Rab15 mediates an early endocytic event in Chinese hamster ovary cells. (17/1961)

Rab GTPases comprise a large family of monomeric proteins that regulate a diverse number of membrane trafficking events, including endocytosis. In this paper, we examine the subcellular distribution and function of the GTPase Rab15. Our biochemical and confocal immunofluorescence studies demonstrate that Rab15 associates with the transferrin receptor, a marker for the early endocytic pathway, but not with Rab7 or the cation-independent mannose 6-phosphate receptor, markers for late endosomal membranes. Furthermore, Rab15 colocalizes with Rab4 and -5 on early/sorting endosomes, as well as Rab11 on pericentriolar recycling endosomes. Consistent with its localization to early endosomal membranes, overexpression of the constitutively active mutant HArab15Q67L reduces receptor-mediated and fluid phase endocytosis. Therefore, our functional studies suggest that Rab15 may function as an inhibitory GTPase in early endocytic trafficking.  (+info)

Genetic interactions in yeast between Ypt GTPases and Arf guanine nucleotide exchangers. (18/1961)

Two families of GTPases, Arfs and Ypt/rabs, are key regulators of vesicular transport. While Arf proteins are implicated in vesicle budding from the donor compartment, Ypt/rab proteins are involved in the targeting of vesicles to the acceptor compartment. Recently, we have shown a role for Ypt31/32p in exit from the yeast trans-Golgi, suggesting a possible function for Ypt/rab proteins in vesicle budding as well. Here we report the identification of a new member of the Sec7-domain family, SYT1, as a high-copy suppressor of a ypt31/32 mutation. Several proteins that belong to the Sec7-domain family, including the yeast Gea1p, have recently been shown to stimulate nucleotide exchange by Arf GTPases. Nucleotide exchange by Arf GTPases, the switch from the GDP- to the GTP-bound form, is thought to be crucial for their function. Sec7p itself has an important role in the yeast secretory pathway. However, its mechanism of action is not yet understood. We show that all members of the Sec7-domain family exhibit distinct genetic interactions with the YPT genes. Biochemical assays demonstrate that, although the homology between the members of the Sec7-domain family is relatively low (20-35%) and limited to a small domain, they all can act as guanine nucleotide exchange factors (GEFs) for Arf proteins, but not for Ypt GTPases. The Sec7-domain of Sec7p is sufficient for this activity. Interestingly, the Sec7 domain activity is inhibited by brefeldin A (BFA), a fungal metabolite that inhibits some of the Arf-GEFs, indicating that this domain is a target for BFA. These results demonstrate that the ability to act as Arf-GEFs is a general property of all Sec7-domain proteins in yeast. The genetic interactions observed between Arf GEFs and Ypt GTPases suggest the existence of a Ypt-Arf GTPase cascade in the secretory pathway.  (+info)

Localization of Bud2p, a GTPase-activating protein necessary for programming cell polarity in yeast to the presumptive bud site. (19/1961)

Yeast cells of different cell type exhibit distinct budding patterns that reflect the organization of the actin cytoskeleton. Bud1p (Rsr1p), a Ras-like GTPase, and Bud2p, a GTPase-activating protein for Bud1p, are essential for proper budding pattern. We show that Bud2p is localized at the presumptive bud site in G(1) cells in all cell types and that this localization is independent of Bud1p. Bud2p subsequently localizes to the mother-bud neck after bud emergence; this localization depends on the integrity of the septins. These observations indicate that Bud2p becomes positioned in G(1) cells by recognizing cell type-specific landmarks at the presumptive bud site.  (+info)

Directed membrane transport is involved in process formation in cultured podocytes. (20/1961)

Mature glomerular visceral epithelial cells, or podocytes, are unique cells with a complex cell architecture. Characteristically, they possess a highly branched array of major processes and foot processes, which are essential for glomerular filtration in the kidney. A podocyte cell line with the potential to exhibit many features of differentiated podocytes, particularly the formation of cell processes, was recently established. In this study, it is shown that directed membrane transport is involved in process formation in cultured podocytes. The well-characterized vesicular stomatitis virus G was used as a marker protein for the biosynthetic pathway in these cells. It seems that newly synthesized vesicular stomatitis virus G is preferentially delivered into the cell processes of the podocytes, where it is colocalized with known regulators of vesicular transport from the Golgi apparatus to the plasma membrane, such as the small GTPase rab8 and the sec6/sec8 complex. To determine the role of vesicular transport in process formation, cells were treated with brefeldin A, a drug that disrupts the trafficking of post-Golgi transport vesicles. As a result, the podocytes reversibly lost their ability to form processes. These findings suggest that podocytes are dependent on a constant fresh source of lipids and proteins to form their processes.  (+info)

The rab7 GTPase controls the maturation of Salmonella typhimurium-containing vacuoles in HeLa cells. (21/1961)

Following entry into non-phagocytic HeLa cells, the facultative pathogen Salmonella typhimurium survives and replicates within a membrane-bound vacuole. Preceding the initiation of intracellular replication there is a lag phase, during which the bacteria modulate their environment. This phase is characterized by the rapid recycling of early endosomal proteins present on the nascent vacuole followed by the acquisition of a subset of lysosomal proteins. To gain a better understanding of the mechanism of intracellular survival, we have followed the biogenesis of the S. typhimurium-containing vacuole (SCV) in HeLa cells expressing different mutant forms of the small GTPase rab7. We demonstrate that the SCV recruits pre-existing lysosomal glycoproteins (Lgps) in a rab7-dependent manner, without directly interacting with lysosomes. We also show the transient accumulation, in the vicinity of the SCV, of novel rab7- and Lgp-containing vesicles containing very low amounts of cathepsin D. The size of these vesicles is dependent on rab7 activity, suggesting a role for rab7 in their homotypic fusion. Taken together, these results indicate that rab7 regulates SCV biogenesis during the phase characterized by the rapid acquisition of lysosomal proteins. We propose that SCV maturation involves its interaction with rab7/Lgp-containing vesicles which are possible intermediate cargo components of the late endocytic pathway.  (+info)

Rab6 is phosphorylated in thrombin-activated platelets by a protein kinase C-dependent mechanism: effects on GTP/GDP binding and cellular distribution. (22/1961)

In platelets and other secretory cells, protein kinase C (PKC) plays a role in exocytosis stimulated by physiological extracellular signals, although its linkage to the secretory machinery is poorly understood. We investigated whether Rab6, a GTP-binding protein that fractionates with platelet alpha-granules, may be involved in linking these processes. We found that Rab6 contains two PKC consensus phosphorylation sites that are evolutionarily conserved. In platelets metabolically labelled with [(32)P]P(i), Rab6 phosphorylation was induced by phorbol esters or by thrombin. This phosphorylation was blocked by a specific PKC inhibitor (Ro-31-8220), but not by a p38 mitogen-activated protein kinase inhibitor (PD-169316). Physiological stimulation of platelets caused a PKC-dependent translocation of Rab6 from platelet particulate fractions, nearly doubling the fraction of Rab6 in the cytosol. A human Rab6 isoform (Rab6C) that is preferentially expressed in human platelet RNA was cloned and its phosphorylation by PKC was characterized. Rab6C incorporated up to 2 mol of [(32)P]P(i) per mol of active protein. Rab6C bound GDP and GTP with K(d) values of 113+/-12 and 119+/-27 nM respectively, and hydrolysed GTP at a rate of 100+/-15 micromol of GTP/mol of Rab6C per min. PKC phosphorylation of Rab6C increased the affinity for GTP by 3-fold, although it had lesser effects on GDP (1.6-fold). Phosphorylation did not alter the GTPase activity. In summary, thrombin activation of platelets leads to PKC-dependent phosphorylation of Rab6 and a translocation of Rab6 to the cytosol. We suggest that PKC phosphorylation may be an important mechanism through which Rab functional interactions in vesicle trafficking and secretion can be altered in response to an external stimulus.  (+info)

The developmental role of warthog, the notch modifier encoding Drab6. (23/1961)

The warthog (wrt) gene, recovered as a modifier for Notch signaling, was found to encode the Drosophila homologue of rab6, Drab6. Vertebrate and yeast homologues of this protein have been shown to regulate Golgi network to TGN trafficking. To study the function of this protein in the development of a multicellular organism, we analyzed three different warthog mutants. The first was an R62C point mutation, the second a genomic null, and the third was an engineered GTP-bound form. Our studies show, contrary to yeast, that the Drosophila homologue of rab6 is an essential gene. However, it has limited effects on development beyond the larval stage. Only the mechanosensory bristles on the head, notum, and scutellum are affected by warthog mutations. We present models for the modifying effect of Drab6 on Notch signaling.  (+info)

Rab11BP/Rabphilin-11, a downstream target of rab11 small G protein implicated in vesicle recycling. (24/1961)

Rab11 small G protein has been implicated in vesicle recycling, but its upstream regulators or downstream targets have not yet been identified. We isolated here a downstream target of Rab11, named rabphilin-11, from bovine brain. Moreover, we isolated from a rat brain cDNA library its cDNA, which encoded a protein with a M(r) of 100,946 and 908 amino acids (aa). Rabphilin-11 bound GTP-Rab11 more preferentially than GDP-Rab11 at the N-terminal region and was specific for Rab11 and inactive for other Rab and Rho small G proteins. Both GTP-Rab11 and rabphilin-11 were colocalized at perinuclear regions, presumably the Golgi complex and recycling endosomes, in Madin-Darby canine kidney cells. In HeLa cells cultured on fibronectin, both the proteins were localized not only at perinuclear regions but also along microtubules, which were oriented toward membrane lamellipodia. Treatment of HeLa cells with nocodazole caused disruption of microtubules and dispersion of GTP-Rab11 and rabphilin-11. Overexpression of the C-terminal fragment of rabphilin-11 (aa 607-730), lacking the GTP-Rab11 binding domain, in HeLa cells reduced accumulation of transferrin at perinuclear regions and cell migration. Rabphilin-11 turned out to be a rat counterpart of recently reported bovine Rab11BP. These results indicate that rabphilin-11 is a downstream target of Rab11 which is involved in vesicle recycling.  (+info)