Roles of lysosomotropic agents on LRRK2 activation and Rab10 phosphorylation.

Leucine-rich repeat kinase 2 (LRRK2), the major causative gene product of autosomal-dominant Parkinson's disease, is a protein kinase that phosphorylates a subset of Rab GTPases. Since pathogenic LRRK2 mutations increase its ability to phosphorylate Rab GTPases, elucidating the mechanisms of how Rab phosphorylation is regulated by LRRK2 is of great importance. We have previously reported that chloroquine-induced lysosomal stress facilitates LRRK2 phosphorylation of Rab10 to maintain lysosomal homeostasis. Here we reveal that Rab10 phosphorylation by LRRK2 is potently stimulated by treatment of cells with a set of lysosome stressors and clinically used lysosomotropic drugs. These agents commonly promoted the formation of LRRK2-coated enlarged lysosomes and extracellular release of lysosomal enzyme cathepsin B, the latter being dependent on LRRK2 kinase activity. In contrast to the increase in Rab10 phosphorylation, treatment with lysosomotropic drugs did not increase the enzymatic activity of LRRK2, as monitored by its autophosphorylation at Ser1292 residue, but rather enhanced the molecular proximity between LRRK2 and its substrate Rab GTPases on the cytosolic surface of lysosomes. Lysosomotropic drug-induced upregulation of Rab10 phosphorylation was likely a downstream event of Rab29 (Rab7L1)-mediated enzymatic activation of LRRK2. These results suggest a regulated process of Rab10 phosphorylation by LRRK2 that is associated with lysosomal overload stress, and provide insights into the novel strategies to halt the aberrant upregulation of LRRK2 kinase activity.

Effects of agonists and phorbol esters on α1A-adrenergic receptor-Rab protein interactions.

In a cell line, stably expressing α1A-adrenoceptors fused to the mCherry red fluorescent protein, noradrenaline, methoxamine, and oxymetazoline induced concentration-dependent increases in intracellular calcium. All of these agents increase α1A-adrenoceptor phosphorylation and internalization. Transient co-expression of these receptors with Rab proteins tagged with the enhanced Green Fluorescent Protein was employed to estimate α1A-adrenoceptor-Rab interaction using Förster Resonance Energy Transfer. Noradrenaline and methoxamine increased α1A-adrenoceptor interaction with Rab5 and Rab7 but did not modify it with Rab9. Oxymetazoline induced adrenoceptor interaction with Rab5 and Rab9 and only an insignificant increase in Rab7 signal. Phorbol myristate acetate increased α1A-adrenoceptor interaction with Rab5 and Rab9 but did not modify it with Rab7. The agonists and the active phorbol ester, all of which induce receptor phosphorylation and internalization, favor receptor interaction with Rab5, i.e., association with early endosomes. Cell stimulation with phorbol myristate acetate induced the α1A-adrenoceptors to interact with the late endosomal marker, Rab9, suggesting that the receptors are directed to slow recycling endosomes once they have transited to the Trans-Golgi network to be retrieved to the plasma membrane. The agonists noradrenaline and methoxamine likely induce a faster recycling and might direct some of the adrenoceptors toward degradation and/or very slow recycling to the plasma membrane. Oxymetazoline produced a mixed pattern of interaction with the Rab proteins. These data indicate that α1A-adrenoceptor agonists can trigger different vesicular traffic and receptor fates within the cells.

Danshensu Ameliorates Cardiac Ischaemia Reperfusion Injury through Activating Sirt1/FoxO1/Rab7 Signal Pathway.

OBJECTIVE: To explore the specific molecular mechanisms of Danshensu (DSS) in the treatment of ischemia reperfusion injury (IRI). METHODS: IRI model was established with isolated rat hearts by performing global ischaemia for 30 min, and then followed by 60 min reperfusion. Also, H9C2 cells were subjected to 4-h hypoxia followed by 3-h reoxygenation. Then 10 µmol/L DSS were added in the reperfusion/reoxygenation step to intervene IRI. Cardiac function, structural change and apoptosis were respectively tested by Langendorff System, hematoxylin and eosin (HE) and terminal-deoxynucleotidyl transferase mediated nick endabeling (TUNEL) stainings. Then lactate dehydrogenase (LDH), reactive oxygen species (ROS), superoxide gasification enzyme (SOD) and glutathione peroxidase (GSH-PX) were detected by enzyme-linked immunosorbent assay (ELISA). Sirt1/FoxO1/Rab7 Signal Pathway was monitored at both protein and mRNA levels. RESULTS: The results showed that IRI not only greatly attenuated cardiac function (LVDP and ±dp/dtmax, P<0.01, P<0.05) and increased the level of the marker enzymes (cardiac troponin T, LDH, P<0.01) from the coronary effluents, but also markedly induced changes in the structure of cardiomyocytes and contributed to apoptosis, which were mediated by boosted endogenous ROS. However, after treatment with DSS all above indexes were improved, which was related to activating Sirt1/FoxO1/Rab7 signal pathway accompanied with the enhancement of antioxidant defense system, such as superoxide gasification enzyme and glutathione peroxidase. CONCLUSION: DSS is able to protect hearts from IRI, which may be attributable to inhibiting excessive ROS through Sirt1/FoxO1/Rab7 signaling.

Two pore channel 2 (TPC2) inhibits autophagosomal-lysosomal fusion by alkalinizing lysosomal pH.

Autophagy is an evolutionarily conserved lysosomal degradation pathway, yet the underlying mechanisms remain poorly understood. Nicotinic acid adenine dinucleotide phosphate (NAADP), one of the most potent Ca(2+) mobilizing messengers, elicits Ca(2+) release from lysosomes via the two pore channel 2 (TPC2) in many cell types. Here we found that overexpression of TPC2 in HeLa or mouse embryonic stem cells inhibited autophagosomal-lysosomal fusion, thereby resulting in the accumulation of autophagosomes. Treatment of TPC2 expressing cells with a cell permeant-NAADP agonist, NAADP-AM, further induced autophagosome accumulation. On the other hand, TPC2 knockdown or treatment of cells with Ned-19, a NAADP antagonist, markedly decreased the accumulation of autophagosomes. TPC2-induced accumulation of autophagosomes was also markedly blocked by ATG5 knockdown. Interestingly, inhibiting mTOR activity failed to increase TPC2-induced autophagosome accumulation. Instead, we found that overexpression of TPC2 alkalinized lysosomal pH, and lysosomal re-acidification abolished TPC2-induced autophagosome accumulation. In addition, TPC2 overexpression had no effect on general endosomal-lysosomal degradation but prevented the recruitment of Rab-7 to autophagosomes. Taken together, our data demonstrate that TPC2/NAADP/Ca(2+) signaling alkalinizes lysosomal pH to specifically inhibit the later stage of basal autophagy progression.

Fluvastatin modulates renal water reabsorption in vivo through increased AQP2 availability at the apical plasma membrane of collecting duct cells.

X-linked nephrogenic diabetes insipidus (XNDI), a severe pathological condition characterized by greatly impaired urine-concentrating ability of the kidney, is caused by inactivating mutations in the V2 vasopressin receptor (V2R) gene. The lack of functional V2Rs prevents vasopressin-induced shuttling of aquaporin-2 (AQP2) water channels to the apical plasma membrane of kidney collecting duct principal cells, thus promoting water reabsorption from urine to the interstitium. At present, no specific pharmacological therapy exists for the treatment of XNDI. We have previously reported that the cholesterol-lowering drug lovastatin increases AQP2 membrane expression in renal cells in vitro. Here we report the novel finding that fluvastatin, another member of the statins family, greatly increases kidney water reabsorption in vivo in mice in a vasopressin-independent fashion. Consistent with this observation, fluvastatin is able to increase AQP2 membrane expression in the collecting duct of treated mice. Additional in vivo and in vitro experiments indicate that these effects of fluvastatin are most likely caused by fluvastatin-dependent changes in the prenylation status of key proteins regulating AQP2 trafficking in collecting duct cells. We identified members of the Rho and Rab families of proteins as possible candidates whose reduced prenylation might result in the accumulation of AQP2 at the plasma membrane. In conclusion, these results strongly suggest that fluvastatin, or other drugs of the statin family, may prove useful in the therapy of XNDI.

Molecular characterization and expression analysis of cDNAs encoding four Rab and two Arf GTPases in the latex of Hevea brasiliensis.

In plants, Rab and Arf GTPases are key regulators of vesicle trafficking. To investigate whether these small GTPases (SG) play a role in the regulation of the regeneration of latex (the cytoplasm of the rubber-producing laticifer cell) in Hevea brasiliensis (Hevea hereafter), full-length cDNAs that encode four HbRab and two HbArf GTPases were cloned. The four HbRab proteins showed specific GTP-binding activity when expressed in Escherichia coli. Transcript expression of the six SG genes was investigated by real-time RT-PCR. All genes revealed to be expressed in each of the six Hevea tissues examined, but the expression patterns were different. Four genes, HbArf1, HbRab2, HbRab3 and HbRab4, displayed a preferential expression in latex. The expression of all genes was upregulated by the act of latex exploitation (tapping), and HbRab1 had the highest level of upregulation. Wounding markedly upregulated the expression of two SG genes (HbRab1 and HbArf2), and exogenous methyl jasmonate upregulated all six SG genes. Wounding might upregulate the expression of HbRab1 and HbArf2 through a jasmonic acid-mediated signaling pathway. None of the genes were markedly upregulated by Ethrel (an ethylene releaser and latex stimulator); instead, HbArf2 and HbRab4 were downregulated significantly after a 24 h treatment with Ethrel. This paper gives the first description of Rab and Arf GTPases in Hevea and provides clues for their involvement, HbRab1 in particular, in latex regeneration.

C5a- and ASP-mediated C5L2 activation, endocytosis and recycling are lost in S323I-C5L2 mutation.

UNLABELLED: C5L2, a G-protein-coupled receptor (GPCR), has been identified as an ASP (C3adesArg) and C5a receptor. Controversy exists regarding both ligand binding and functionality. ASP activation of C5L2 is proposed to regulate fat storage. C5L2 is also proposed as a decoy receptor for C5a, an inflammatory mediator, based on absence of Ca(2+) or chemotaxis changes. AIMS: (i) to evaluate C5L2 receptor activation and recycling using recombinant ASP (rASP) and rC5a and (ii) assess receptor trafficking of S323I-C5L2 mutation previously identified in a family and demonstrated to have altered functionality. RESULTS: stably transfected C5L2-HEK cells were sorted using fluorescent-ASP (Fluos-ASP) binding. Following 2-h serum-free pretreatment, C5L2 was typically localized to the cell-surface. beta-Arrestin-2-GFP transiently transfected C5L2-HEK cells demonstrated rASP and rC5a-dependent beta-arrestin-2-GFP translocation, which showed time-dependent intracellular colocalization with C5L2. Without ligand or C5L2 transfection, no translocation was identified at any time point. Ligand-dependent (rASP and rC5a) C5L2 endocytosis was time-dependent with a 1-h nadir, and was clathrin- and cholesterol-dependent. Transiently transfected Rab-GFP proteins (Rabs 5, 7 and 11) demonstrated time-dependent colocalization of Rab5, Rab7, and Rab11 with C5L2. In contrast to C5L2, a large proportion of stably transfected S323I-C5L2 did not localize to the cell-surface. While S323I-C5L2 was competent for Fluos-ASP and (125)I-ASP binding, although at a reduced level, there was no ligand-mediated receptor phosphorylation. Further, there was no ligand-mediated activation of beta-arrestin-2-GFP translocation, and no downstream functional activation of glucose transport or triglyceride synthesis. CONCLUSION: C5L2 is a functional metabolic receptor, and serine 323 is important for ASP induced functionality.

Redistribution of small GTP-binding protein, Rab27B, in rat parotid acinar cells after stimulation with isoproterenol.

Small GTP-binding protein, Rab27, has been implicated in the regulation of different types of membrane trafficking, including melanosome transport in melanocytes and regulated secretion events in a wide variety of secretory cells. We have previously shown that Rab27 is involved in the control of isoproterenol (IPR)-induced amylase release from rat parotid acinar cells. Although Rab27 is predominantly localized on secretory granules under resting conditions, changes to its intracellular localization after beta-stimulation have never been elucidated. The present study investigated IPR-induced redistribution of Rab27B in the parotid acinar cells, revealing translocation from secretory granules to the subapical region after 5 min of IPR treatment and then diffusion into the cytosol after 30 min of IPR treatment. Dissociation of Rab27B from the apical plasma membrane is probably mediated through the Rab GDP dissociation inhibitor (GDI) in the cytosol extracting GDP-bound Rab protein from membranes, as a dramatic increase in the amount of the Rab27B-GDI complex in the cytosol was observed 30 min after stimulation with IPR. These results indicate that, in parotid acinar cells, Rab27B is translocated, in a time-dependent manner, from secretory granules into the apical plasma membrane as a result of exposure to IPR, and then into the cytosol through binding with the GDI.

Rab23 is a potential biological target for treating hepatocellular carcinoma.

AIM: To elucidate the role of Rab23 in hepatocellular carcinoma (HCC) by assessing the expression of Rab23 in HCC tissue and in HCC cell lines. METHODS: Primary tumors (n = 100) were stained with Rab23 antibodies using immunohistochemistry and in situ hybridization in tissue microarrays. Relationships between gene expression and pathology parameters were analysed. The biological significance of Rab23 in Hep-3B cells was examined by knocking down Rab23 gene expression. We designed a pair of double-stranded RNAs against human rab23 and transfected siRNA into Hep-3B cells. Rab23 expression in these cells was examined using RT-PCR and Western blots. We investigated cell growth by MTT assays and fluorescence-activated cell sorting. RESULTS: High cytoplasmic and nuclear expression of Rab23 was found in 38 of 71 (53.5%) and in 49 of 68 HCC patients (72%) respectively, which correlated with tumor size. HCC cell lines expressed Rab23. In Hep3B cells, siRNA for Rab23 decreased Rab23 mRNA by 4.5-fold and protein expression by 2-fold. Survival rates at 24 and 48 h for Hep-3B cells transfected with siRNA were lower and about 30% Hep-3B cells were apoptotic. Knocking down rab23 suppressed Hep3B cell growth, suggesting that rab23 could play an important role in Hep3B cell growth. CONCLUSION: Rab23 is overexpressed and/or activated in HCC. Rab23 may be both a HCC predictor and a target for treating HCC.

Cytokine stimulation promotes glucose uptake via phosphatidylinositol-3 kinase/Akt regulation of Glut1 activity and trafficking.

Cells require growth factors to support glucose metabolism for survival and growth. It is unclear, however, how noninsulin growth factors may regulate glucose uptake and glucose transporters. We show that the hematopoietic growth factor interleukin (IL)3, maintained the glucose transporter Glut1 on the cell surface and promoted Rab11a-dependent recycling of intracellular Glut1. IL3 required phosphatidylinositol-3 kinase activity to regulate Glut1 trafficking, and activated Akt was sufficient to maintain glucose uptake and surface Glut1 in the absence of IL3. To determine how Akt may regulate Glut1, we analyzed the role of Akt activation of mammalian target of rapamycin (mTOR)/regulatory associated protein of mTOR (RAPTOR) and inhibition of glycogen synthase kinase (GSK)3. Although Akt did not require mTOR/RAPTOR to maintain surface Glut1 levels, inhibition of mTOR/RAPTOR by rapamycin greatly diminished glucose uptake, suggesting Akt-stimulated mTOR/RAPTOR may promote Glut1 transporter activity. In contrast, inhibition of GSK3 did not affect Glut1 internalization but nevertheless maintained surface Glut1 levels in IL3-deprived cells, possibly via enhanced recycling of internalized Glut1. In addition, Akt attenuated Glut1 internalization through a GSK3-independent mechanism. These data demonstrate that intracellular trafficking of Glut1 is a regulated component of growth factor-stimulated glucose uptake and that Akt can promote Glut1 activity and recycling as well as prevent Glut1 internalization.

Co-resistance to retinoic acid and TRAIL by insertion mutagenesis into RAM.

Retinoic acid (RA), used as first-line therapy for acute promyelocytic leukemia (APL), exerts its antileukemic activity by inducing blast differentiation and activating tumor-selective TNF-related apoptosis-inducing ligand (TRAIL) signaling. To identify downstream mediators of RA signaling, we used retrovirus-mediated insertion mutagenesis in PLB985 leukemia cells and established the RA-resistant cell line WY-1. In PLB985, but not WY-1 cells, RA induced TRAIL and its DR4 and DR5 receptors. Knocking down TRAIL expression by RNA interference blocked RA-induced apoptosis. WY-1 cells are defective for RA-induced differentiation, G1 arrest and exhibit co-resistance to TRAIL. In WY-1 cells, a single virus copy is integrated into a novel RA-regulated gene termed RAM (retinoic acid modulator). RAM is expressed in the myelomonocytic lineage and extinguished by RA in PLB985, but not WY-1 cells. Whereas knocking down RAM expression by RNA interference promoted RA-induced differentiation and TRAIL-triggered apoptosis of PLB985 and WY-1 cells, overexpression of the predicted 109 amino-acid RAM open reading frame did not alter RA signaling in PLB985 cells. This indicates that, apart from encoding the putative RAM protein, RAM RNA may exert additional functions that are impaired by the retrovirus insertion. Our study demonstrates that RA induction of the TRAIL pathway is also operative in leukemia cells lacking an RARalpha oncofusion protein and identifies RAM as a novel RA-dependent modulator of myeloid differentiation and death.

Ethanol perturbs the secretory pathway in astrocytes.

Ethanol exposure induces retention of glycoproteins in growing astrocytes. We examined the intracellular sites at which this retention occurs and investigated whether this effect is accompanied by alterations in the Golgi complex and microtubular system. We studied the effects of ethanol on the Golgi complex structure, as well as on the secretory pathway functionality by monitoring both the transport of the VSV-G protein and the protein levels of several molecules involved in the regulation of this pathway. Ethanol was found to delay VSV-G transport, modify Golgi complex morphology, and reduce the number of secretory vesicles. Moreover, ethanol affected the levels of mannosidase II, p58, betaCOP, rbet1, and several Rab GTPases. It also affected microtubule organization and polymerization and the levels of the motor proteins kinesin and dynein. Most of these effects were dose-dependent. These alterations, together with those previously reported concerning biosynthesis of glycoconjugates, provide novel insights into how ethanol impairs brain development.

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.

Sucrose-induced vacuolation results in increased expression of cholesterol biosynthesis and lysosomal genes.

Mammalian cells cultured in the presence of high concentrations of sucrose demonstrate large, phase-lucent, osmotically swollen vacuoles. Three normal human fibroblast cell lines exposed to 100 mM of sucrose for 24 h demonstrated increased expression of lysosomal, intracellular vesicle trafficking, cholesterol biosynthesis, and fatty acid metabolism genes. Most steps of the cholesterol biosynthesis pathway were upregulated including HMG CoA reductase, which catalyzes the rate-limiting step of cholesterol biosynthesis. The lysosomal genes neuraminidase, CLN3, and CLCN5 and the small GTP-binding proteins Rab7L1 and Arl7 were also increased. A Rab7L1-GFP fusion protein was overexpressed in human fibroblasts and was demonstrated to localize primarily to the Golgi apparatus, and in some cells to the membranes bounding vesicles in the perinuclear region. Increased levels of the transcription factor C/EBP were found in nuclear extracts from cells exposed to sucrose for 12 h, relative to matched controls suggesting regulation of gene expression following sucrose-induced vacuolation may be coordinated, at least in part, by the transcription factor C/EBP. Sucrose-induced vacuolation is a useful model in which to study the regulation of lysosomal gene expression and biogenesis.

Diverting intracellular trafficking of Salmonella to the lysosome through activation of the late endocytic Rab7 by intracellular delivery of muramyl dipeptide.

Previously, we showed that live Salmonella-containing phagosomes (LSP) recruit early acting Rab5 and promote fusion with early endosomes, thus avoiding transport to the lysosomes. Therefore, live Salmonella survive in a specialized compartment. Here we show that scavenger-receptor-mediated intracellular delivery of muramyl dipeptide (MDP) to macrophages leads to efficient killing of Salmonella both in vitro and in vivo. To understand the intracellular trafficking modulation of Salmonella by delivery of MDP, we investigated the levels of endocytic Rab proteins, which are the major regulators of vesicular transport. Western blot analysis reveals reduced Rab5 and enhanced Rab7 content in the maleylated bovine serum albumin-MDP (MBSA-MDP)-treated cells. The reduced content of Rab5 in the treated cells and on phagosomes inhibits the fusion of Salmonella-containing phagosomes with early endosomes, and the enhanced Rab7 content in these cells facilitated targeting of LSP to lysosomes, which contain cathepsin D and vacuolar ATPase, for killing. In vitro reconstitution of lysosomal transport demonstrated that a reduced content of Rab5 and an enhanced level of Rab7 in MBSA-MDP-treated cells is primarily responsible for targeting Salmonella to lysosomes. Intracellular delivery of MDP thus offers a general strategy against macrophage-associated infections caused by intracellular pathogens that survive in the host cell by resisting transport to lysosomes.

Grtp1, a novel gene regulated by growth hormone.

An in vitro model of GH-responsive cells was subjected to microarray analysis to identify a novel gene regulated by GH. This 258 amino acid protein, we term GH Regulated TBC Protein-1 (GRTP1), contains the TBC signature motif of GTPase activator proteins of Rab-like small GTPases. Northern blot analysis revealed a 1.3 kb major mRNA species, most abundant in testes. TaqMan assay confirmed that in the mouse, Grtp1 is expressed at highest levels in testes, with lesser abundance in intestine, kidney, lung, and liver. In the testis, expression of Grtp1 significantly increases post-pubertally. Administration of GH to mice increased levels of GRTP1 mRNA in testes (140%), but decreased GRTP1 mRNA abundance in kidney (50%) and liver (25%). Grtp1 was localized to mouse proximal chromosome 8. Orthologs of this protein are present in human, mouse, rat, and drosophila suggesting that GRTP1 has an important biological role(s).

Low levels of Ypt protein prenylation cause vesicle polarization defects and thermosensitive growth that can be suppressed by genes involved in cell wall maintenance.

The Rab/Ypt small G proteins are essential for intracellular vesicle trafficking in mammals and yeast. The vesicle-docking process requires that Ypt proteins are located in the vesicle membrane. C-terminal geranylgeranyl anchors mediate the membrane attachment of these proteins. The Rab escort protein (REP) is essential for the recognition of Rab/Ypt small G proteins by geranylgeranyltransferase II (GGTase II) and for their delivery to acceptor membranes. What effect an alteration in the levels of prenylated Rab/Ypt proteins has on vesicle transport or other cellular processes is so far unknown. Here, we report the characterization of a yeast REP mutant, mrs6-2, in which reduced prenylation of Ypt proteins occurs even at the permissive temperature. A shift to the restrictive temperature does not alter exponential growth during the first 3 h. The amount of Sec4p, but not Ypt1p, bound to vesicle membranes is reduced 2.5 h after the shift compared with wild-type or mrs6-2 cells incubated at 25 degrees C. In addition, vesicles fail to be polarized towards the bud and small budded binucleate cells accumulate at this time point. Growth in 1 M sorbitol or overexpression of MLC1, encoding a myosin light chain able to bind the unconventional type V myosin Myo2, or of genes involved in cell wall maintenance, such as SLG1, GFA1 and LRE1, suppresses mrs6-2 thermosensitivity. Our data suggest that, at least at high temperature, a critical minimal level of Ypt protein prenylation is required for maintaining vesicle polarization.

Rab11 is associated with GLUT4-containing vesicles and redistributes in response to insulin.

AIMS/HYPOTHESIS: To identify a GTPase of 24,000 M(r) which we recently found to co-localize with GLUT4 in cardiac muscle. METHODS: A 24,000 M(r)-GTP-binding fraction was purified from pig heart by a three-step chromatographic procedure, followed by two-dimensional electrophoresis and electrospray ionization-mass spectrometry. Subcellular distribution of the GTPase was assessed by western blotting. Co-localization with GLUT4 was assessed by continuous sucrose density gradient fractionation and immunoadsorption of GLUT4-containing vesicles. RESULTS: The Rab11 protein was identified as a major component of the GTP-binding fraction and its expression in rat cardiac muscle was confirmed. In vivo insulin treatment resulted in the recruitment of Rab11 from the microsomal fraction to the plasma membrane. Subcellular fractionation indicated two immunoreactive GLUT4 pools. Most of the intracellular pool of Rab11 overlapped with the high-density GLUT4 pool and most of the transferrin receptor pool. The Rab11 protein also co-sedimented with the low-density, non-endosomal GLUT4 pool and substantially increased in this fraction after insulin treatment. It was specifically present in GLUT4-containing vesicles and insulin increased its abundance in these vesicles 2.2-fold relative to the amount of GLUT4. These vesicles also containend Rab4 and Akt-2, the latter being only associated after insulin stimulation. Insulin was unable to alter the cellular localization of Rab11 in insulin-resistant obese Zucker rats. CONCLUSION/INTERPRETATION: These results support the hypothesis that at least two GTPases of the Rab family participate in GLUT4-vesicle trafficking. We suggest that Rab11 is involved in the endosomal recycling, sorting and exocytotic movement of the glucose transporter.