Salmonella enterica requires a type III secretion system, designated Spi/Ssa, to survive and proliferate within macrophages. The Spi/Ssa system is encoded within the SPI-2 pathogenicity island and appears to function intracellularly. Here, we establish that the SPI-2-encoded SpiC protein is exported by the Spi/Ssa type III secretion system into the host cell cytosol where it interferes with intracellular trafficking. In J774 macrophages, wild-type Salmonella inhibited fusion of Salmonella-containing phagosomes with lysosomes and endosomes, and interfered with trafficking of vesicles devoid of the microorganism. These inhibitory activities required living Salmonella and a functional spiC gene. Purified SpiC protein inhibited endosome-endosome fusion in vitro. A Sindbis virus expressing the SpiC protein interfered with normal trafficking of the transferrin receptor in vivo. A spiC mutant was attenuated for virulence, suggesting that the ability to interfere with intracellular trafficking is essential for Salmonella pathogenesis.

RIN1 was originally identified by its ability to inhibit activated Ras and likely participates in multiple signaling pathways because it binds c-ABL and 14-3-3 proteins, in addition to Ras. RIN1 also contains a region homologous to the catalytic domain of Vps9p-like Rab guanine nucleotide exchange factors (GEFs). Here, we show that this region is necessary and sufficient for RIN1 interaction with the GDP-bound Rabs, Vps21p, and Rab5A. RIN1 is also shown to stimulate Rab5 guanine nucleotide exchange, Rab5A-dependent endosome fusion, and EGF receptor-mediated endocytosis. The stimulatory effect of RIN1 on all three of these processes is potentiated by activated Ras. We conclude that Ras-activated endocytosis is facilitated, in part, by the ability of Ras to directly regulate the Rab5 nucleotide exchange activity of RIN1.

Phosphatidylinositol (PI) 3-kinases have been implicated in several aspects of intracellular membrane trafficking, although a detailed mechanism is yet to be established. In this study we demonstrated that wortmannin, a specific inhibitor of PI 3-kinases, inhibited constitutive endocytosis of horseradish peroxidase and transferrin in BHK-21 and TRVb-l cells. The IC50 was "40 ng/ml (93 nM). In addition, wortmannin blocked the stimulation of horseradish peroxidase uptake by the Endocytosis occurs in most eukaryotic cells and is essential for uptake of nutrients, downregulation of cell surface receptors, and maintenance of cell homeostasis (1). Generally there are two types of endocytosis: receptor-mediated endocytosis and fluid-phase endocytosis. Receptor-mediated endocytosis can be either a constitutive process (e.g., the process mediated by transferrin receptor) or a ligand-induced event (e.g., the downregulation of activated platelet-derived growth factor receptor). The internalization of cell surface receptors occurs at specific areas (coated pits) that are coated with clathrin molecules. Fluid-phase endocytosis is a constitutive cellular process that internalizes liquids and solutes from extracellular environments. After internalization, fusion between endosomal vesicles occurs with delivery of endocytosed contents into a population of early endosomes where they are further sorted to other intracellular destinations. Receptor-mediated endocytosis and fluid-phase endocytosis possibly share a common machinery at the early endosome fusion step. For example, the well-characterized small GTPase RabS regulates membrane fusion between early endosomes that can be derived from either receptor-mediated endocytosis or fluid-phase endocytosis (2-5).In the past several years, there has been an explosive discovery of molecules that control distinct membrane trafficking steps. Among the newest additions are phosphatidylinositol (PI) 3-kinases and their lipid products: phosphatidylinositol 3-phosphate (PI-3-P), phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate. The first suggestion of PI 3-kinase involvement came from the study of VPS34p, a yeast protein essential for protein targeting to the yeast vacuole and vacuole morphogenesis (6). VPS34p shares sequence homology with the catalytic subunit (p110) of a mammalian PI 3-kinase and indeed exhibits PI 3-kinase activity (7). PI 3-kinase is also involved in IgE-mediated histamine secretion (8) and insulinregulated glucose transport (9-11). PI 3-kinase is necessary for the downregulation (endocytosis) of the activated platelet-derived growth factor receptor (12). Although a detailed mechanism has not been established, these lines of evidence strongly suggest the involvement of PI 3-kinase in several aspects of intracellular membrane trafficking.In this study, we address the importance of PI 3-kinase activity in constitutive endocytosis and endosome fusion. A very useful tool in the study of PI 3-kinases is the fungal metabolite wortman...

Activated epidermal growth factor receptors recruit various intracellular proteins leading to signal generation and endocytic trafficking. Although activated receptors are rapidly internalized into the endocytic compartment and subsequently degraded in lysosomes, the linkage between signaling and endocytosis is not well understood. Here we show that EGF stimulation of NR6 cells induces a specific, rapid and transient activation of Rab5a. EGF also enhanced translocation of the Rab5 effector, early endosomal autoantigen 1 (EEA1), from cytosol to membrane. The activation of endocytosis, fluid phase and receptor mediated, by EGF was enhanced by Rab5a expression, but not by Rab5b, Rab5c, or Rab5a truncated at the NH2 and/or COOH terminus. Dominant negative Rab5a (Rab5:N34) blocked EGF-stimulated receptor-mediated and fluid-phase endocytosis. EGF activation of Rab5a function was dependent on tyrosine residues in the COOH-terminal domain of the EGF receptor (EGFR). Removal of the entire COOH terminus by truncation (c'973 and c'991) abrogated ligand-induced Rab5a activation of endocytosis. A “kinase-dead” EGFR failed to stimulate Rab5a function. However, another EGF receptor mutant (c'1000), with the kinase domain intact and a single autophosphorylation site effectively signaled Rab5 activation. These results indicate that EGFR and Rab5a are linked via a cascade that results in the activation of Rab5a and that appears essential for internalization. The results point to an interdependent relationship between receptor activation, signal generation and endocytosis.

ALS2 is an autosomal recessive form of spastic paraparesis (motor neuron disease) with juvenile onset and slow progression caused by loss of function of alsin, an activator of Rac1 and Rab5 small GTPases. To establish an animal model of ALS2 and derive insights into the pathogenesis of this illness, we have generated alsin-null mice. Cytosol from brains of Als2 ؊/؊ mice shows marked diminution of Rab5-dependent endosome fusion activity. Furthermore, primary neurons from Als2 ؊/؊ mice show a disturbance in endosomal transport of insulin-like growth factor 1 (IGF1) and BDNF receptors, whereas neuronal viability and endocytosis of transferrin and dextran seem unaltered. There is a significant decrease in the size of cortical motor neurons, and Als2 ؊/؊ mice are mildly hypoactive. Altered trophic receptor trafficking in neurons of Als2 ؊/؊ mice may underlie the histopathological and behavioral changes observed and the pathogenesis of ALS2.ALS ͉ alsin ͉ knockout mouse ͉ motor neuron ͉ Rab5

Rab22a is a member of the Rab family of small GTPases that localizes in the endocytic pathway. In CHO cells, expression of canine Rab22a (cRab22a) causes a dramatic enlargement of early endocytic compartments. We wondered whether transferrin recycling is altered in these cells. Expression of the wild-type protein and a GTP hydrolysis-deficient mutant led to the redistribution of transferrin receptor to large cRab22a-positive structures in the periphery of the cell and to a significant decrease in the plasma membrane receptor. Kinetic analysis of transferrin uptake indicates that internalization and early recycling were not affected by cRab22a expression. However, recycling from large cRab22a-positive compartments was strongly inhibited. A similar effect on transferrin transport was observed when human but not canine Rab22a was expressed in HeLa cells. After internalization for short periods of time (5 to 8 min) or at a reduced temperature (16°C), transferrin localized with endogenous Rab22a in small vesicles that did not tubulate with brefeldin A, suggesting that the endogenous protein is present in early/sorting endosomes. Rab22a depletion by small interfering RNA disorganized the perinuclear recycling center and strongly inhibited transferrin recycling. We speculate that Rab22a controls the transport of the transferrin receptor from sorting to recycling endosomes.

1,25(OH)2

Activated epidermal growth factor receptors (EGFRs) recruit intracellular proteins that mediate receptor signaling and endocytic trafficking. Rin1, a multifunctional protein, has been shown to regulate EGFR internalization (1). Here we show that EGF stimulation induces a specific, rapid, and transient membrane recruitment of Rin1 and that recruitment is dependent on the Src homology 2 (SH2) domain of Rin1. Immunoprecipitation of EGFR is accompanied by co-immunoprecipitation of Rin1 in a time-and ligand-dependent manner. Association of Rin1 and specifically the SH2 domain of Rin1 with the EGFR was dependent on tyrosine phosphorylation of the intracellular domain of the EGFR. The recruitment of Rin1, observed by light microscopy, indicated that although initially cytosolic, Rin1 was recruited to both plasma membrane and endosomes following EGF addition. Moreover, the expression of the SH2 domain of Rin1 substantially impaired the internalization of EGF without affecting internalization of transferrin. Finally, we found that Rin1 co-immunoprecipitated with a number of tyrosine kinase receptors but not with cargo endocytic receptors. These results indicate that Rin1 provides a link via its SH2 domain between activated tyrosine kinase receptors and the endocytic pathway through the recruitment and activation of Rab5a.