Rab Family Of GTP-binding Proteins Research Articles

JM receives German regulatory approval of Argillon acquisition

Vesicular transport between the endoplasmic reticulum (ER) and the Golgi in the yeast Saccharomyces cerevisiae requires a Ras-like, small GTP-binding protein, Sarip [1–3]. Whether a functional homologue operates in export from the ER in mammalian cells is unknown, nor is it clear if transport in other branches of the secretory pathway requires member(s) of a gene family. In this study, we used a PCR approach to examine the complexity of SAR1-related sequences expressed in mammalian cells that possess multiple secretory pathways. Amplification of cDNA sequences from rodent pituitary cells with primers corresponding to two conserved GTP binding domains of Sarlp yielded several clones with sequences homologous to Sarl and/or the closely related ADP-ribosylation factor (ARF) family. Of these, only two showed closer homologies to S. cerevisiae Sar1 than members of the ARF family and are designated as mSARa and mSARb. Northern blot analysis shows that mSARa is expressed in most tissues including liver, heart, brain, skeletal muscle and kidney. In contrast, mSARb is preferentially expressed in skeletal muscle and liver. The full-length cDNA of mSARa isolated from a mouse pituitary AtT-20 cDNA library encodes a protein of 198 amino acids, and is 61.6% identical to Sarlp from S. cerevisiae. Thus in contrast to the large rab family of GTP-binding proteins, vesicular transport in mammalian cells appears to be mediated by a relatively small number of Sar1-related proteins.

Rip11 is a Rab11- and AS160-RabGAP-binding protein required for insulin-stimulated glucose uptake in adipocytes

The translocation of GLUT4 to the plasma membrane underlies the ability of insulin to stimulate glucose uptake, an event that involves the activation of protein kinase B, several members of the Rab family of GTP-binding proteins and the phosphorylation of the Rab GTPase-activating protein AS160. Here, we explored the regulation by insulin of the class I Rab11-interacting proteins Rip11, RCP and FIP2. We show that Rip11, but not RCP or FIP2, translocates to the plasma membrane of 3T3-L1 adipocytes in response to insulin. This unique response of Rip11 prompted us to explore the role of this protein in more detail. We found that Rip11 partially colocalises with GLUT4 in intracellular compartments. siRNA-mediated knockdown of Rip11 inhibits insulin-stimulated uptake of 2-deoxyglucose, and overexpression of Rip11 blocks insulin-stimulated insertion of translocated GLUT4 vesicles into the plasma membrane. We additionally show that Rip11 forms a complex with AS160 in a Rab11-independent manner and that insulin induces dissociation of AS160 from Rip11. We propose that Rip11 is an AS160- and Rab-binding protein that coordinates the protein kinase signalling and trafficking machinery required to stimulate glucose uptake in response to insulin.

EHD1 and Eps15 Interact with Phosphatidylinositols via Their Eps15 Homology Domains

The C-terminal Eps15 homology domain-containing protein, EHD1, regulates the recycling of receptors from the endocytic recycling compartment to the plasma membrane. In cells, EHD1 localizes to tubular and spherical recycling endosomes. To date, the mode by which EHD1 associates with endosomal membranes remains unknown, and it has not been determined whether this interaction is direct or via interacting proteins. Here, we provide evidence demonstrating that EHD1 has the ability to bind directly and preferentially to an array of phospholipids, preferring phosphatidylinositols with a phosphate at position 3. Previous studies have demonstrated that EH domains coordinate calcium binding and interact with proteins containing the tripeptide asparagine-proline-phenylalanine (NPF). Using two-dimensional nuclear magnetic resonance analysis, we now describe a new function for the Eps15 homology (EH) domain of EHD1 and show that it is capable of directly binding phosphatidylinositol moieties. Moreover, we have expanded our studies to include the C-terminal EH domain of EHD4 and the second of the three N-terminal EH domains of Eps15 and demonstrated that phosphatidylinositol binding may be a more general property shared by certain other EH domains. Further studies identified a positively charged lysine residue (Lys-483) localized within the third helix of the EH domain, on the opposite face of the NPF-binding pocket, as being critical for the interaction with the phosphatidylinositols.

Ethanol-induced alterations in Rab proteins: possible implications for pituitary dysfunction

Chronic exposure of pubertal male rats to ethanol results in a decline in serum testosterone, increased gonadotropins, pituitary luteinizing hormone (LH) and follicle stimulating hormone (FSH) content, and decreased or inappropriately normal serum LH and FSH levels, suggesting impaired secretory release of gonadotropins. The molecular mechanisms behind this disorder are undefined, but a disruption of vesicle-mediated secretory processes is possible because intracellular protein trafficking pathways are involved in secretion of glycoproteins such as FSH and LH. Because small GTP-binding proteins of Rab family have been implicated as key regulators of membrane and protein trafficking in mammalian cells, this study was designed to test if ethanol-impaired pituitary FSH and LH secretion is associated with changes in Rab proteins, particularly Rab1B, Rab3B, Rab6, and Rab11. Male Sprague–Dawley rats 35 days old were pair-fed a Lieber–DeCarli diet with ethanol or without ethanol for 5 to 60 days. After ethanol exposure, serum testosterone levels decreased while LH and FSH were inappropriately unchanged. Immunohistochemical staining showed decreased Rab1B, Rab3B, and Rab11 protein levels in ethanol-treated pituitaries. Immunoblotting showed that ethanol induced a transient reduction in Rab6 after 5 days of ethanol exposure, whereas Rab3B decreased after 20 days, Rab11 after 30 days, and Rab1B after 60 days. Despite these changes in Rab proteins, mRNA levels were unaffected by ethanol exposure. We concluded that reductions in key Rab proteins may lead to altered vesicle trafficking and may play a role in disruption of pituitary FSH and LH secretion caused by ethanol.

Intracellular membrane trafficking pathways in bone-resorbing osteoclasts revealed by cloning and subcellular localization studies of small GTP-binding rab proteins

A variety of intracellular membrane trafficking pathways are involved in establishing the polarization of resorbing osteoclasts and regulating bone resorption activities. Small GTP-binding proteins of rab family have been implicated as key regulators of membrane trafficking in mammalian cells. Here we used a RT-PCR-based cloning method and confocal laser scanning microscopy to explore the expression array and subcellular localization of rab proteins in osteoclasts. Rab1B, rab4B, rab5C, rab7, rab9, rab11B, and rab35 were identified from rat osteoclasts in this study. Rab5C may be associated with early endosomes, while rab11B is localized at perinuclear recycling compartments and may function in the ruffled border membrane turnover and osteoclast motility. Interestingly, late endosomal rabs, rab7, and rab9, were found to localize at the ruffled border membrane indicating a late endosomal nature of this specialized plasma membrane domain in resorbing osteoclasts. This also suggests that late endocytotic pathways may play an important role in the secretion of lysosomal enzymes, such as cathepsin K, during bone resorption.

Rab3a is involved in transport of synaptic vesicles to the active zone in mouse brain nerve terminals.

The rab family of GTP-binding proteins regulates membrane transport between intracellular compartments. The major rab protein in brain, rab3A, associates with synaptic vesicles. However, rab3A was shown to regulate the fusion probability of synaptic vesicles, rather than their transport and docking. We tested whether rab3A has a transport function by analyzing synaptic vesicle distribution and exocytosis in rab3A null-mutant mice. Rab3A deletion did not affect the number of vesicles and their distribution in resting nerve terminals. The secretion response upon a single depolarization was also unaffected. In normal mice, a depolarization pulse in the presence of Ca(2+) induces an accumulation of vesicles close to and docked at the active zone (recruitment). Rab3A deletion completely abolished this activity-dependent recruitment, without affecting the total number of vesicles. Concomitantly, the secretion response in the rab3A-deficient terminals recovered slowly and incompletely after exhaustive stimulation, and the replenishment of docked vesicles after exhaustive stimulation was also impaired in the absence of rab3A. These data indicate that rab3A has a function upstream of vesicle fusion in the activity-dependent transport of synaptic vesicles to and their docking at the active zone.

Rapid identification of cysteine-linked isoprenyl groups by metabolic labeling with [3H]farnesol and [3H]geranylgeraniol.

The posttranslational modification of proteins by the covalent attachment of farnesyl and geranylgeranyl groups to cysteine residues at or near the C-terminus via a thioether bond is now well established in mammalian cells (1–6). Most isoprenylated proteins are thought to serve as regulators of cell signaling and membrane trafficking. Farnesylation and geranylgeranylation of the cysteinyl residues have been shown to promote both protein–protein and protein–membrane interactions (6–8). Isoprenylation, and, in some cases, the subsequent palmitoylation, provide a mechanism for the membrane association of polypeptides, which lack a transmembrane domain, and appear to be prerequisite for their in vivo activity (6,9,10). Three distinct protein prenyltransferases catalyzing these modifications have been identified (1–5). Two geranylgeranyltransferases (GGTases) have been characterized, and are known to modify distinct protein substrates. The CaaX GGTase (also known as GGTase-1) geranylgeranylates proteins that end in a CaaL(F) sequence, where C is cysteine, a is usually an aliphatic amino acid, and the C-terminal amino acyl group is leucine (L) or phenylalanine (F). Rab GGTase (also known as GGTase-2) catalyzes the attachment of two geranylgeranyl groups to paired carboxyl-terminal cysteines in most members of the Rab family of GTP-binding proteins (11). These proteins terminate in a Cys-Cys, Cys-X-Cys or Cys-Cys-X-X motif, where X is a small hydrophobic amino acid. Another set

A Human Homologue of the Rat Rab Geranylgeranyl Transferase β Subunit on Chromosome 1p22–p31

This report describes the localization of a human homologue of the rat rab geranylgeranyl transferase {beta} subunit, a member of the small rab family of GTP-binding proteins, to human chromosome 1p22-p31 using Southern blot analysis of somatic cell hybrids. 8 refs., 3 figs.

Molecular analysis of SAR1 -related cDNAs from a mouse pituitary cell line

Vesicular transport between the endoplasmic reticulum (ER) and the Golgi in the yeast Saccharomyces cerevisiae requires a Ras-like, small GTP-binding protein, Sarip [1–3]. Whether a functional homologue operates in export from the ER in mammalian cells is unknown, nor is it clear if transport in other branches of the secretory pathway requires member(s) of a gene family. In this study, we used a PCR approach to examine the complexity of SAR1-related sequences expressed in mammalian cells that possess multiple secretory pathways. Amplification of cDNA sequences from rodent pituitary cells with primers corresponding to two conserved GTP binding domains of Sarlp yielded several clones with sequences homologous to Sarl and/or the closely related ADP-ribosylation factor (ARF) family. Of these, only two showed closer homologies to S. cerevisiae Sar1 than members of the ARF family and are designated as mSARa and mSARb. Northern blot analysis shows that mSARa is expressed in most tissues including liver, heart, brain, skeletal muscle and kidney. In contrast, mSARb is preferentially expressed in skeletal muscle and liver. The full-length cDNA of mSARa isolated from a mouse pituitary AtT-20 cDNA library encodes a protein of 198 amino acids, and is 61.6% identical to Sarlp from S. cerevisiae. Thus in contrast to the large rab family of GTP-binding proteins, vesicular transport in mammalian cells appears to be mediated by a relatively small number of Sar1-related proteins.