Abstract
In muscle and adipose cells, the insulin-responsive aminopeptidase (IRAP) is localized to intracellular storage sites and undergoes insulin-dependent redistribution to the cell surface. Following expression, the newly synthesized IRAP protein traffics to the perinuclear insulin-sensitive compartment and acquires insulin sensitivity 6-9 h following biosynthesis. Knockdown of GGA1 by RNA interference prevented IRAP from entering, but not exiting, the insulin-responsive compartment. Mutation of the dileucine motif at positions 76 and 77 (EGFP-IRAP/AA(76,77)), but not the dileucine motif at positions 53 and 54, resulted in the rapid default of the reporter to the cell surface beginning at 3 h following biosynthesis. Alanine substitution of 9 residues amino- or carboxyl-terminal to LL(76,77) did not perturb basal intracellular sequestration or abrogate insulin-stimulated IRAP translocation. Moreover, a dominant interfering GGA mutant (VHS-GAT) potently inhibited insulin-stimulated translocation of EGFP-IRAP/WT but did not block the constitutive exocytotic trafficking of EGFP-IRAP/AA(76,77). In addition, the EGFP-IRAP/WT and EGFP-IRAP/AA(76,77) constructs occupied morphologically distinct tubulovesicular compartments in the perinuclear region. Taken together, these data indicate that LL(76,77) functions during the GGA-dependent sorting of newly made IRAP into the insulin-responsive storage compartment.
Highlights
Pathway for both membrane and secreted proteins
Entry of Newly Synthesized insulin-regulated aminopeptidase (IRAP) into, but Not Exit from, the Insulin-responsive Compartment Is Dependent upon GGA Function—We showed previously that the intracellular compartmentalization and insulin-stimulated translocation of the EGFP-IRAP/WT reporter construct is indistinguishable from that of endogenous IRAP and GLUT4 [54, 60]
The siRNA-mediated knockdown of GGA1 protein required 24 – 48 h (Fig. 1B), providing us with a window of opportunity to determine whether GGA functions in either the biosynthetic entry and/or exit of IRAP from the insulin-responsive compartment (IRC). 3T3L1 adipocytes were first transfected with the EGFP-IRAP/WT reporter
Summary
Pathway for both membrane and secreted proteins. Having cleared the quality control system of the endoplasmic reticulum, correctly folded cargo proteins are efficiently incorporated into COPII carrier vesicles prior to anterograde transport to the Golgi apparatus [4, 5]. Entry of Newly Synthesized IRAP into, but Not Exit from, the Insulin-responsive Compartment Is Dependent upon GGA Function—We showed previously that the intracellular compartmentalization and insulin-stimulated translocation of the EGFP-IRAP/WT reporter construct is indistinguishable from that of endogenous IRAP and GLUT4 [54, 60].
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