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1.  Insulin-regulated Aminopeptidase Is a Key Regulator of GLUT4 Trafficking by Controlling the Sorting of GLUT4 from Endosomes to Specialized Insulin-regulated Vesicles 
Molecular Biology of the Cell  2010;21(12):2034-2044.
IRAP is a key regulator of GLUT4 trafficking by controlling sorting from endosomes to specialized insulin-regulated vesicles.
Insulin stimulates glucose uptake by regulating translocation of the GLUT4 glucose transporter from intracellular compartments to the plasma membrane. In the absence of insulin GLUT4 is actively sequestered away from the general endosomes into GLUT4-specialized compartments, thereby controlling the amount of GLUT4 at the plasma membrane. Here, we investigated the role of the aminopeptidase IRAP in GLUT4 trafficking. In unstimulated IRAP knockdown adipocytes, plasma membrane GLUT4 levels are elevated because of increased exocytosis, demonstrating an essential role of IRAP in GLUT4 retention. Current evidence supports the model that AS160 RabGAP, which is required for basal GLUT4 retention, is recruited to GLUT4 compartments via an interaction with IRAP. However, here we show that AS160 recruitment to GLUT4 compartments and AS160 regulation of GLUT4 trafficking were unaffected by IRAP knockdown. These results demonstrate that AS160 is recruited to membranes by an IRAP-independent mechanism. Consistent with a role independent of AS160, we showed that IRAP functions in GLUT4 sorting from endosomes to GLUT4-specialized compartments. This is revealed by the relocalization of GLUT4 to endosomes in IRAP knockdown cells. Although IRAP knockdown has profound effects on GLUT4 traffic, GLUT4 knockdown does not affect IRAP trafficking, demonstrating that IRAP traffics independent of GLUT4. In sum, we show that IRAP is both cargo and a key regulator of the insulin-regulated pathway.
doi:10.1091/mbc.E10-02-0158
PMCID: PMC2883947  PMID: 20410133
2.  GLUT4 Is Sorted to Vesicles Whose Accumulation Beneath and Insertion into the Plasma Membrane Are Differentially Regulated by Insulin and Selectively Affected by Insulin Resistance 
Molecular Biology of the Cell  2010;21(8):1375-1386.
GLUT4 is sorted to vesicles whose accumulation beneath and insertion into the plasma membrane are differentially regulated by insulin and selectively affected by insulin resistance.
Insulin stimulates glucose transport by recruiting the GLUT4 glucose transporter to the plasma membrane. Here we use total internal reflection fluorescence microscopy to show that two trafficking motifs of GLUT4, a FQQI motif and a TELE-based motif, target GLUT4 to specialized vesicles that accumulate adjacent to the plasma membrane of unstimulated adipocytes. Mutations of these motifs redistributed GLUT4 to transferrin-containing recycling vesicles adjacent to the plasma membrane, and the degree of redistribution correlated with the increases of the GLUT4 mutants in the plasma membrane of basal adipocytes. These results establish that GLUT4 defaults to recycling endosomes when trafficking to specialized vesicles is disrupted, supporting the hypothesis that the specialized vesicles are derived from an endosomal compartment. Insulin stimulates both the accumulation of GLUT4 in the evanescent field and the fraction of this GLUT4 that is inserted into the plasma membrane. Unexpectedly, these two steps are differentially affected by the development of insulin resistance. We ascribe this selective insulin resistance to inherent differences in the sensitivities of GLUT4 vesicle accumulation and insertion into the plasma membrane to insulin. Differences in insulin sensitivities of various processes may be a general mechanism for the development of the physiologically important phenomenon of selective insulin resistance.
doi:10.1091/mbc.E09-08-0751
PMCID: PMC2854095  PMID: 20181829
3.  Activation of endosomal dynein motors by stepwise assembly of Rab7–RILP–p150Glued, ORP1L, and the receptor βlll spectrin 
The Journal of Cell Biology  2007;176(4):459-471.
The small GTPase Rab7 controls late endocytic transport by the minus end–directed motor protein complex dynein–dynactin, but how it does this is unclear. Rab7-interacting lysosomal protein (RILP) and oxysterol-binding protein–related protein 1L (ORP1L) are two effectors of Rab7. We show that GTP-bound Rab7 simultaneously binds RILP and ORP1L to form a RILP–Rab7–ORP1L complex. RILP interacts directly with the C-terminal 25-kD region of the dynactin projecting arm p150Glued, which is required for dynein motor recruitment to late endocytic compartments (LEs). Still, p150Glued recruitment by Rab7–RILP does not suffice to induce dynein-driven minus-end transport of LEs. ORP1L, as well as βIII spectrin, which is the general receptor for dynactin on vesicles, are essential for dynein motor activity. Our results illustrate that the assembly of microtubule motors on endosomes involves a cascade of linked events. First, Rab7 recruits two effectors, RILP and ORP1L, to form a tripartite complex. Next, RILP directly binds to the p150Glued dynactin subunit to recruit the dynein motor. Finally, the specific dynein motor receptor Rab7–RILP is transferred by ORP1L to βIII spectrin. Dynein will initiate translocation of late endosomes to microtubule minus ends only after interacting with βIII spectrin, which requires the activities of Rab7–RILP and ORP1L.
doi:10.1083/jcb.200606077
PMCID: PMC2063981  PMID: 17283181
4.  Dynein-mediated Vesicle Transport Controls Intracellular Salmonella Replication 
Molecular Biology of the Cell  2004;15(6):2954-2964.
Salmonella typhimurium survives and replicates intracellular in a membrane-bound compartment, the Salmonella-containing vacuole (SCV). In HeLa cells, the SCV matures through interactions with the endocytic pathway, but Salmonella avoids fusion with mature lysosomes. The exact mechanism of the inhibition of phagolysosomal fusion is not understood. Rab GTPases control several proteins involved in membrane fusion and vesicular transport. The small GTPase Rab7 regulates the transport of and fusion between late endosomes and lysosomes and associates with the SCV. We show that the Rab7 GTPase cycle is not affected on the SCV. We then manipulated a pathway downstream of the small GTPase Rab7 in HeLa cells infected with Salmonella. Expression of the Rab7 effector RILP induces recruitment of the dynein/dynactin motor complex to the SCV. Subsequently, SCV fuse with lysosomes. As a result, the intracellular replication of Salmonella is inhibited. Activation of dynein-mediated vesicle transport can thus control intracellular survival of Salmonella.
doi:10.1091/mbc.E03-08-0614
PMCID: PMC420117  PMID: 15064357

Results 1-4 (4)