Identification of amino acid residues within the C terminus of the Glut4 glucose transporter that are essential for insulin-stimulated redistribution to the plasma membrane

The Glut4 glucose transporter undergoes complex insulin-regulated subcellular trafficking in adipocytes. Much effort has been expended in an attempt to identify targeting motifs within Glut4 that direct its subcellular trafficking, but an amino acid motif responsible for the targeting of the transporter to insulin-responsive intracellular compartments in the basal state or that is directly responsible for its insulin-stimulated redistribution to the plasma membrane has not yet been delineated. In this study we define amino acid residues within the C-terminal cytoplasmic tail of Glut4 that are essential for its insulin-stimulated translocation to the plasma membrane. The residues were identified based on sequence similarity (LXXLXPDEXD) between cytoplasmic domains of Glut4 and the insulin-responsive aminopeptidase (IRAP). Alteration of this putative targeting motif (IRM, insulin-responsive motif) resulted in the targeting of the bulk of the mutant Glut4 molecules to dispersed membrane vesicles that lacked detectable levels of wild-type Glut4 in either the basal or insulin-stimulated states and completely abolished the insulin-stimulated translocation of the mutant Glut4 to the plasma membrane in 3T3L1 adipocytes. The bulk of the dispersed membrane vesicles containing the IRM mutant did not contain detectable levels of any subcellular marker tested. A fraction of the total IRM mutant was also detected in a wild-type Glut4/Syntaxin 6-containing perinuclear compartment. Interestingly, mutation of the IRM sequence did not appreciably alter the subcellular trafficking of IRAP. We conclude that residues within the IRM are critical for the targeting of Glut4, but not of IRAP, to insulin-responsive intracellular membrane compartments in 3T3-L1 adipocytes.     

Syntaxin 16 controls the intracellular sequestration of GLUT4 in 3T3-L1 adipocytes

The regulated delivery of Glut4-containing vesicles to the plasma membrane is a specialised example of regulated membrane trafficking. Present models favour the transporter trafficking through two inter-related endosomal cycles. The first is the proto-typical endosomal system. This is a fast trafficking event that, in the absence of insulin, serves to internalise Glut4 from the plasma membrane. Once in this pathway, Glut4 is further sorted into a slowly recycling pathway that operates between recycling endosomes, the trans Golgi network, and a population of vesicles often referred to as Glut4-storage vesicles. Little is known about the molecules that regulate these distinct sorting steps. Here, we have studied the role of Stx16 in Glut4 trafficking. Using two independent strategies, we show that Stx16 plays a crucial role in Glut4 traffic in 3T3-L1 adipocytes. Over-expression of a mutant form of Stx16 devoid of a transmembrane anchor was found to significantly slow the reversal of insulin-stimulated glucose transport. Depletion of Stx16 using antisense approaches profoundly reduced insulin-stimulated glucose transport but was without effect on cell surface transferrin receptor levels, and also reduced the extent of Glut4 translocation to the plasma membrane in response to insulin. These data support a model in which Stx16 is crucial in the sorting of Glut4 from the fast cycling to the slow cycling intracellular trafficking pathways in adipocytes.     

p115 Interacts with the GLUT4 vesicle protein, IRAP, and plays a critical role in insulin-stimulated GLUT4 translocation

Insulin-regulated aminopeptidase (IRAP) is an abundant cargo protein of Glut4 storage vesicles (GSVs) that traffics to and from the plasma membrane in response to insulin. We used the amino terminus cytoplasmic domain of IRAP, residues 1-109, as an affinity reagent to identify cytosolic proteins that might be involved in GSV trafficking. In this way, we identified p115, a peripheral membrane protein known to be involved in membrane trafficking. In murine adipocytes, we determined that p115 was localized to the perinuclear region by immunofluorescence and throughout the cell by fractionation. By immunofluorescence, p115 partially colocalizes with GLUT4 and IRAP in the perinuclear region of cultured fat cells. The amino terminus of p115 binds to IRAP and overexpression of a N-terminal construct results in its colocalization with GLUT4 throughout the cell. Insulin-stimulated GLUT4 translocation is completely inhibited under these conditions. Overexpression of p115 C-terminus has no significant effect on GLUT4 distribution and translocation. Finally, expression of the p115 N-terminus construct has no effect on the distribution and trafficking of GLUT1. These data suggest that p115 has an important and specific role in insulin-stimulated Glut4 translocation, probably by way of tethering insulin-sensitive Glut4 vesicles at an as yet unknown intracellular site.     

Glut 4 content in the plasma membrane of rat skeletal muscle: comparative studies of the subcellular fractionation method and the exofacial photolabelling technique using ATB-BMPA

Employing subcellular membrane fractionation methods it has been shown that insulin induces a 2-fold increase in the Glut 4 protein content in the plasma membrane of skeletal muscle from rats. Data based upon this technique are, however, impeded by poor plasma membrane recovery and cross-contamination with intracellular membrane vesicles. The present study was undertaken to compare the subcellular fractionation technique with the technique using [³H]ATB-BMPA exofacial photolabelling and immunoprecipitation of Glut 4 on soleus muscles from 3-week-old Wistar rats. Maximal insulin stimulation resulted in a 6-fold increase in 3-O-methylglucose uptake, and studies based on the subcellular fractionation method showed a 2-fold increase in Glut 4 content in the plasma membrane, whereas the exofacial photolabelling demonstrated a 6- to 7-fold rise in cell surface associated Glut 4 protein. Glucose transport activity was positively correlated with cell surface Glut 4 content as estimated by exofacial labelling. In conclusion: (1) the increase in glucose uptake in muscle after insulin exposure is caused by an augmented concentration of Glut 4 protein on the cell surface membrane, (2) at maximal insulin stimulation (20 mU/ml) approximately 40% of the muscle cell content of Glut 4 is at the cell surface, and (3) the exofacial labelling technique is more sensitive than the subcellular fractionation technique in measuring the amount of glucose transporters on muscle cell surface.     

Single Point Mutations Result in the Miss-Sorting of Glut4 to a Novel Membrane Compartment Associated with Stress Granule Proteins

Insulin increases cellular glucose uptake and metabolism in the postprandial state by acutely stimulating the translocation of the Glut4 glucose transporter from intracellular membrane compartments to the cell surface in muscle and fat cells. The intracellular targeting of Glut4 is dictated by specific structural motifs within cytoplasmic domains of the transporter. We demonstrate that two leucine residues at the extreme C-terminus of Glut4 are critical components of a motif (IRM, insulin responsive motif) involved in the sorting of the transporter to insulin responsive vesicles in 3T3L1 adipocytes. Light microscopy, immunogold electron microscopy, subcellular fractionation, and sedimentation analysis indicate that mutations in the IRM cause the aberrant targeting of Glut4 to large dispersed membrane vesicles that are not insulin responsive. Proteomic characterization of rapidly and slowly sedimenting membrane vesicles (RSVs and SSVs) that were highly enriched by immunoadsorption for either wild-type Glut4 or an IRM mutant revealed that the major vesicle fraction containing the mutant transporter (IRM-RSVs) possessed a relatively small and highly distinct protein population that was enriched for proteins associated with stress granules. We suggest that the IRM is critical for an early step in the sorting of Glut4 to insulin-responsive subcellular membrane compartments and that IRM mutants are miss-targeted to relatively large, amorphous membrane vesicles that may be involved in a degradation pathway for miss-targeted or miss-folded proteins or represent a transitional membrane compartment that Glut4 traverses en route to insulin responsive storage compartments.     

Domain wise docking analyses of the modular chitin binding protein CBP50 from Bacillus thuringiensis serovar konkukian S4

This paper presents an in silico characterization of the chitin binding protein CBP50 from B. thuringiensis serovar konkukian S4 through homology modeling and molecular docking. The CBP50 has shown a modular structure containing an N-terminal CBM33 domain, two consecutive fibronectin-III (Fn-III) like domains and a C-terminal CBM5 domain. The protein presented a unique modular structure which could not be modeled using ordinary procedures. So, domain wise modeling using MODELLER and docking analyses using Autodock Vina were performed. The best conformation for each domain was selected using standard procedure. It was revealed that four amino acid residues Glu-71, Ser-74, Glu-76 and Gln-90 from N-terminal domain are involved in protein-substrate interaction. Similarly, amino acid residues Trp-20, Asn-21, Ser-23 and Val-30 of Fn-III like domains and Glu-15, Ala-17, Ser-18 and Leu-35 of C-terminal domain were involved in substrate binding. Site-directed mutagenesis of these proposed amino acid residues in future will elucidate the key amino acids involved in chitin binding activity of CBP50 protein.     

The role of small G-proteins in the regulation of glucose transport (review).

Insulin increases the rate of glucose transport into fat and muscle cells by stimulating the translocation of intracellular Glut 4-containing vesicles to the plasma membrane. This results in a marked increase in the amount of the facilitative glucose transporter Glut 4 at the cell surface, allowing for an enhanced glucose uptake. This process requires a continuous cycling through the early endosomes, a Glut 4 specific storage compartment and the plasma membrane. The main effect of insulin is to increase the rate of Glut 4 trafficking from its specific storage compartment to the plasma membrane. The whole phenomenon involves signal transduction from the insulin receptor, vesicle trafficking (sorting and fusion processes) and actin cytoskeleton modifications, which are all supposed to require small GTPases. This review describes the potential role of the various members of the Ras, Rad, Rho, Arf and Rab families in the traffic of the Glut 4-containing vesicles.     

The role of small G-proteins in the regulation of glucose transport

Insulin increases the rate of glucose transport into fat and muscle cells by stimulating the translocation of intracellular Glut 4-containing vesicles to the plasma membrane. This results in a marked increase in the amount of the facilitative glucose transporter Glut 4 at the cell surface, allowing for an enhanced glucose uptake. This process requires a continuous cycling through the early endosomes, a Glut 4 specific storage compartment and the plasma membrane. The main effect of insulin is to increase the rate of Glut 4 trafficking from its specific storage compartment to the plasma membrane. The whole phenomenon involves signal transduction from the insulin receptor, vesicle trafficking (sorting and fusion processes) and actin cytoskeleton modifications, which are all supposed to require small GTPases. This review describes the potential role of the various members of the Ras, Rad, Rho, Arf and Rab families in the traffic of the Glut 4-containing vesicles.     

Insulin regulation of protein traffic in rat adipose cells.

Rat adipocytes were biotinylated with cell-impermeable reagents, sulfo-N-hydroxysuccinimide-biotin and sulfo-N-hydroxysuccinimide-S-S-biotin in the absence and presence of insulin. Biotinylated and nonbiotinylated populations of the insulin-like growth factor-II/mannose 6-phosphate receptor, the transferrin receptor, and insulin-responsive aminopeptidase were separated by adsorption to streptavidin-agarose to determine the percentage of the biotinylated protein molecules versus their total amount in different subcellular compartments. Results indicate that adipose cells possess at least two distinct cell surface recycling pathways for insulin-like growth factor-II/mannose 6-phosphate receptor (MPR) and transferrin receptor (TfR): one which is mediated by glucose transporter isoform 4(Glut4)-vesicles and another that bypasses this compartment. Under basal conditions, the first pathway is not active, and cell surface recycling of TfR and, to a lesser extent, MPR proceeds via the second pathway. Insulin dramatically stimulates recycling through the first pathway and has little effect on the second. Within the Glut4-containing compartment, insulin has profoundly different effects on intracellular trafficking of insulin-responsive aminopeptidase on one hand and MPR and TfR on the other. After insulin administration, insulin-responsive aminopeptidase is redistributed from Glut4-containing vesicles to the plasma membrane and stays there for at least 30 min with minimal detectable internalization and recycling, whereas MPR and TfR rapidly shuttle between Glut4 vesicles and the plasma membrane in such a way that after 30 min of insulin treatment, virtually every receptor molecule in this compartment completes at least one trafficking cycle to the cell surface. Thus, different recycling proteins, which compose Glut4-containing vesicles, are internalized into this compartment at their own distinctive rates.     

Syntaxin 6 regulates Glut4 trafficking in 3T3-L1 adipocytes

Insulin stimulates the movement of glucose transporter-4 (Glut4)-containing vesicles to the plasma membrane of adipose cells. We investigated the role of post-Golgi t-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in the trafficking of Glut4 in 3T3-L1 adipocytes. Greater than 85% of syntaxin 6 was found in Glut4-containing vesicles, and this t-SNARE exhibited insulin-stimulated movement to the plasma membrane. In contrast, the colocalization of Glut4 with syntaxin 7, 8, or 12/13 was limited and these molecules did not translocate to the plasma membrane. We used adenovirus to overexpress the cytosolic domain of these syntaxin's and studied their effects on Glut4 traffic. Overexpression of the cytosolic domain of syntaxin 6 did not affect insulin-stimulated glucose transport, but increased basal deGlc transport and cell surface Glut4 levels. Moreover, the syntaxin 6 cytosolic domain significantly reduced the rate of Glut4 reinternalization after insulin withdrawal and perturbed subendosomal Glut4 sorting; the corresponding domains of syntaxins 8 and 12 were without effect. Our data suggest that syntaxin 6 is involved in a membrane-trafficking step that sequesters Glut4 away from traffic destined for the plasma membrane. We speculate that this is at the level of traffic of Glut4 into its unique storage compartment and that syntaxin 16 may be involved.     

Polymyxin B inhibits insulin-induced glucose transporter and IGF II receptor translocation in isolated adipocytes.

In isolated adipocytes, polymyxin B inhibited insulin-induced glucose incorporation into lipids in a dose-dependent manner, while polymyxin E, a structurally related antibiotic, was ineffective. To approach the mechanism of this effect, the subcellular distribution of the glucose transporter Glut 4 was investigated. Adipocytes were pretreated without or with polymyxin B before insulin stimulation, subcellular fractionation was performed and Glut 4 was detected by immunodetection. Incubation of adipocytes with polymyxin B prevented the insulin-induced appearance of Glut 4 in the plasma membranes, but did not prevent their decrease from the low-density microsomal fraction. A lower purity of the plasma membrane fractions, a detergent effect of polymyxin B on the membranes or an interference of the substance with the immunodetection of the Glut 4 molecules were excluded. These results suggest that polymyxin B was interfering with the Glut 4 translocation process stimulated by insulin in adipocytes. In a similar fashion, polymyxin B inhibited the insulin-induced increase in IGF II binding to adipocytes. This resulted from a blockade of the appearance of IGF II receptors in the plasma membranes. Since low-molecular-mass GTP-binding proteins have been implicated in the regulation of vesicular trafficking, we have used [alpha-32P]GTP binding to analyze such proteins in adipocyte fractions, after SDS/PAGE and transfer to nitrocellulose. Specific and distinct subsets of GTP-binding proteins were revealed in plasma membrane and low-density microsomal fractions of control adipocytes, whether they were stimulated or not with insulin. Polymyxin B treatment of adipocytes markedly modified the profile of the low-molecular-mass GTP-binding proteins in plasma membranes, but not in low-density microsomal fractions. Our results suggest that polymyxin B was interfering with the exocytotic process of the Glut 4 and IGF II receptor-containing vesicles, perhaps at the fusion step between vesicles and plasma membranes.     

Insulin releases Glut4 from static storage compartments into cycling endosomes and increases the rate constant for Glut4 exocytosis

In adipocytes, insulin triggers the redistribution of Glut4 from intracellular compartments to the plasma membrane. Two models have been proposed to explain the effect of insulin on Glut4 localization. In the first, termed dynamic exchange, Glut4 continually cycles between the plasma membrane and intracellular compartments in basal cells, and the major effect of insulin is through changes in the exocytic and endocytic rate constants, k(ex) and k(en). In the second model, termed static retention, Glut4 is packaged in specialized storage vesicles (GSVs) in basal cells and does not traffic through the plasma membrane or endosomes. Insulin triggers GSV exocytosis, increasing the amount of Glut4 in the actively cycling pool. Using a flow cytometry-based assay, we found that Glut4 is regulated by both static and dynamic retention mechanisms. In basal cells, 75-80% of the Glut4 is packaged in noncycling GSVs. Insulin increased the amount of Glut4 in the actively cycling pool 4-5-fold. Insulin also increased k(ex) in the cycling pool 3-fold. After insulin withdrawal, Glut4 is rapidly cleared from the plasma membrane (t((1/2)) of 20 min) by rapid adjustments in k(ex) and k(en) and recycled into static compartments. Complete recovery of the static pool required more than 3 h, however. We conclude that in fully differentiated confluent adipocytes, both the dynamic and static retention mechanisms are important for the regulation of plasma membrane Glut4 content. However, cell culture conditions affect Glut4 trafficking. For example, replating after differentiation inhibited the static retention of Glut4, which may explain differences in previous reports.     

Insulin-stimulated plasma membrane fusion of Glut4 glucose transporter-containing vesicles is regulated by phospholipase D1

Insulin stimulates glucose uptake in fat and muscle by mobilizing Glut4 glucose transporters from intracellular membrane storage sites to the plasma membrane. This process requires the trafficking of Glut4-containing vesicles toward the cell periphery, docking at exocytic sites, and plasma membrane fusion. We show here that phospholipase D (PLD) production of the lipid phosphatidic acid (PA) is a key event in the fusion process. PLD1 is found on Glut4-containing vesicles, is activated by insulin signaling, and traffics with Glut4 to exocytic sites. Increasing PLD1 activity facilitates glucose uptake, whereas decreasing PLD1 activity is inhibitory. Diminished PA production does not substantially hinder trafficking of the vesicles or their docking at the plasma membrane, but it does impede fusion-mediated extracellular exposure of the transporter. The fusion block caused by RNA interference-mediated PLD1 deficiency is rescued by exogenous provision of a lipid that promotes fusion pore formation and expansion, suggesting that the step regulated by PA is late in the process of vesicle fusion.     

Activation of RalA is required for insulin-stimulated Glut4 trafficking to the plasma membrane via the exocyst and the motor protein Myo1c

Insulin stimulates glucose transport in muscle and adipose tissue by producing translocation of the glucose transporter Glut4. The exocyst, an evolutionarily conserved vesicle tethering complex, is crucial for targeting Glut4 to the plasma membrane. Here we report that insulin regulates this process via the G protein RalA, which is present in Glut4 vesicles and interacts with the exocyst in adipocytes. Insulin stimulates the activity of RalA in a PI 3-kinase-dependent manner. Disruption of RalA function by dominant-negative mutants or siRNA-mediated knockdown attenuates insulin-stimulated glucose transport. RalA also interacts with Myo1c, a molecular motor implicated in Glut4 trafficking. This interaction is modulated by Calmodulin, which functions as the light chain for Myo1c during insulin-stimulated glucose uptake. Thus, RalA serves two functions in insulin action: as a cargo receptor for the Myo1c motor, and as a signal for the unification of the exocyst to target Glut4 vesicles to the plasma membrane.     

Identification of amino acids in the tetratricopeptide repeat and C-terminal domains of protein phosphatase 5 involved in autoinhibition and lipid activation

Protein phosphatase 5 (PP5) exhibits low basal activity due to the autoinhibitory properties of its N-terminal and C-terminal domains but can be activated approximately 40-fold in vitro by polyunsaturated fatty acids. To identify residues involved in regulating PP5 activity, we performed scanning mutagenesis of its N-terminal tetratricopeptide repeat (TPR) domain and deletion mutagenesis of its C-terminal domain. Mutating residues in a groove of the TPR domain that binds to heat shock protein 90 had no effect on basal phosphatase activity. Mutation of Glu-76, however, whose side chain projects away from this groove, resulted in a 10-fold elevation of basal activity without affecting arachidonic acid-stimulated activity. Thus, the interface of the TPR domain involved in PP5 autoinhibition appears to be different from that involved in heat shock protein 90 binding. We also observed a 10-fold elevation of basal phosphatase activity upon removing the C-terminal 13 amino acids of PP5, with a concomitant 50% decrease in arachidonic acid-stimulated activity. These two effects were accounted for by two distinct amino acid deletions: deleting the four C-terminal residues (496-499) of PP5 had no effect on its activity, but removing Gln-495 elevated basal activity 10-fold. Removal of a further three amino acids had no additional effect, but deleting Asn-491 resulted in a 50% reduction in arachidonic acid-stimulated activity. Thus, Glu-76 in the TPR domain and Gln-495 at the C-terminus were implicated in maintaining the low basal activity of PP5. While the TPR domain alone has been thought to mediate fatty acid activation of PP5, our data suggest that Asn-491, near its C-terminus, may also be involved in this process.     

The Association of ClipR-59 Protein with AS160 Modulates AS160 Protein Phosphorylation and Adipocyte Glut4 Protein Membrane Translocation

ClipR-59 is a membrane-associated protein and has been implicated in membrane signaling and vesicle trafficking. Recently, we have identified ClipR-59 as an Akt-interacting protein, and we have found that, by interacting with Akt, ClipR-59 modulates Akt subcellular compartmentalization and Akt substrate AS160 phosphorylation, thereby promoting Glut4 membrane translocation. Here, we have further investigated the regulatory effects of ClipR-59 on AS160 phosphorylation and subsequent adipocyte glucose transport. Our data showed that ClipR-59 interacted with AS160, which was mediated by the ankyrin repeats of ClipR-59 and regulated by insulin signaling. Moreover, the data also demonstrated that the interaction of ClipR-59 with AS160 was required for ClipR-59 to modulate Glut4 membrane translocation as ΔANK-ClipR-59, an AS160 interaction-defective mutant, failed to promote AS160 phosphorylation, Glut4 membrane translocation, and glucose transport induced by insulin in 3T3-L1 adipocytes. Because ClipR-59 also interacts with Akt and enhances the interaction between Akt and AS160, we suggest that ClipR-59 functions as a scaffold protein to facilitate Akt-mediated AS160 phosphorylation, thereby regulating glucose transport.     

Kinetic evidence that Glut4 follows different endocytic pathways than the receptors for transferrin and alpha2-macroglobulin

Insulin regulates glucose uptake through effects on the trafficking of the glucose transporter Glut4. To investigate the degree of overlap between Glut4 and the general endocytic pathways, the kinetics of trafficking of Glut4 and the receptors for transferrin (Tf) and α(2)-macroglobulin (α-2-M; LRP-1) were compared using quantitative flow cytometric assays. Insulin increased the exocytic rate constant (k(ex)) for both Glut4 and Tf. However, the k(ex) of Glut4 was 5-15 times slower than Tf in both basal and insulin-stimulated cells. The endocytic rate constant (k(en)) of Glut4 was also five times slower than Tf. Insulin did not affect the k(en) of either protein. In basal cells, the k(en) for α-2-M/LRP-1 was similar to Glut4 but 5-fold slower than Tf. Insulin increased k(en) for α-2-M/LRP-1 by 30%. In contrast, the k(ex) for LRP-1 was five times faster than Glut4 in basal cells, and insulin did not increase this rate constant. Thus, although there is overlap in the protein machineries/compartments utilized, the differences in trafficking kinetics indicate that Glut4, the Tf receptor, and LRP-1 are differentially processed both within the cell and at the plasma membrane. It has been reported that insulin decreases the k(en) of Glut4 in adipocytes. However, the effect of exocytosis on the "internalization" assays was not considered. Because it is counterintuitive, the effect of exocytosis on these assays is often overlooked in endocytosis studies. Using mathematical modeling and simulation, we show that the reported decrease in Glut4 k(en) can be entirely accounted for by the well established increase in Glut4 k(ex).     

Amino acid sequence of a ferredoxin from Bumilleriopsis filiformis, a yellow-green alga: relationship with red algae, protoflorideophyceae, and filamentous blue-green algae

The amino acid sequence of a [2Fe-2S] ferredoxin isolated from Bumilleriopsis filiformis, a yellow-green alga, was determined by using conventional techniques. It consisted of 98 amino acid residues with a microheterogeneity at the amino-terminus: Ala/Glu-Thr-Tyr-Ser-Val-Thr-Leu-Val-Asn-Glu-Glu-Lys-Asn-Ile-Asn-Ala-Val- Ile- Lys-Cys-Pro-Asp-Asp-Gln-Phe-Ile-Leu-Asp-Ala-Ala-Glu-Glu-Gln-Gly-Ile-Glu- Leu- Pro-Tyr-Ser-Cys-Arg-Ala-Gly-Ala-Cys-Ser-Thr-Cys-Ala-Gly-Lys-Val-Leu-Ser- Gly- Thr-Ile-Asp-Gln-Ser-Glu-Gln-Ser-Phe-Leu-Asp-Asp-Asp-Gln-Met-Gly-Ala-Gly- Phe- Leu-Leu-Thr-Cys-Val-Ala-Tyr-Pro-Thr-Ser-Asp-Cys-Lys-Val-Gln-Thr-His-Ala- Glu- Asp-Asp-Leu-Tyr. No prominent structural feature was noted in this ferredoxin in comparison with other homologous ferredoxins. From the structural comparison, B. filiformis was placed taxonomically close to filamentous blue-green algae and red algae.     

Insulin signaling in microdomains of the plasma membrane

Although the effects of insulin on glucose and lipid metabolism are well documented, gaps remain in our understanding of the precise molecular mechanisms of signal transduction. Recent evidence suggests that compartmentalization of signaling molecules and metabolic enzymes may explain the unique cellular effects of the hormone. Signal initiation from the insulin receptor is restricted in part to caveolae microdomains of the plasma membrane. A fraction of the insulin receptor directly interacts with caveolin, thus directing the protein to caveolae. Following its activation by insulin, the receptor recruits a series of adapter proteins, resulting in the activation of the G protein TC10, which also resides in caveolae. TC10 can influence a number of cellular processes, including changes in the actin cytoskeleton, recruitment of effector including the adapter protein CIP4, and assembly of the exocyst complex. These events play crucial roles in the trafficking, docking and fusion of vesicles containing the insulin-responsive glucose transporter Glut4 at the plasma membrane.     

Gapex-5, a Rab31 guanine nucleotide exchange factor that regulates Glut4 trafficking in adipocytes

Insulin stimulates glucose uptake by promoting translocation of the Glut4 glucose transporter from intracellular storage compartments to the plasma membrane. In the absence of insulin, Glut4 is retained intracellularly; the mechanism underlying this process remains uncertain. Using the TC10-interacting protein CIP4 as bait in a yeast two-hybrid screen, we cloned a RasGAP and VPS9 domain-containing protein, Gapex-5/RME-6. The VPS9 domain is a guanine nucleotide exchange factor for Rab31, a Rab5 subfamily GTPase implicated in trans-Golgi network (TGN)-to-endosome trafficking. Overexpression of Rab31 blocks insulin-stimulated Glut4 translocation, whereas knockdown of Rab31 potentiates insulin-stimulated Glut4 translocation and glucose uptake. Gapex-5 is predominantly cytosolic in untreated cells; its overexpression promotes intracellular retention of Glut4 in adipocytes. Insulin recruits the CIP4/Gapex-5 complex to the plasma membrane, thus reducing Rab31 activity and permitting Glut4 vesicles to translocate to the cell surface, where Glut4 docks and fuses to transport glucose into the cell.