**2.1 Modulation of EAAC1-mediated glutamate uptake by addicsin**

As an introduction to addicsin/GTRAP3-18-mediated regulation of EAAC1 activity, we discuss two early papers in detail. Lin et al. demonstrated that addicsin/GTRAP3-18 binds to EAAC1 and inhibits EAAC1-mediated glutamate uptake by this direct interaction (Lin et al., 2001). The second is our study showing that addicsin inhibits EAAC1-mediated glutamate uptake in a PKC activity-dependent manner while Arl6ip1 promotes glutamate uptake (also in a PKC activity-dependent manner) by inhibiting the interaction of addicsin with EAAC1 (Akiduki & Ikemoto, 2008). Lin et al. first identified addicsin/GTRAP3-18 as an EAAC1-interacting protein by yeast two-hybrid screening of a rat brain cDNA library. To evaluate whether addicsin/GTRAP3-18 modulates EAAC1 function, they examined the effect of increasing addicsin/GTRAP3-18 expression on EAAC1-mediated glutamate uptake *in vitro* and *in vivo*. First, they showed that glutamate uptake decreased progressively with increasing expression of addicsin/GTRAP3-18 in HEK293 cells. Subsequent kinetic analyses in HEK293, C6BU-1, and COS7 cells revealed that elevated expression of addicsin decreased the glutamate affinity of EAAC1 without altering the maximal transport velocity (correlated with expression). Furthermore, HEK293 cells coexpressing addicsin/GTRAP3-18 and a truncated EAAC1 missing the addicsin/GTRAP3-18 association region showed higher glutamate uptake than cells expressing wild-type EAAC1. In addition, this truncated EAAC1 had a higher affinity for glutamate, suggesting that addicsin/GTRAP3-18 normally reduces EAAC1-mediated glutamate uptake by binding to this association region and reducing transporter glutamate affinity. Next, they evaluated the effect of intraventricular injection of an addicsin/GTRAP3-18 antisense mRNA on EAAC1-mediated glutamate uptake *in vivo*. The antisense treatment resulted in reduced addicsin/GTRAP3-18 expression, a significant increase in cortical EAAC1-mediated glutamate uptake, and an increase in glutamate affinity compared to saline-treated or sense mRNA-treated control animals. In conclusion, addicsin/GTRAP3-18 can negatively modulate EAAC1-mediated glutamate uptake by a direct interaction with EAAC1.

We first isolated addicsin as a novel protein richly expressed in the amygdala of mice under chronic morphine treatment. Addicsin has a tendency to form the multimeric complex *in vitro* (Ikemoto et al., 2002; Lin et al., 2001). The initial discovery of addicsin prompted us to perform yeast two-hybrid screening of an amygdala cDNA library constructed from chronic morphine-administered mice. From this screen, we identified Arl6ip1 as a candidate addicsin-interacting protein. As described in section 1.3, Arl6ip1 is an anti-apoptotic protein located in the ER. As previously described, addicsin inhibits EAAC1-mediated glutamate uptake by direct association at the plasma membrane (Lin et al., 2001), so we speculated that Arl6ip1 upregulates EAAC1-mediated glutamate transport by inhibiting the interaction between addicsin and EAAC1 (Fig. 3).

As a first step to verify this hypothesis, we investigated whether addicsin could bind Arl6ip1 *in vitro* and *in vivo*. To eliminate the possibility of false-positive clones, reconfirmation tests using a full length mouse Arl6ip1 as prey or bait were performed. This tests revealed the specific interaction with addicsin in the yeast AH109 strain. We next examined the reproducibility of this screening result by yeast two-hybrid screening using a different cDNA library prepared from whole brains of 7-week-old mice. We obtained 20 positive clones that clearly displayed α-galactosidase activity (the gene driven by the protein–protein interaction in the two-hybrid screen). Among these positive clones, 11 were identical to *Arl6ip1* cDNA (M.J. Ikemoto et al., unpublished data), confirming the interaction with addicsin and Arl6ip1 in the yeast AH109 strain. We then performed immunoprecipitation analysis, glycerol gradient analysis, and immunocytochemical analysis to directly test the interaction between Arl6ip1 and addicsin *in vitro*. For this purpose, we prepared cell lysates from NG108-15 cells expressing FLAG-tagged Arl6ip1 (Arl6ip1-FLAG), Myc-tagged addicsin (addicsin-myc), or both. Immunoprecipitation analysis of these cell lysates demonstrated that Arl6ip1-FLAG specifically interacted with addicsin-myc in the cell lysates prepared from coexpressing cells, but not from cells expressing Arl6ip1-FLAG or addicsin-myc alone. Glycerol gradient analysis revealed that the elution profile of Arl6ip1-FLAG was similar to that of addicsin-myc. The elution peaks of both proteins were observed in the fraction with a deduced molecular mass of 24 kDa. Moreover, the elution peak of the addicsin homodimer was present in the 44-kDa fraction,

increasing expression of addicsin/GTRAP3-18 in HEK293 cells. Subsequent kinetic analyses in HEK293, C6BU-1, and COS7 cells revealed that elevated expression of addicsin decreased the glutamate affinity of EAAC1 without altering the maximal transport velocity (correlated with expression). Furthermore, HEK293 cells coexpressing addicsin/GTRAP3-18 and a truncated EAAC1 missing the addicsin/GTRAP3-18 association region showed higher glutamate uptake than cells expressing wild-type EAAC1. In addition, this truncated EAAC1 had a higher affinity for glutamate, suggesting that addicsin/GTRAP3-18 normally reduces EAAC1-mediated glutamate uptake by binding to this association region and reducing transporter glutamate affinity. Next, they evaluated the effect of intraventricular injection of an addicsin/GTRAP3-18 antisense mRNA on EAAC1-mediated glutamate uptake *in vivo*. The antisense treatment resulted in reduced addicsin/GTRAP3-18 expression, a significant increase in cortical EAAC1-mediated glutamate uptake, and an increase in glutamate affinity compared to saline-treated or sense mRNA-treated control animals. In conclusion, addicsin/GTRAP3-18 can negatively modulate EAAC1-mediated

We first isolated addicsin as a novel protein richly expressed in the amygdala of mice under chronic morphine treatment. Addicsin has a tendency to form the multimeric complex *in vitro* (Ikemoto et al., 2002; Lin et al., 2001). The initial discovery of addicsin prompted us to perform yeast two-hybrid screening of an amygdala cDNA library constructed from chronic morphine-administered mice. From this screen, we identified Arl6ip1 as a candidate addicsin-interacting protein. As described in section 1.3, Arl6ip1 is an anti-apoptotic protein located in the ER. As previously described, addicsin inhibits EAAC1-mediated glutamate uptake by direct association at the plasma membrane (Lin et al., 2001), so we speculated that Arl6ip1 upregulates EAAC1-mediated glutamate transport by inhibiting the interaction

As a first step to verify this hypothesis, we investigated whether addicsin could bind Arl6ip1 *in vitro* and *in vivo*. To eliminate the possibility of false-positive clones, reconfirmation tests using a full length mouse Arl6ip1 as prey or bait were performed. This tests revealed the specific interaction with addicsin in the yeast AH109 strain. We next examined the reproducibility of this screening result by yeast two-hybrid screening using a different cDNA library prepared from whole brains of 7-week-old mice. We obtained 20 positive clones that clearly displayed α-galactosidase activity (the gene driven by the protein–protein interaction in the two-hybrid screen). Among these positive clones, 11 were identical to *Arl6ip1* cDNA (M.J. Ikemoto et al., unpublished data), confirming the interaction with addicsin and Arl6ip1 in the yeast AH109 strain. We then performed immunoprecipitation analysis, glycerol gradient analysis, and immunocytochemical analysis to directly test the interaction between Arl6ip1 and addicsin *in vitro*. For this purpose, we prepared cell lysates from NG108-15 cells expressing FLAG-tagged Arl6ip1 (Arl6ip1-FLAG), Myc-tagged addicsin (addicsin-myc), or both. Immunoprecipitation analysis of these cell lysates demonstrated that Arl6ip1-FLAG specifically interacted with addicsin-myc in the cell lysates prepared from coexpressing cells, but not from cells expressing Arl6ip1-FLAG or addicsin-myc alone. Glycerol gradient analysis revealed that the elution profile of Arl6ip1-FLAG was similar to that of addicsin-myc. The elution peaks of both proteins were observed in the fraction with a deduced molecular mass of 24 kDa. Moreover, the elution peak of the addicsin homodimer was present in the 44-kDa fraction,

glutamate uptake by a direct interaction with EAAC1.

between addicsin and EAAC1 (Fig. 3).

suggesting that addicsin forms Arl6ip1–addicsin heterodimers and addicsin–addicsin homodimers *in vitro*. Immunocytochemical analysis in NG108-15 cells overexpressing Arl6ip1-FLAG and addicsin-myc demonstrated subcellular colocalization (M.J. Ikemoto et al., unpublished data). To examine the interaction of both proteins *in vivo*, we performed *in vivo* immunoprecipitation assays of whole brain lysates using an anti-Arl6ip1 polyclonal antibody (generated from a synthetic peptide spanning amino acids 185–199 of mouse Arl6ip1) that again revealed a specific interaction between Arl6ip1 and addicsin. Western blot analysis demonstrated that Arl6ip1 was widely expressed in the mature brain and showed substantial regional overlap with addicsin. In addition, immunohistochemical staining confirmed that Arl6ip1 was widely expressed in the mature brain and localized in neuron-like cells. The neural expression pattern of Arl6ip1 was the same as addicsin, suggesting that Arl6ip1 is colocalized with addicsin in the mature CNS. We concluded that addicsin specifically interacted with Arl6ip1 *in vitro* and *in vivo*.

As a second step, we then determined the Arl6ip1- and addicsin-binding regions on addicsin. If Arl6ip1 does regulate EAAC1 activity by competitively binding to addicsin molecules and thus preventing the formation of addicsin homodimers that downregulate EAAC1 activity, the Arl6ip1- and addicsin-binding regions on addicsin should be located close enough for such a competitive interaction. Immunoprecipitation assays using several addicsin truncation mutants indicated that Arl6ip1 associated with full length addicsin (wt), a truncation lacking the C-terminal region at amino acids 145–188 (d1), a deletion mutant of the N-terminal domain at amino acids 1–102 (d2), and a mutant missing the region containing the C-terminal phosphorylation motif at amino acids 136–144 (d3). However, Arl6ip1 could not interact with a mutant lacking a portion of the hydrophobic region at amino acids 103–117 (d4). As expected, addicsin was able to associate with the wt, d1, d2, or d3 mutant, but not the d4 truncation mutant, indicating that the hydrophobic region at amino acids 103–117 of addicsin is a crucial domain for the formation of addicsin–addicsin homodimers and addicsin-Arl6ip1 heterodimers (Fig. 1). These results strongly support our hypothesis that Arl6ip1 antagonizes addicsin-mediated downregulation of EAAC1 activity by sequestering free addicsin.

As a third step, we investigated whether Arl6ip1 had a positive effect on EAAC1-mediated glutamate uptake. For this purpose, we selected C6BU-1 glioma cells that expressed EAAC1 as the principal or only EAAT (Palos et al., 1996). We created two stably expressing C6BU-1 cell lines, designated C6BU-1-pSw-addicsin and C6BU-1-pSw-Arl6ip1. In these cell lines, we could strictly control the expression levels of V5-tagged addicsin (addicsin-V5) or V5-tagged Arl6ip1 (Arl6ip1-V5) by exposure to 10 nM mifepristone (11β-[4-dimethylamino]phenyl-17β-hydroxy-17-[1-propynyl]estra-4,9-dien-3-one), a synthetic 19-norsteroid. In addition, a cell viability assay demonstrated that upregulation of Arl6ip1-V5 or addicsin-V5 by exposure to 10 nM mifepristone was not cytotoxic, making these cell lines excellent models to evaluate the effects of changing Arl6ip1 and addicsin expression on the functional activity of EAAC1. Compared to control cells untreated with mifepristone or the PKC agonist PMA, the upregulation of Arl6ip-V5 or addicsin-V5 by 10 nM mifepristone alone did not change EAAC1-mediated glutamate uptake. When these cells were stimulated with 100 nM PMA alone, the glutamate uptake activity in C6BU-1-pSw-addicsin cells and C6BU-1-pSw-Arl6ip1 cells increased about two-fold compared to untreated controls. EAAC1-mediated glutamate uptake was significantly lower in C6BU-1-pSw-addicsin cells stimulated with both mifepristone and PMA compared to C6BU-1-pSw-addicsin cells treated with PMA alone, indicating that activation of addicsin expression inhibited PKC-dependent EAAC1 activity. In contrast, C6BU-1-pSw-Arl6ip1 cells treated with PMA and mifepristone exhibited a threefold increase in glutamate uptake compared to the same line treated with PMA alone, indicating that Arl6ip1 overexpression enhanced PKC-dependent EAAC1 activity. On the other hand, the nonstimulating PMA analog 4α phorbol did not increase glutamate uptake relative to controls.

To further support these conclusions, we performed a knockdown experiment by transient transfection of double-stranded siRNAs into C6BU-1-pSw-Arl6ip1 cells to investigate the effect of decreased addicsin expression on EAAC1-mediated glutamate uptake. As expected, cells transfected with either of two alternative addicsin siRNAs showed about a two-fold increase in glutamate uptake in response to PMA exposure compared to cells treated with control scrambled siRNA. The elevated glutamate uptake concomitant with addicsin knockdown strongly supported the proposed mechanism for EAAC1 regulation by addicsin and Arl6ip1.

To investigate the molecular mechanisms for altered EAAC1-mediated glutamate uptake in C6BU-1-pSw-Arl6ip1 cells, we performed kinetic analysis of glutamate flux across C6BU-1 pSw-Arl6ip1 cell membranes. When Arl6ip1 was conditionally overexpressed using mifepristone, PMA treatment increased the glutamate affinity but not the maximal velocity compared to vehicle-treated controls (PMA: *Km* = 647 μM, *Vmax* = 1.5 × 103 pmole/mg/min; vehicle: *Km* = 824 μM, *Vmax* = 1.5 × 103 pmole/mg/min) with no change of addicsin expression levels. Thus, Arl6ip1 promoted EAAC1-mediated glutamate uptake by increasing the catalytic efficacy of EAAC1. Specifically, Arl6ip1 blocked the addicsinmediated reduction in EAAC1 glutamate affinity.

As a fourth step, we then examined the subcellular localization of Arl6ip1 in C6BU-1-pSw-Arl6ip1 cells. Western blot analysis revealed that Arl6ip1-V5 expression levels were unaffected by 100 nM PMA exposure. Immunocytochemical analysis demonstrated that Arl6ip1-V5 was predominantly localized to cytoplasmic structures such as the ER and that this subcellular expression pattern was not changed by PMA. Furthermore, cell biotinylation analysis indicated that Arl6ip1 did not interact with the plasma membrane, consistent with our previous result that Arl6ip1 failed to interact with EAAC1 by immunoprecipitation. Therefore, Arl6ip1 was localized to the ER under all conditions tested and acted to "trap" addicsin molecules in Arl6ip1–addicsin heterodimers, thus preventing the direct interaction of addicsin with EAAC1. To confirm our hypothesis, we produced an addicsin mutant that lacked interaction with Arl6ip1 but not with other addicsin molecules. Fine mutational analysis was used to separate the Arl6ip1- and addicsin-binding regions within the addicsin d4 region. We compared addicsin sequences among various species and noted that two amino acids at positions 110 and 112 of mouse addicsin were completely conserved from fruit fly to human. We created a double-mutated form of addicsin that substituted both the native tyrosine at amino acid 110 and the leucine at amino acid 112 with alanine. The mutant, designated addicsin Y110A/L112A (or addicsinYL), showed markedly less binding to Arl6ip1 (40% of wild-type addicsin) but normal wild-type binding to addicsin, as revealed by immunoprecipitation. In addition, a cell biotinylation assay indicated that addicsinYL was unable to localize to the plasma membrane, suggesting that addicsinYL lost EAAC1-binding activity. To evaluate the effect of addicsinYL on EAAC1-mediated

mifepristone and PMA compared to C6BU-1-pSw-addicsin cells treated with PMA alone, indicating that activation of addicsin expression inhibited PKC-dependent EAAC1 activity. In contrast, C6BU-1-pSw-Arl6ip1 cells treated with PMA and mifepristone exhibited a threefold increase in glutamate uptake compared to the same line treated with PMA alone, indicating that Arl6ip1 overexpression enhanced PKC-dependent EAAC1 activity. On the other hand, the nonstimulating PMA analog 4α phorbol did not increase glutamate uptake

To further support these conclusions, we performed a knockdown experiment by transient transfection of double-stranded siRNAs into C6BU-1-pSw-Arl6ip1 cells to investigate the effect of decreased addicsin expression on EAAC1-mediated glutamate uptake. As expected, cells transfected with either of two alternative addicsin siRNAs showed about a two-fold increase in glutamate uptake in response to PMA exposure compared to cells treated with control scrambled siRNA. The elevated glutamate uptake concomitant with addicsin knockdown strongly supported the proposed mechanism for

To investigate the molecular mechanisms for altered EAAC1-mediated glutamate uptake in C6BU-1-pSw-Arl6ip1 cells, we performed kinetic analysis of glutamate flux across C6BU-1 pSw-Arl6ip1 cell membranes. When Arl6ip1 was conditionally overexpressed using mifepristone, PMA treatment increased the glutamate affinity but not the maximal velocity compared to vehicle-treated controls (PMA: *Km* = 647 μM, *Vmax* = 1.5 × 103 pmole/mg/min; vehicle: *Km* = 824 μM, *Vmax* = 1.5 × 103 pmole/mg/min) with no change of addicsin expression levels. Thus, Arl6ip1 promoted EAAC1-mediated glutamate uptake by increasing the catalytic efficacy of EAAC1. Specifically, Arl6ip1 blocked the addicsin-

As a fourth step, we then examined the subcellular localization of Arl6ip1 in C6BU-1-pSw-Arl6ip1 cells. Western blot analysis revealed that Arl6ip1-V5 expression levels were unaffected by 100 nM PMA exposure. Immunocytochemical analysis demonstrated that Arl6ip1-V5 was predominantly localized to cytoplasmic structures such as the ER and that this subcellular expression pattern was not changed by PMA. Furthermore, cell biotinylation analysis indicated that Arl6ip1 did not interact with the plasma membrane, consistent with our previous result that Arl6ip1 failed to interact with EAAC1 by immunoprecipitation. Therefore, Arl6ip1 was localized to the ER under all conditions tested and acted to "trap" addicsin molecules in Arl6ip1–addicsin heterodimers, thus preventing the direct interaction of addicsin with EAAC1. To confirm our hypothesis, we produced an addicsin mutant that lacked interaction with Arl6ip1 but not with other addicsin molecules. Fine mutational analysis was used to separate the Arl6ip1- and addicsin-binding regions within the addicsin d4 region. We compared addicsin sequences among various species and noted that two amino acids at positions 110 and 112 of mouse addicsin were completely conserved from fruit fly to human. We created a double-mutated form of addicsin that substituted both the native tyrosine at amino acid 110 and the leucine at amino acid 112 with alanine. The mutant, designated addicsin Y110A/L112A (or addicsinYL), showed markedly less binding to Arl6ip1 (40% of wild-type addicsin) but normal wild-type binding to addicsin, as revealed by immunoprecipitation. In addition, a cell biotinylation assay indicated that addicsinYL was unable to localize to the plasma membrane, suggesting that addicsinYL lost EAAC1-binding activity. To evaluate the effect of addicsinYL on EAAC1-mediated

relative to controls.

EAAC1 regulation by addicsin and Arl6ip1.

mediated reduction in EAAC1 glutamate affinity.

glutamate uptake, we created a conditional C6BU-1 cell line, designated C6BU-1-pSwaddicsinYL. This cell line exhibited mifepristone-dependent upregulation of V5-tagged addicsinYL and increased glutamate uptake in response to PMA that was unchanged by mifepristone-induced upregulation of addicsinYL. That is, glutamate uptake was not reduced by induced addicsinYL expression. These data strongly suggest that addicsin is a key negative regulator of EAAC1 in the plasma membrane and that Arl6ip1 is a negative regulator of addicsin.

As a final step, we examined the effect of addicsin PKC phosphorylation sites on EAAC1 mediated glutamate uptake in C6BU-1 cells. Addicsin has putative PKC phosphorylation motifs at amino acids 18-20 and 138-140, and PKC activation increases EAAC1-mediated glutamate uptake. We established conditional C6BU-1 cell lines, designated C6BU-1-pSwaddicsinS18A and C6BU-1-pSw-addicsinS138A. C6BU-1-pSw-addicsinS18A cells expressed a V5-tagged addicsin point mutant that substituted native serine 18 for alanine in the Nterminal motif in response to mifepristone, while C6BU-1-pSw-addicsinS138A cells expressed a V5-tagged addicsin point mutant that substituted native serine 138 for alanine in the C-terminal motif. These cells showed no cytotoxicity in response to 10 nM mifepristone. In contrast to cells expressing wild-type addicsin, expression of addicsinS18A did not suppress the PMA-induced increase in EAAC1-mediated glutamate uptake. Moreover, increased expression of addicsinS18A caused a significant increase in glutamate uptake even without PMA stimulation by a dominant negative effect. Similarly, addicsinS138A expression did not suppress the PMA-induced increase in EAAC1-mediated glutamate uptake. Thus, these mutations abolished the inhibitory effect of addicsin. However, in contrast to addicsinS18A, addicsinS138A expression had no influence on EAAC1-mediated glutamate uptake activity in the absence of PMA stimulation. Both serine 18 and serine 138 within the putative PKC phosphorylation motifs are critical for the negative regulation of EAAC1-mediated glutamate uptake and suggest that the PKC phosphorylation site at serine 138 is functional under physiological conditions.

Based on these data, we proposed the regulatory model of EAAC1-mediated glutamate uptake illustrated in Fig. 3. If addicsin expression is high enough relative to Arl6ip1 to form many more addicsin homodimers than addicsin–Arl6ip1 heterodimers, EAAC1-mediated glutamate uptake is reduced. Furthermore, activation of the PKC isozyme that phosphorylates addicsin at S18 or S138 may further potentiate this negative regulation. On the other hand, if addicsin expression is low enough or Arl6ip1 expression high enough that formation of heterodimers predominates, fewer addicsin homodimers are available to suppress EAAC1 activity. The resulting decrease in addicsin–EAAC1 binding will enhance the catalytic efficacy of EAAC1, in a PKC-activity dependent manner. In sum, Arl6ip1 acts as a positive regulator of EAAC1-mediated glutamate uptake (Fig. 3) and may therefore possess significant neuroprotective efficacy against neurodegenerative diseases linked to excitotoxicity and oxidative stress.

#### **2.2 Modulation of ER protein trafficking by addicsin**

Addicsin is a member of the PRAF protein family with homology to PRA1 and PRAF2 (JM4) (Schweneker et al., 2005). PRA1 is associated with the Golgi membrane and interacts with Rab, a member of the Ras superfamily of small GTP-binding proteins, which regulates intracellular protein trafficking (Bucci et al., 1999; Liang & Li, 2000; Martincic et al., 1997). Immunocytochemical studies reveal that mature addicsin is present in both the plasma membrane and the intracellular compartment, including the ER (Ikemoto et al., 2002; Watabe et al., 2007, 2008). Thus, addicsin may also be involved in intracellular protein trafficking. To investigate this possibility, we examined EAAC1 oligosaccharide residues under conditions of varying addicsin expression. The oligosaccharide residues on EAAC1 are an excellent indicator of the extent of ER-to-Golgi trafficking and plasma membrane localization because the newly synthesized EAAC1 is *N*-glycosylated with high mannose oligosaccharide chains that are subsequently processed into more complex sugar chains by resident Golgi enzymes (Yang & Kilberg, 2002). In HEK293T cells coexpressing addicsin, EAAC1 is predominantly modified by high mannose oligosaccharides, suggesting that EAAC1 proteins are largely confined to the ER. Furthermore, addicsin delays oligosaccharide maturation of EAAC1 but does not induce EAAC1 degradation (Ruggiero et al., 2008). These data suggest that addicsin delays ER-to-Golgi trafficking of EAAC1. Moreover, addicsin inhibits ER-to-Golgi trafficking of dopamine transporter, GABA transporter 1, and several G-protein-coupled receptors, including β2-adrenergic receptor, α1 β receptor, and D2 receptor (Ruggiero et al., 2008). Furthermore, addicsin inhibits the function of RTN2B, a member of the reticulon protein family localized in the ER, which enhances ER-to-Golgi trafficking of EAAC1 (Liu et al., 2008). As addicsin, RTN2B, and EAAC1 are coexpressed in neurons, they may interact in one complex. Indeed, addicsin and EAAC1 can interact with RTN2B by binding to different regions of the protein. In addition, coexpression of RTN2B and EAAC1 in HEK293 cells increases EAAC1 cell surface expression, while increasing addicsin expression blocks this effect. Thus, EAAC1 trafficking is inhibited by addicsin and facilitated by RTN2B (Liu et al., 2008). Based on these data, Liu et al. proposed a model in which the regulation of ER trafficking governs the activity and density of EAAC1 at the plasma membrane. Under normal conditions, RTN2B facilitates EAAC1 trafficking from the ER because basal expression of addicsin is too low to have an inhibitory effect. Under stressful conditions, such as oxidative and chemical stress, addicsin expression is upregulated and the inhibitory effect on EAAC1 trafficking predominates over the facilitating effect of RTN2B (Liu et al., 2008). Addicsin can delay ER-to-Golgi trafficking of structurally and functionally distinct proteins in addition to EAAC1. Thus, addicsin is a stress-induced multifunctional protein that participates in various physiological and pathological functions by regulating ER trafficking of many membrane effector proteins, including receptors and transporters.
