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Originally published In Press as doi:10.1074/jbc.M601583200 on August 31, 2006

J. Biol. Chem., Vol. 281, Issue 44, 33457-33466, November 3, 2006
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A Specific Dileucine Motif Is Required for the GGA-dependent Entry of Newly Synthesized Insulin-responsive Aminopeptidase into the Insulin-responsive Compartment*

June Chunqiu Hou, Naoko Suzuki, Jeffrey E. Pessin, and Robert T. Watson1

From the Department of Pharmacological Sciences, Stony Brook University, Stony Brook, New York 11794-8651

Received for publication, February 17, 2006 , and in revised form, August 1, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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/AA76,77), 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 LL76,77 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/AA76,77. In addition, the EGFP-IRAP/WT and EGFP-IRAP/AA76,77 constructs occupied morphologically distinct tubulovesicular compartments in the perinuclear region. Taken together, these data indicate that LL76,77 functions during the GGA-dependent sorting of newly made IRAP into the insulin-responsive storage compartment.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The secretory pathway of eukaryotic cells is functionally organized into a series of discrete subcellular membrane compartments, each uniquely outfitted with a relatively stable population of resident proteins (1, 2). Against this fairly constant backdrop, many proteins transiently occupy a succession of membrane compartments before reaching their final destinations. In addition, a select few proteins are initially sequestered within subcellular compartments until an appropriate extracellular signal triggers their regulated exocytosis. Regardless of the specific trafficking itinerary, cargo proteins harbor intrinsic targeting information that directs their subcellular localization (3). Protein targeting begins at the level of the endoplasmic reticulum, which represents the entry point into the secretory 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). Although the mechanism for intra-Golgi transport is still under debate, it is generally believed that cargo proteins lacking specific endoplasmic reticulum retrieval signals arrive at the Golgi complex and are subsequently transported through the stacked Golgi cisternae (6, 7).

The trans-Golgi network (TGN)2 is the final subcompartment of the Golgi complex and functions as a key sorting station for proteins and lipids. In this capacity, recent work has shown that the TGN is subdivided into functional domains that recruit distinct coat complexes (8, 9). Indeed, a new family of clathrin adaptors, the GGA (Golgi-localized, {gamma}-ear-containing, ADP-ribosylation factor-binding proteins), have recently been identified as key players in transmembrane cargo selection at the TGN (10-14). The three mammalian GGA isoforms are modular adaptors, each comprised of an amino-terminal VHS domain, a middle GAT domain, a hinge region, and a carboxyl-terminal GAE domain. The VHS domain interacts with a consensus DXXLL sorting motif found in the cytosolic tails of a subset of transmembrane cargo proteins, including the cation-dependent and independent mannose-6-phosphate receptors, sortilin, and LRP3, among others. The GAT domain binds GTP-loaded ADP-ribosylation factor during GGA-mediated coat recruitment to the TGN, the hinge region interacts with clathrin, and the GAE domain binds several accessory coat proteins (15, 16).

Recently, we investigated the sorting of the insulin-responsive glucose transporter GLUT4 in the Golgi region of adipocytes (17, 18). Our results implicated a key functional role for GGA proteins during the entry of newly synthesized GLUT4 into the insulin-responsive compartment (IRC). Although the molecular identity and protein components required for the entry and exit from the IRC remain elusive, the general trafficking itinerary of GLUT4 has been intensely studied (19-30). In the basal state, GLUT4 resides predominantly in intracellular membrane compartments, where it may be actively tethered (31, 32) or dynamically retained (33). Insulin induces the translocation of GLUT4 to the cell surface, a complex process that involves the coordinated activity of many cellular proteins (34-38). Moreover, recent in vivo (39) and in vitro (40) results indicate that insulin regulates the fusion of GLUT4 vesicles with the plasma membrane.

Other than GLUT4, the insulin-regulated aminopeptidase (IRAP) is the only known transmembrane protein that undergoes a dramatic redistribution to the cell surface in response to acute insulin stimulation. IRAP was identified as a major protein that colocalizes with GLUT4 in insulin-responsive storage vesicles (27, 32, 41-48). Although the physiological function of IRAP remains unclear (49), it has nevertheless been used successfully as a reporter molecule to analyze insulin-regulated intracellular trafficking (50-53). In the present study, we investigated the initial trafficking and sorting properties of the newly synthesized IRAP protein as it undergoes the transition from insulin-insensitive to insulin-sensitive compartmentalization in adipocytes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Brefeldin A was purchased from Sigma. Secondary Alexa 488 and 594 IgGs were obtained from Molecular Probes. Horseradish peroxidase-conjugated secondary antibodies were from Pierce. The rabbit anti-GFP antibody was from Abcam, the sheep anti-TGN38 antibody was from Serotec, the rabbit anti-protein kinase B antibody was from Santa Cruz, and the monoclonal anti-syntaxin 6 antibody was from Transduction Laboratories. The rabbit anti-GGA1 antibody was made using a glutathione S-transferase fusion peptide corresponding to residues 289-508 from rat GGA1 (Covance). The EGFP-IRAP and IRAP (1-109)-TR cDNA clones have been described previously (17, 50, 54, 55). The HA3-tagged syntaxin 3 construct (HA-Stx3) was prepared by cloning the full-length Stx3 cDNA into the pKH3 vector. The dsRed-GGA1 and dsRed-VHS-GAT (Vps27, Hrs, Stam, GGA, and TOM (target of Myb)) constructs were prepared by subcloning the GGA1 and VHS-GAT constructs into the dsRed-C1 vector (Clontech).

Preparation of IRAP Amino-terminal Point Mutations—Alanine substitutions were introduced into the cytosolic tail of IRAP by PCR overlap extension, using oligonucleotides encoding the corresponding base substitutions as previously described (56). The resulting PCR products were cloned into the EGFP-C1 vector (Clontech) using standard molecular biology techniques. Final clones were fully sequenced to verify the inserts.

Culture and Electroporation of 3T3L1 Adipocytes—Murine 3T3L1 preadipocytes were cultured and differentiated as described (56). Differentiated 3T3L1 adipocytes were transiently transfected by electroporation (160 V, 950 microfarads) as described (57). After electroporation, the cells were seeded on collagen-coated glass coverslips and allowed to recover for various times as described in the figure legends. For siRNA-mediated knockdown of GGA1, three different siRNA oligonucleotides were used: GGG CGA CCA GAC CUA CAA UGA, GCA GUG AGG ACC UCA UGA AGG, and GAC CAG AAG CGG AUG GAA AUG; 1 nM of each of the three siRNAs were electroporated into adipocytes (170 V, 950 microfarads). For the corresponding control experiments, 1 nM of each of the following three scrambled siRNA oligonucleotides were used: UCA CCA CGC GAG CGG AAA UGA, ACA CUU CCG AGG GAG GAA UGG, and AAC AGC CGG AAG UGA GGA AUG.

Time Course Experiments—Transfected adipocytes were allowed to recover for 3-24 h prior to insulin stimulation as previously described (17). The cells that were treated with insulin at the 3-h time point post-transfection were plated directly into serum-free medium. For all other time points the cells were first plated in complete medium and then switched to serum-free medium 2-3 h prior to insulin stimulation. The incubation with or without 100 nM insulin for 30 min was done at the determined time to end the experiments exactly 3, 6, 9, and 12 h after transfection, at which time the cells were fixed with 4% paraformaldehyde and processed for wide field fluorescent microscopy. Translocation data in 3T3L1 adipocytes are expressed as the means ± S.E. of cells showing a plasma membrane ring, obtained by counting 50 cells/condition in three to five independent experiments.

Confocal Fluorescence Microscopy and Image Analysis—Following the individual experimental procedure, the cells were washed in phosphate-buffered saline and fixed for 15 min in 4% paraformaldehyde containing 0.2% Triton X-100, washed in phosphate-buffered saline, and blocked with 5% donkey serum plus 1% bovine serum albumin for 1 h at room temperature. Primary and secondary antibodies were used at 1:100 dilutions (unless otherwise indicated) in blocking solution and samples were mounted on glass slides with Vectashield (Vector Laboratories). The cells were imaged using a Zeiss LSM 510 confocal fluorescent microscope.

Subcellular Fractionation and Western Blotting—Adipocytes were electroporated with 50 µg of EGFP-IRAP DNA and allowed to recover for various times prior to serum starvation and insulin stimulation as indicated in the figure legend. The cells were then washed in phosphate-buffered saline, resuspended in HES buffer (20 mM HEPES, pH 7.4, 1 mM EDTA, 225 mM sucrose, 2 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin, 10 µg/ml aprotinin, 5 µg/ml leupeptin), and sheared by 10 passes through a ball-bearing homogenizer. Differential centrifugation was used to fractionate adipocytes as described previously (58, 59). Total protein in the high speed pellet (HSP), low speed pellet (LSP), and plasma membrane (PM) fractions was quantified using the BCA protein assay kit (Pierce). Equal protein amounts from each of the subcellular fractions were loaded onto 4-12% gradient SDS-polyacrylamide gels, subjected to electrophoresis, transferred to nitrocellulose membranes, and immunoblotted with a polyclonal GFP antibody.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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). In addition, it has been reported that the GGA clathrin adaptors function during the entry of newly synthesized GLUT4 into the insulin responsive storage compartment (17). To examine the potential role of GGA1 function in the initial sorting and regulated trafficking IRAP into and out of the IRC in adipocytes, we first determined the time course of expression of GGA1 during the differentiation of 3T3L1 adipocytes (Fig. 1A). Interestingly, GGA1 expression levels showed a marked induction between days 2 and 3 of differentiation, a time frame that is coincident with the formation of the insulin-responsive compartment (61). We next utilized siRNA-mediated gene silencing to reduce the expression levels of GGA1 (Fig. 1B). Although GGA1 protein levels remained unchanged relative to scrambled siRNA controls at 6 and 12 h following transfection (Fig. 1B, lanes 1-4), at the 24-h time point following transfection with GGA1 siRNAs, a substantial reduction of GGA1 protein levels was observed compared with cells transfected with scrambled siRNA (Fig. 1B, lanes 5 and 6). GGA1 protein levels were further decreased at 48 and 72 h following siRNA transfection (Fig. 1B, lanes 7-10). This reduction in GGA1 protein levels was specific because there was no significant effect on the expression of protein kinase B (also referred to as Akt) (Fig. 1B) and TUG (data not shown).


Figure 1
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  		FIGURE 1.
Targeted knockdown of GGA1 blocks IRAP entry into, but not insulin-stimulated exit from, the IRC. A, time course of expression of GGA1 during the differentiation of 3T3L1 adipocytes. The lysates were collected on the indicated days following the addition of adipocyte differentiation media and subjected to immunoblotting using a GGA1 antibody. B, differentiated 3T3L1 adipocytes were electroporated with 1 nmol each of siRNAs 1, 2, and 3 directed against GGA1 (lanes 2, 4, 6, 8, and 10) or with scrambled siRNAs (lanes 1, 3, 5, 7, and 9) as described under "Experimental Procedures. " At 6, 12, 24, 48, and 72 h post-transfection, the cells were analyzed for GGA1 protein expression by Western blot. Protein kinase B (PKB) was used as a protein loading control. C, differentiated 3T3L1 adipocytes were transfected with 50µg EGFP-IRAP/WT plasmid DNA at time t = 0 h. At t = 12 h, the cells were transfected a second time with 1 nmol each of siRNAs 1, 2, and 3, or with scrambled siRNAs (Scram.). The cells were allowed to recover for 60 h and treated without or with 100 nM insulin for 30 min. The cells were then fixed, and the cell surface accumulation of EGFP-IRAP/WT was determined as described under "Experimental Procedures." A timeline is shown above the panel for clarity. D, differentiated 3T3L1 adipocytes were electroporated with 1 nmol each of siRNAs 1, 2, and 3, or scrambled siRNAs at t = 0 h and allowed to recover for 36 h. The cells were then transfected again with 50 µg of EGFP-IRAP/WT plasmid DNA and allowed to recover for 12 h and then treated without or with 100 nM insulin for 30 min. In a separate set of experiments, adipocytes were transfected with 1 nmol each of siRNAs 1, 2, and 3 or scrambled siRNAs at t = 0 h. The cells were then allowed to recover for 60 h. The cells were then retransfected with 50 µg of EGFP-IRAP/WT plasmid DNA and allowed to recover for 12 h and treated without or with 100 nM insulin for 30 min. The translocation of EGFP-IRAP/WT was then determined as described under "Experimental Procedures." Timelines are shown above the panel for clarity. The data in C and D are from counting 50 cells/experiment (means ± S.E.) from three independent experiments.

 


Figure 2
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  		FIGURE 2.
A minimal 30-amino acid subregion of the IRAP cytosolic domain recapitulates efficient perinuclear sorting. Differentiated 3T3L1 adipocytes were electroporated with 50 µg of the indicated IRAP-TR construct, either without (A, C, and E) or with (B, D, and F) 100 µg of dsRed-VHS-GAT. Following transfection, the cells were allowed to recover for various times and either left untreated (circles) or stimulated for 30 min with 100 nM insulin (squares). The data are from counting 50 cells/experiment (means ± S.E.) from three independent experiments.

 
Previously, we observed that newly synthesized IRAP protein requires 6-9 h following expression to acquire insulin responsiveness (Fig. 2 and Ref. 60). In contrast, 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 IRC. 3T3L1 adipocytes were first transfected with the EGFP-IRAP/WT reporter. Twelve h later, the cells were transfected a second time with GGA1 siRNA (or scrambled siRNA control), and 60 h later treated without or with 100 nM insulin. Under these experimental conditions, EGFP-IRAP/WT has been synthesized and reached its steady-state intracellular distribution prior to the loss of GGA1 protein. Expression of the IRAP reporter before the decrease in GGA1 protein had no significant effect on the extent of insulin-stimulated translocation of EGFP-IRAP/WT (Fig. 1C). These data demonstrate that once IRAP enters into the IRC, insulin-stimulated exit is independent of GGA1 function.

We next knocked down GGA1 protein levels first, prior to the expression of the EGFP-IRAP/WT reporter (Fig. 1D). In one set of experiments, adipocytes were transfected with GGA1 siRNA, and then 36 h later they were transfected again with EGFP-IRAP/WT. The cells were then allowed to recover for 12 h and treated without and with insulin. In a second set of experiments, adipocytes were transfected with the GGA1 siRNA, and then 60 h later were transfected again with the EGFP-IRAP/WT reporter. The cells were then allowed to recover for 12 h and treated without or with insulin. Under both of these conditions, a specific and marked inhibition of insulin-stimulated IRAP translocation was observed (Fig. 1D). These data demonstrate that GGA1 is required for the biosynthetic acquisition of insulin responsiveness. Taken together, these data establish that GGA1 functions in the initial biosynthetic entry of IRAP into the IRC but does not affect its ability to exit from this compartment.

A Dileucine Motif at Positions 76 and 77 (LL76,77) Is Required for Insulin-responsive Compartmentalization of Newly Synthesized IRAP—Having established a role for GGA in the biosynthetic sorting of IRAP into the IRC, we next compared the time course and GGA sensitivity of various IRAP mutations. The cytosolic domain of IRAP, when fused to the transmembrane and extracellular regions of the human transferrin receptor, IRAP (1-109)-TR, displays all the established trafficking characteristics of the IRAP protein (50-53). Moreover, a 30-amino acid minimal region (amino acids 55-84) of IRAP was found to be functionally sufficient and to faithfully recapitulate the intracellular trafficking and insulin responsiveness of the parent IRAP (1-109)-TR construct under steady-state conditions (50-53). We found that IRAP (1-109)-TR was efficiently retained in intracellular compartments at the 3-, 6-, 9-, and 12-h time points following transient expression in the absence of insulin (Fig. 2A). Beginning at 6 h after expression, IRAP (1-109)-TR displayed a small but readily observable insulin-stimulated translocation to the plasma membrane (Fig. 2A). At the 9- and 12-h time points, IRAP (1-109)-TR exhibited a robust cell surface accumulation following acute insulin stimulation. In addition, the insulin-stimulated translocation of IRAP (1-109)-TR was potently inhibited at all time points examined when coexpressed with the dominant interfering VHS-GAT domains of GGA (Fig. 2B).

As expected, the IRAP minimal unit, IRAP (55-84)-TR, also resulted in a trafficking behavior and insulin responsiveness that was indistinguishable from IRAP (1-109)-TR (Fig. 2C). In addition, the ability of IRAP (55-84)-TR to respond to insulin was potently inhibited when coexpressed with the VHS-GAT dominant interfering GGA construct (Fig. 2D).


Figure 3
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  		FIGURE 3.
A dileucine motif at positions 76 and 77 is required for GGA-sensitive intracellular sorting of newly made IRAP. Differentiated 3T3L1 adipocytes were electroporated with 50 µg of the indicated EGFP-IRAP construct, either without (A and C) or with (B and D) 100 µg of dsRed-VHS-GAT. Following transfection, the cells were allowed to recover for various times and either left untreated (circles) or stimulated for 30 min with 100 nM insulin (squares). Differentiated 3T3L1 adipocytes were electroporated with 50 µgof HA-syntaxin 3 cDNA, either without (E) or with (F) 100 µg of dsRed-VHS-GAT. Following transfection, the cells were allowed to recover for various times and either left untreated (circles) or stimulated for 30 min with 100 nM insulin (squares). The data are from counting 50 cells/experiment (means ± S.E.) from three independent experiments.

 
We next undertook to identify potential sorting motifs present within the cytosolic domain of IRAP. Previous work had identified a dileucine motif at positions 76 and 77 (LL76,77) that was critical for endocytosis of IRAP from the plasma membrane and subsequent endosomal sorting (53). Because the IRAP (55-84)-TR chimera contains the LL76,77 motif, we mutated these residues to alanines, generating the IRAP (55-84)-TR/AA76,77 construct (Fig. 2, E and F). The IRAP (55-84)-TR/AA76,77 mutant reporter showed a consistent increase in basal cell surface localization at the 3-h time point that dramatically increased over the time course examined (Fig. 2E). Because the plasma membrane accumulation was quite pronounced, there was no additional significant increase in response to insulin stimulation (Fig. 2E). Moreover, coexpression of VHS-GAT had no significant effect on the cell surface localization of the IRAP (55-84)-TR/AA76,77 construct (Fig. 2F). These data further demonstrate that the minimal IRAP (55-84)-TR construct contains all the necessary targeting information to direct efficient GGA-sensitive perinuclear sorting into the insulin-responsive storage compartment.

To address the question of whether the LL76,77 motif functions in the context of the full-length 109-amino acid tail region of IRAP, we utilized a EGFP-IRAP/WT construct. Previous work showed that EGFP-IRAP/WT is sequestered in perinuclear compartments in the absence of insulin (54, 60). Consistent with these earlier observations, EGFP-IRAP/WT was excluded from the plasma membrane at all time points under basal conditions (Fig. 3A). Insulin stimulation of cells expressing IRAP for 3 h resulted in no significant difference in the distribution of the newly synthesized IRAP protein (Fig. 3A). However, at 6 h post-transfection, there was a small but readily detectable insulin-stimulated cell surface localization of EGFP-IRAP/WT (Fig. 3A). At 9 and 12 h after transfection, acute insulin stimulation resulted in a dramatic redistribution of EGFP-IRAP/WT to the cell surface (Fig. 3A), an effect that was strongly inhibited by the coexpression of the dominant interfering GGA mutant, VHS-GAT (Fig. 3B).


Figure 4
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  		FIGURE 4.
A dileucine motif at positions 53 and 54 is dispensable for GGA-sensitive intracellular sorting of newly made IRAP. Differentiated 3T3L1 adipocytes were electroporated with 50 µg of the indicated EGFP-IRAP construct, either without (A, C, and E) or with (B, D, and F) 100 µg of dsRed-VHS-GAT. Following transfection, the cells were allowed to recover for various times and either left untreated (circles) or stimulated for 30 min with 100 nM insulin (squares). The data are from counting 50 cells/experiment (means ± S.E.) from three independent experiments.

 
We made a EGFP-tagged reporter construct in which the dileucine at position 76,77 was mutated to alanines (EGFP-IRAP/AA76,77). Transient expression of EGFP-IRAP/AA76,77 resulted in the rapid default of the reporter to the cell surface at all time points following expression (Fig. 3C). Indeed, even at the 3-h time point there was a small but significant increase in the basal cell surface accumulation of EGFP-IRAP/AA76,77 compared with the EGFP-IRAP/WT construct. Moreover, the plasma membrane accumulation of EGFP-IRAP/AA76,77 was insensitive to the presence of the dominant interfering GGA mutant, VHS-GAT (Fig. 3D). As a control for constitutive exocytosis, we also analyzed the time-dependent intracellular localization of Stx3. Syntaxin 3 is a resident protein of the plasma membrane and, like IRAP, is a Type II integral membrane protein (55). Similar to EGFP-IRAP/AA76,77, the HA-Stx3 protein displayed a small but significant accumulation at the cell surface at 3 h post-transfection (Fig. 3E). The plasma membrane localization of HA-Stx3 was quite pronounced by 6 h and remained persistent throughout the time course examined (Fig. 3E). As observed for EGFP-IRAP/AA76,77, the plasma membrane localization of HA-Stx3 was also refractory to the coexpression of VHS-GAT (Fig. 3F).

In addition to the LL76,77 motif, the cytosolic region of IRAP contains another dileucine sequence at positions 53 and 54 (LL53,54). To investigate the possible redundant function of LL53,54 in targeting newly synthesized IRAP, we mutated these residues to alanine, generating the EGFP-IRAP/AA53,54 reporter (Fig. 4). Expression of EGFP-IRAP/AA53,54 resulted in its efficient retention in perinuclear compartments with little or no observable cell surface accumulation in the absence of insulin that was essentially identical to EGFP-IRAP/WT (Fig. 4, A and C). Beginning at 6 h after transfection, a small but readily apparent insulin-stimulated translocation of EGFP-IRAP/AA53,54 was observed, and this increased at the 9- and 12-h time points (Fig. 4, A and C). Similar to EGFP-IRAP/WT, the insulin-induced translocation of EGFP-IRAP/AA53,54 was efficiently inhibited by coexpression of VHS-GAT (Fig. 4, B and D). We further generated a combined mutant wherein the dileucines at positions 53 and 54 and positions 76 and 77 were all mutated to alanine residues (EGFP-IRAP/AA53,54, AA76,77). When expressed in adipocytes, this reporter trafficked to the cell surface in the absence of insulin in a manner indistinguishable from the EGFP-IRAP/AA76,77 construct (Fig. 4E). Furthermore, the cell surface accumulation of EGFP-IRAP/AA53,54, AA76,77 was not inhibited by coexpression of the VHS-GAT domain (Fig. 4F).


Figure 5
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  		FIGURE 5.
Subcellular compartmentalization of IRAP reporter constructs determined by membrane fractionation. A, differentiated 3T3L1 adipocytes were electroporated with 50 µg of EGFP-IRAP/WT plasmid DNA. Beginning 3.5 h after transfection cells were serum-starved for 2 h, followed by treatment without (lanes 1, 3, and 5) or with (lanes 2, 4, and 6) 100 nM insulin for 30 min. For B and C, differentiated 3T3L1 adipocytes were electroporated with 50 µg of EGFP-IRAP/WT (B) or 50 µg of EGFP-IRAP/AA76,77 (C) plasmid DNA. Beginning at 9.5 h following transfection, the cells were serum-starved for 2 h, followed by treatment without (lanes 1, 3, and 5) or with (lanes 2, 4, and 6) 100 nM insulin for 30 min. The cells in A-C were then collected, lysed, and subjected to differential centrifugation as described under "Experimental Procedures." D, the membrane fractions collected above were subjected to Western blotting for the detection of endogenous GLUT4 protein. These are representative immunoblots from four independent experiments.

 


Figure 6
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  		FIGURE 6.
Alanine-scanning mutagenesis of residues flanking LL76,77. A, differentiated 3T3L1 adipocytes were electroporated with 50 µg of EGFP-IRAP/WT cDNA or with mutant constructs carrying the indicated triplet amino acids substituted with alanine. The cells were treated with or without insulin 12 h following transfection. B, differentiated 3T3L1 adipocytes were electroporated with 50 µg of EGFP-IRAP/WT cDNA or the indicated mutant and 100 µg of dsRed-VHS-GAT. The cells were treated with or without insulin 12 h following transfection. A diagram of IRAP reporter constructs illustrating the relevant amino acids is shown above. The data are from counting 50 cells/experiment (means ± S.E.) from three to five independent experiments.

 


Figure 7
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  		FIGURE 7.
IRAP/WT and IRAP/AA76,77 undergo distinct compartmentalization processes within tubulovesicular structures in the region of the Golgi ribbon. A, differentiated 3T3L1 adipocytes were fixed and labeled with mouse anti-syntaxin 6 monoclonal antibody followed by Alexa 488 secondary antibody (panel a) and sheep anti-TGN38 polyclonal antibody followed by Alexa 594 secondary (panel b). The merged images are shown in panel c, and the zoomed image is in panel d. B, differentiated adipocytes were electroporated with 50 µg of either EGFP-IRAP/WT (panels a-d), EGFP-IRAP/AA76,77 (panels e-h), or EGFP-IRAP/AA53,54 (panels i-l) cDNAs, and 12 h later the cells were fixed and labeled with anti-syntaxin 6 monoclonal antibody followed by Alexa 594 secondary (panels a, e, and i). The merged images are shown in panels c, g, and k. The zoomed images are shown in panels d, h, and l. C, differentiated adipocytes were electroporated with 50 µg of HA-syntaxin 3 and EGFP-IRAP/WT (panels a-d) or HA-syntaxin 3 and EGFP-IRAP/AA76,77 (panels e-h) cDNAs, and 12 h later the cells were fixed and labeled with anti-HA monoclonal antibody followed by Alexa 594 secondary (panels a and e). The merged images are shown in panels c and g. The zoomed images are shown in panels d and h. Bar, 5 µm. These are representative images from two or three independent experiments. D, differentiated 3T3L1 adipocytes were electroporated with 1 nmol of GGA1 siRNA 3 or with scrambled siRNA. Forty-eight h later, the cells were transfected again with EGFP-IRAP/WT reporter (panels b and f), and 12 h later the cells were fixed and labeled with the syntaxin 6 monoclonal antibody followed by Alexa 594 secondary antibody (panels a and e). The merged images are shown in panels c and g, and the zoomed images in panels d and h. Bar, 5 µm.

 
The above identification of LL76,77 as an essential motif in the trafficking of IRAP to the IRC was based upon the localization of IRAP by confocal fluorescent microscopy. To assure that these apparent trafficking differences could be confirmed biochemically, we next preformed subcellular fractionation of transfected EGFP-IRAP/WT and EGFP-IRAP/AA76,77 (Fig. 5). Subcellular fractionation of the EGFP-IRAP/WT protein 6 h following synthesis demonstrated a preponderance of protein in the LSP fraction compared with the HSP and PM fractions (Fig. 5A, lanes 1, 3, and 5). Insulin stimulation resulted in a small reduction of EGFP-IRAP/WT in the LSP and HSP fractions concomitant with a small increase in the PM fraction (Fig. 5A, lanes 2, 4, and 6). The small insulin-stimulated increase at the plasma membrane by subcellular fractionation following 6 h of expression is consistent with the relatively small amount of insulin-stimulated translocation observed by confocal fluorescent microscopy. Consistent with the time-dependent acquisition of insulin responsiveness, 12 h following expression the EGFP-IRAP/WT protein displayed a robust extent of plasma membrane translocation with a marked decrease in the HSP fraction and increase in the PM fraction (Fig. 5B, lanes 1-6). In contrast, 12 h following expression, the EGFP-IRAP/AA76,77 protein was predominantly found in the LSP and PM fractions with reduced amounts in the HSP in the basal state (Fig. 5C, lanes 1, 3, and 5). Moreover, insulin was unable to decrease the amount of EGFP-IRAP/AA76,77 protein in the HSP fraction or to induce an increase in the PM fraction (Fig. 5C, lanes 2, 4, and 6). As a control, the endogenous GLUT4 protein displayed the typical insulin-stimulated translocation and redistributed from the HSP fraction to the PM fraction (Fig. 5D). Thus, these data confirm the confocal microscopy results and demonstrate that following biosynthesis the IRAP protein slowly acquires insulin responsiveness and that the IRAP/AA76,77 mutant undergoes a default transport to the plasma membrane that is also unresponsive to insulin.

Dileucine-based sorting signals are present in many proteins, and two general categories have been recognized, defined by the consensus (D/E)XXXL(LI) and DXXLL sequences (where X is any amino acid) (3). However, the IRAP functional LL76,77 motif does not fall within either of these contexts. Moreover, we have not been able to detect a direct binding interaction between GGA and IRAP (data not shown). Thus, in an effort to delineate the functional context of LL76,77, we made a series of alanine substitutions both upstream and downstream of LL76,77 (Fig. 6). Surprisingly, alanine substitution of three amino acid groups at a time for a total of 9 amino acids amino-terminal and 9 amino acids carboxyl-terminal to LL76,77 had no significant effect on basal state perinuclear sequestration or insulin-stimulated translocation (Fig. 6A). As expected, the insulin-stimulated translocation of EGFP-IRAP/WT was potently inhibited by coexpression of VHS-GAT (Fig. 6B). In addition, coexpression of VHS-GAT potently inhibited insulin-stimulated translocation of all the other constructs carrying alanine point mutations flanking LL76,77 (Fig. 6B). Although we consistently observed a modest impairment of insulin-stimulated translocation of one of the mutants (SAK-AAA), this construct was nevertheless efficiently sequestered in the basal state, and coexpression of VHS-GAT potently inhibited insulin-stimulated translocation of this construct (Fig. 6).

EGFP-IRAP/WT and EGFP-IRAP/AA76,77 Traffic through Morphologically Distinct Tubulovesicular Compartments in the Perinuclear Region of Adipocytes—Taken together, the data presented above suggest that EGFP-IRAP/WT and EGFP-IRAP/AA76,77 undergo separate sorting events at the level of the Golgi and most likely the TGN. To further characterize these differential sorting processes, we examined the perinuclear compartmentalization of EGFP-IRAP/WT, EGFP-IRAP/AA76,77, and EGFP-IRAP/AA53,54 by confocal immunofluorescence microscopy (Fig. 7). Although light microscopy is limited in resolving power relative to electron microscopy, it has nevertheless been used successfully to study differential sorting events at the TGN (62). Emerging evidence indicates that the TGN is functionally subdivided into discrete domains with regional specializations (8). Consistent with this model, we observed that two TGN resident proteins, syntaxin 6 and TGN38, showed distinct, nonoverlapping distributions in the perinuclear region of adipocytes (Fig. 7A). Previous studies have interpreted this separation between syntaxin 6 and TGN38 as evidence for distinct subcompartments within the TGN (66). In any case, the expressed EGFP-IRAP/WT protein displayed substantial overlap with the endogenous syntaxin 6 protein (Fig. 7B, panels a-d) but had very little colocalization with TGN38 (data not shown). In contrast, the EGFP-IRAP/AA76,77 dileucine mutant had only minimal overlap with syntaxin 6 (Fig. 7B, panels e-h), whereas IRAP/AA53,54 overlapped substantially with syntaxin 6 (Fig. 7B, panels i-l). Because the EGFP-IRAP/AA76,77 mutant rapidly defaulted to the plasma membrane following biosynthesis and did not colocalize with syntaxin 6, we next compared the Golgi compartmentalization of the EGFP-IRAP constructs relative to the constitutive exocytic HA-syntaxin 3 reporter (Fig. 7C). Although the EGFP-IRAP/WT construct showed only slight colocalization with HA-syntaxin 3 (Fig. 7C, panels a-d), the IRAP/AA76,77 construct had substantial overlap with HA-syntaxin 3 (Fig. 7C, panels e-h). It is clearly technically impossible to define the precise carrier compartments occupied by EGFP-IRAP/WT and EGFP-IRAP/AA76,77 by light microscopy approaches. Nevertheless, the observation that these two reporters are present in distinct perinuclear compartments complements the functional results, demonstrating differential insulin-responsive compartmentalization for EGFP-IRAP/WT and EGFP-IRAP/AA76,77.

We next examined the localization of the EGFP-IRAP/WT reporter in GGA1 knockdown cells and control siRNA cells (Fig. 7D). As expected, in control (scrambled) siRNA transfected cells, the EGFP-IRAP/WT reporter colocalized substantially with syntaxin 6 (Fig. 7D, panels a-d). In GGA1 knock-down cells, the IRAP reporter also displayed a similar degree of colocalization with syntaxin 6 (Fig. 7D, panels e-h). These data suggest that the IRC may be closely apposed or is a specialized subdomain of the TGN itself that is not sufficiently separate to allow resolution at the light microscopy level.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
GLUT4 and IRAP undergo subcellular compartmentalization and insulin-stimulated translocation properties that are essentially indistinguishable from each other (43-47, 63). Unlike GLUT4, which has 12 transmembrane domains, IRAP is a type II transmembrane protein with one linear cytoplasmic region (49). IRAP is therefore an excellent model protein for elucidating the molecular mechanisms responsible for targeting cargo molecules into the insulin responsive GLUT4 storage compartment. Indeed, we show here that a dileucine motif at position 76,77 (LL76,77) functions at the level of the perinuclear Golgi region to sort newly synthesized IRAP into the IRC. Previous work showed that LL76,77 is a dual function motif that operates both at the cell surface to allow efficient endocytosis of IRAP and also at the level of endosomes to promote the dynamic retention of IRAP within intracellular compartments (53). In these studies, chimeras consisting of the extracellular domain of the human transferrin receptor and the cytosolic domain of IRAP were used to study the internalization of IRAP from the cell surface and its subsequent endosomal routing. In contrast, the present study has focused on identifying the intrinsic sorting motifs critical for targeting the newly synthesized IRAP protein from the perinuclear Golgi region directly into the insulin-responsive GLUT4 storage compartment.

Dileucine-based sorting signals fall into two described categories, defined by the consensus (D/E)XXXL(L/I) and DXXLL sequences, where X is any amino acid (3). Through interactions with subunits of the AP1 and AP3 heterotetrameric clathrin adaptor complexes, (D/E)XXXL(L/I) motifs target proteins to endosomes and lysosomes. In contrast, DXXLL signals interact with the GGA monomeric clathrin adaptors and sort transmembrane proteins such as the mannose-6-phosphate receptor from the TGN to endosomes (15). The 109-amino acid cytosolic region of IRAP contains two dileucine motifs, LL53,54 and LL76,77. Interestingly, the IRAP LL53,54 appears to be a variant of the (D/E)XXXL(L/I) type motif with the acidic Asp or Glu residue substituted for a basic arginine residue (RXXXLL). Moreover, GLUT4 also contains a RXXXLL sequence, and in this context the arginine residue has been suggested to play an important role in routing endocytosed GLUT4 from endosomal compartments to the insulin-responsive storage compartment (64). However, we found that mutation of LL53,54 to AA53,54 resulted in no observable differences in the trafficking and subsequent insulin-responsive compartmentalization of the newly synthesized IRAP protein. In contrast, mutation of the LL76,77 residues to AA76,77 resulted in the rapid default of the reporter to the cell surface, as determined both by confocal immunofluorescence microscopy and subcellular fractionation of adipocytes. We showed previously that newly made IRAP traffics directly from the Golgi complex to the insulin-responsive storage compartment, without first traveling to the cell surface (60). Thus, our current observation that the IRAP/AA76,77 construct rapidly defaulted to the cell surface during the 3-6-h time frame immediately following expression is consistent with the LL76,77 dileucine playing a key sorting role at the level of the Golgi complex during the compartmentalization of IRAP within the insulin-responsive storage depot.

Several adaptor complexes function at the TGN to sort transmembrane cargo molecules into carrier compartments (7, 9). For example, the heterotetrameric adaptors AP1 and AP3 mediate transmembrane cargo selection at the level of the TGN and/or endosomes (3). Moreover, the GGA proteins are a relatively new family of monomeric TGN clathrin adaptors that mediate the sorting of cargo proteins destined for endosomes. Although GGAs were initially shown to function specifically during the entry of newly synthesized GLUT4 into the IRC (17), additional work has suggested that GGAs may also function during the recycling of GLUT4 from the cell surface back into the IRC (65). Consistent with the former observations, we demonstrate here that siRNA-mediated knockdown of GGA1 blocks the initial sorting of IRAP into the IRC but does not impair the insulin-stimulated exit of IRAP out of this compartment. Moreover, the dominant interfering VHS-GAT domains of GGA potently inhibited insulin-stimulated IRAP translocation but had no effect on the constitutive exocytotic marker Stx3. This differential requirement for GGA function allowed us to distinguish between proteins trafficking to the IRC versus those undergoing alternative Golgi/TGN sorting pathways. Indeed, the observation that the cell surface localization of IRAP/AA76,77 was not inhibited by VHS-GAT coexpression strongly suggests that the LL76,77 motif operates at the TGN during the sorting of IRAP into the IRC.

The LL76,77 motif is embedded in a distinct sequence, 67EEDYESSAKLLGMSFMNRSS86, that is not easily categorized into established consensus dileucine-type targeting motifs (3). This could reflect the fact that IRAP appears to interact indirectly through an as yet unidentified binding partner with the GGA sorting machinery. Importantly, alanine substitution of the nine upstream or downstream residues surrounding LL76,77 did not disrupt normal IRAP trafficking or insulin responsiveness. These data suggest that the immediate context surrounding the LL76,77 motif is not the critical determinant for function. This begs the question why is LL53,54 unable to convey the same trafficking information. One possibility is that the linear sequence position with respect to the transmembrane is a critical determinant. Alternatively, the sequence context surrounding LL53,54 may provide a negative signal that impairs function. Although these two possibilities are not necessarily mutually exclusive, further mutational analyses will be required to address this issue.

We observed that two TGN resident proteins, syntaxin 6 and TGN38, showed distinct, nonoverlapping distributions in the perinuclear region of adipocytes. In addition, the IRAP/WT and IRAP/AA53,54 constructs colocalized quite well with the TGN resident protein syntaxin 6 but rather poorly with the HA-syntaxin 3 constitutive exocytotic marker. These findings are consistent with the observation that GLUT4 appears to more closely colocalize with syntaxin 6 than with TGN38 (66). In contrast, the IRAP/AA76,77 reporter showed strong overlap with HA-syntaxin 3, but very weak overlap with syntaxin 6. Although light microscopy is limited in its resolving power, the fact that these reporter molecules show clear separation in their trafficking properties relative to TGN marker proteins is consistent with multi-domain models of the TGN (8). However, although the TGN has traditionally been viewed as the key-sorting compartment from which transmembrane proteins are targeted to post-Golgi destinations, some evidence also supports the view that vesicular traffic can exit from trans-cisternae of the Golgi stack as well (67). According to this hypothesis, proteins are sorted into specific trans-cisternae, which in turn provide distinct exit routes for various trafficking destinations. Thus, although our data indicate a pivotal role for LL76,77 in the sorting of IRAP into the IRC, the precise subcompartment of the Golgi apparatus in which the LL76,77 targeting information is deciphered remains an open question.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants DK33823 and DK55811 (to J. E. P.) and the American Cancer Society Grant PF-03-133-01-TBE (to R. T. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 631-444-3059; Fax: 631-444-9749; E-mail: Watson{at}pharm.stonybrook.edu.

2 The abbreviations used are: TGN, trans-Golgi network; IRAP, insulin-responsive aminopeptidase; IRC, insulin-responsive compartment; WT, wild type; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; HA, hemagglutinin; siRNA, small interfering RNA; HSP, high speed pellet; LSP, low speed pellet; PM, plasma membrane; Stx3, syntaxin 3. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Timothy McGraw for the generous gift of IRAP-TR constructs and for helpful comments on the manuscript.



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R. T. Watson and J. E. Pessin
Recycling of IRAP from the plasma membrane back to the insulin-responsive compartment requires the Q-SNARE syntaxin 6 but not the GGA clathrin adaptors
J. Cell Sci., April 15, 2008; 121(8): 1243 - 1251.
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