with Caenorhabditis elegans ZK632. These fingers also show limited conservation with S. cerevisiae VAC1, Vps11, and Vps18p proteins implicated in vacuolar transport. We propose that EEA1 is required for vesicular transport of proteins through early endosomes and that its finger motifs are required for this activity.

We have recently identified conventional B2 cells as atherogenic and B1a cells as atheroprotective in hypercholesterolemic ApoE−/− mice. Here, we examined the development of atherosclerosis in BAFF-R deficient ApoE−/− mice because B2 cells but not B1a cells are selectively depleted in BAFF-R deficient mice. We fed BAFF-R−/− ApoE−/− (BaffR.ApoE DKO) and BAFF-R+/+ApoE−/− (ApoE KO) mice a high fat diet (HFD) for 8-weeks. B2 cells were significantly reduced by 82%, 81%, 94%, 72% in blood, peritoneal fluid, spleen and peripheral lymph nodes respectively; while B1a cells and non-B lymphocytes were unaffected. Aortic atherosclerotic lesions assessed by oil red-O stained-lipid accumulation and CD68+ macrophage accumulation were decreased by 44% and 50% respectively. B cells were absent in atherosclerotic lesions of BaffR.ApoE DKO mice as were IgG1 and IgG2a immunoglobulins produced by B2 cells, despite low but measurable numbers of B2 cells and IgG1 and IgG2a immunoglobulin concentrations in plasma. Plasma IgM and IgM deposits in atherosclerotic lesions were also reduced. BAFF-R deficiency in ApoE−/− mice was also associated with a reduced expression of VCAM-1 and fewer macrophages, dendritic cells, CD4+ and CD8+ T cell infiltrates and PCNA+ cells in lesions. The expression of proinflammatory cytokines, TNF-α, IL1-β and proinflammatory chemokine MCP-1 was also reduced. Body weight and plasma cholesterols were unaffected in BaffR.ApoE DKO mice. Our data indicate that B2 cells are important contributors to the development of atherosclerosis and that targeting the BAFF-R to specifically reduce atherogenic B2 cell numbers while preserving atheroprotective B1a cell numbers may be a potential therapeutic strategy to reduce atherosclerosis by potently reducing arterial inflammation.