RUTBC2 Protein, a Rab9A Effector and GTPase-activating Protein for Rab36*

Background: Rab GTPases control membrane traffic, and identification of Rab regulators is incomplete. Results: RUTBC2 binds Rab9A, enhances GTP hydrolysis by Rab34 and Rab36 in vitro, and is a GAP for Rab36 in cells. Conclusion: These data suggest a connection between Rab9A and Rab36 as part of a cascade of membrane traffic steps. Significance: This represents the first assignment of a molecular function for RUTBC2. Rab GTPases regulate vesicle budding, motility, docking, and fusion. In cells, their cycling between active, GTP-bound states and inactive, GDP-bound states is regulated by the action of opposing enzymes called guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). The substrates for most RabGAPs are unknown, and the potential for cross-talk between different membrane trafficking pathways remains uncharted territory. Rab9A and its effectors regulate recycling of mannose 6-phosphate receptors from late endosomes to the trans Golgi network. We show here that RUTBC2 is a TBC domain-containing protein that binds to Rab9A specifically both in vitro and in cultured cells but is not a GAP for Rab9A. Biochemical screening of Rab protein substrates for RUTBC2 revealed highest GAP activity toward Rab34 and Rab36. In cells, membrane-associated RUTBC2 co-localizes with Rab36, and expression of wild type RUTBC2, but not the catalytically inactive, RUTBC2 R829A mutant, decreases the amount of membrane-associated Rab36 protein. These data show that RUTBC2 can act as a Rab36 GAP in cells and suggest that RUTBC2 links Rab9A function to Rab36 function in the endosomal system.

highly related Rab34 and Rab36 proteins. In cells, Rab36 is a bona fide substrate for RUTBC2.

EXPERIMENTAL PROCEDURES
Yeast Two-hybrid Analysis-Yeast two-hybrid analysis was carried out as described previously (14). Briefly, 56 mutant Rab proteins deficient for GTP hydrolysis (Gln to Ala) were cloned into pGBT9 bait vector (Clontech). RUTBC2 was amplified from cDNA libraries and was cloned into pACT2 prey vector (Clontech); growth on selective medium indicated an interaction between a Rab and RUTBC2.
Plasmids-For mammalian expression, N-terminally 3ϫMyctagged RUTBC2 was obtained by amplification from a cDNA library and ligation into a modified version of pCDNA3.1(ϩ) (Invitrogen) (14). This construct encodes the shortest of four isoforms found in GenBank TM and is missing the first 25 amino acids at the N terminus. GFP-RUTBC2 was constructed by amplification of this isoform with the addition of the missing N terminus by PCR and ligated into pEGFP-C3 (Clontech). The predicted RUTBC2 GAP-inactive mutant (R829A) was created using QuikChange (Stratagene).
Rab proteins tested for GAP activity were previously described (21). Phosphate-binding protein (PBP) from Escherichia coli was amplified by PCR from bacteria and cloned into modified pET15. His-PBP A197C was constructed by site-directed mutagenesis.
Protein Expression and Purification-Either His-RUTBC2-N or His-RUTBC2-C was transformed into Rosetta2 (DE3) cells (Novagen) and grown at 37°C until A 600 ϭ 0.5. The culture was induced with 0.4 mM isopropyl ␤-D-thiogalactoside and grown for an additional 4 h at 22°C. Cell pellets were resuspended in lysis buffer (25 mM HEPES, pH 7.4, 300 mM NaCl, 50 mM imidazole) supplemented with 1 mM PMSF and lysed by two passes at 20,000 p.s.i. through an EmulsiFlex-C5 apparatus (Avestin). Cleared lysates (20,000 rpm for 45 min at 4°C in a JA-20 rotor; Beckman Coulter) were incubated with nickel-nitrilotriacetic acid (Qiagen) for 1 h at 4°C. The resin was then washed with lysis buffer and eluted with 25 mM HEPES, pH 7.4, 300 mM NaCl, and 250 mM imidazole. Fractions containing the required protein were pooled and concentrated using an Amicon Ultra concentrator (Millipore). The sample was dialyzed to remove imidazole and then brought to 10% (v/v) glycerol. The sample was aliquoted, snap-frozen in liquid nitrogen, and stored at Ϫ80°C. GST-RUTBC2, GST-RUTBC2-N, and GST-RUTBC2-C were each transformed into Rosetta2 (DE3) cells and grown at 37°C until A 600 ϭ 0.6. The cells were induced with 100 M isopropyl ␤-D-thiogalactoside and grown for an additional 4.5 h at 30°C. Cell pellets were resuspended in lysis buffer (50 mM HEPES, pH 7.4, 250 mM NaCl, 1 mM DTT, 1 mM EDTA) supplemented with 1 mM PMSF and lysed by two passes at 20,000 p.s.i. through an EmulsiFlex-C5. Cleared lysates (19,000 rpm for 30 min at 4°C in a JA-20 rotor; Beckman Coulter) were incubated with glutathione-Sepharose Fast Flow (GE Healthcare) for 1.5 h at 4°C. The resin was then washed with 25 column volumes lysis buffer and eluted with 50 mM Tris-HCl, pH 8.0, 250 mM NaCl, and 20 mM glutathione. Fractions containing RUTBC2-C were pooled and concentrated using an Amicon Ultra concentrator (Millipore). The sample was dialyzed to remove glutathione and brought to 10%(v/v) glycerol, snapfrozen in liquid nitrogen, and stored at Ϫ80°C.
Antibodies-Mouse monoclonal anti-Myc (9E10), mouse monoclonal anti-Rab9A, mouse monoclonal anti-CI-MPR (2G11), and rabbit anti-CI-MPR were all previously described (23). Rabbit anti-GFP antibody was from Invitrogen; mouse anti-GFP antibody was from Roche Applied Science. Rabbit anti-RUTBC2 was purchased from Sigma. Anti-Penta-His antibody was from Qiagen. Chicken anti-Myc antibody was from Bethyl Laboratories. HRP-conjugated goat anti-mouse and goat anti-rabbit secondary antibodies as well as protein-A-HRP were from Bio-Rad.
Binding Assays-Constructs encoding 3ϫMyc-RUTBC2 were translated in vitro using a TNT quick coupled transcription/translation system (Promega) following the manufacturer's protocol. GST-tagged Rabs were loaded with GTP␥S or GDP as described (15) and mixed with TNT lysate for 1.5 h at 25°C in binding buffer (25 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 5 mM MgCl 2 , 1 mM DTT, 0.1 mM GTP␥S). RUTBC2 constructs bound to GST-Rabs were isolated using glutathione-Sepharose, washed in binding buffer (with 400 mM NaCl), and then eluted by the addition of 25 mM glutathione and analyzed by immunoblot. For binding described for Fig. 2A, 1 M His-Rab2 and His-Rab9A was preloaded with GTP␥S and incubated with 2.5 M GST or full-length GST-RUTBC2 for 90 min at room temperature in binding buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM MgCl 2 , 0.1 mg/ml BSA). Glutathione-Sepharose was added and incubated for 30 min at room temperature, and bound protein complexes were eluted by heating the sam-ples in 2ϫ Laemmli buffer and analyzed by immunoblotting with anti-His antibody. For binding described for Fig. 2, B and F, the Rab concentration was 5 M, and the GST or GST-RUTBC2 concentration was 2.5 M. Binding assays described for Fig. 2D using purified proteins were as described (16). For Fig. 2E, GST alone or GST-Rab9A Q66L (5 M) was mixed with His-RUTBC2-N or His-RUTBC2-C (2.5 M) and incubated for 1 h at 25°C in binding buffer (25 mm HEPES, pH 7.4, 150 mM KCl, 5 mM MgCl 2 , 0.1 mg/ml BSA). RUTBC2 constructs bound to GST and GST-Rab9A were isolated using glutathione-Sepharose, washed in binding buffer, eluted with 2ϫ Laemmli buffer, and analyzed by immunoblotting with anti-His antibody.
Cell Culture and Transfections-HeLa, HEK293T, SK-N-SH, and Vero cells were obtained from American Type Culture Collection and cultured at 37°C and 5% CO 2 in Dulbecco's modified Eagle's medium supplemented with 7.5% fetal calf serum, 100 units of penicillin, and 100 g/ml streptomycin. For overexpression studies, all cells were transfected using FuGENE 6 (Roche Applied Science). Cells were harvested either 24 h or 48 h after transfection as indicated. For siRNA treatment, HEK293T cells were transfected using Lipofectamine 2000 (Invitrogen). The siRNA against RUTBC2 targeted the sequence 5Ј-GCAGAUUGGAGGAGAAACA-3Ј and was purchased from Dharmacon.
GAP Assays-The procedure followed by Pan et al. (21) was used, except that phosphate released during the reaction was bound by His-PBP A197C labeled at position 197 with MDCC (24). Reactions were started by adding a solution containing GAP and MgCl 2 to one of MDCC-PBP and desalted, GTP-exchanged Rabs by a Precision 2000 liquid handling system (BioTek). All reactions contained 2 M Rab GTPase, 5 mM MgCl 2 , and 85 nM MDCC-PBP, whereas the concentration of His-RUTBC2-C was varied. Phosphate production was monitored continuously in a TECAN Sapphire microplate reader using an excitation wavelength of 425 nm and an emission wavelength of 455 nm.
Immunofluorescence Microscopy-HeLa cells were seeded on glass coverslips and transfected the following day with FuGENE 6 (Promega). 18 h after transfection, cells were washed twice in PBS followed by two washes with ice-cold glutamate buffer (25 mM HEPES-NaOH, pH 7.4, 25 mM KCl, 2.5 mM magnesium acetate, 5 mM EGTA, and 150 mM monopotassium glutamate). Cytosol leak was performed by incubating the coverslips for 5 s in liquid N 2 and thawing them at 20°C for 1 min. Coverslips were then washed twice in ice-cold glutamate buffer and fixed for 15 min in 3.7% paraformaldehyde in 200 mM HEPES-NaOH, pH 7.4. After fixation, cells were washed twice in PBS and permeabilized with 0.1% Triton X-100 in PBS for 3 min. Coverslips were washed twice in PBS, blocked with 1% BSA in PBS (blocking buffer) for 30 min, incubated with chicken anti-Myc antibody diluted in blocking buffer for 1 h at 20°C, washed, incubated with Alexa Fluor 555 or Alexa Fluor 594 (Invitrogen) secondary antibody diluted in blocking buffer for 1 h, and then washed and mounted using Mowiol (Polysciences). Imaging was performed with a Leica TCS SP2 SE confocal scanner in conjunction with a Leica DM6000 B upright scope (with attached Leica HCX PL apochromatic 63ϫ/NA 1.4 objective) and a Leica CTR 6000 confocal control box. This setup was controlled by Leica Control software (Leica Microsystems). Images were processed using ImageJ. The quantification of Rab36 puncta and fluorescence intensity as well as Rab9A and RUTBC2 co-localization was performed with CellProfiler.

RESULTS
We are interested in understanding the cellular role of Rab9A GTPase. As a starting point to find regulators of Rab9A, we used a yeast two-hybrid screen consisting of all TBC domain-containing proteins in the human genome as prey against a comprehensive library of hydrolysis-deficient Rab GTPases as bait (14,25). This screen revealed that a TBC domain-containing protein, RUTBC2, interacted with both Rab9A and Rab9B (Fig. 1B). RUTBC2 also interacted with Rab2A/B and Rab7li.
According to the National Center for Biotechnology Information (NCBI) HomoloGene database, there is only one RUTBC1/2 protein in C. elegans (tbc-8), Drosophila (CG1905), and zebrafish (LOC794373), whereas vertebrates have both RUTBC1 and RUTBC2 proteins (9). Unlike most other GAP proteins, the TBC catalytic domains of RUTBC1 and RUTBC2 contain a large insertion between the first two "fingerprint" A and B motifs (Fig. 1A, sequence). These motifs are unambiguously conserved in all TBC domains (1) and comprise the catalytic core of these enzymes. What is the role of the sequences that interrupt the catalytic core? In the structural model for Rab and Rab-GAP interaction, the analogous region of Gyp1 GAP is situated away from the Rab-GAP binding interface (21), thus these large insertions may have important binding interactions related to the functions of these proteins. Most of the dissimilarity between RUTBC1 and RUTBC2 in the TBC domain is found in this insertion; thus, these sequences are likely to contribute to the different functions of RUTBC1 and RUTBC2.
RUTBC2 Is a Rab9A Effector-To confirm the results of the screen and to test the nature of the interactions, we tested whether Rab9A could bind RUTBC2 in vitro. Full-length RUTBC2 was produced both in an in vitro transcription/translation system (with an N-terminal 3ϫMyc tag) as well as in E. coli (with an N-terminal GST tag). As shown in Fig. 2A, His-Rab9A bound well to GST-RUTBC2 and not GST, whereas another Rab that showed interaction in the two-hybrid screen, Rab2A, did not bind either GST or GST-RUTBC2. GST-RUTBC2 bound to His-Rab9A but not to His-Rab6A (Fig. 2B). Next, we asked whether RUTBC2 preferred either the GTPbound or the GDP-bound form of Rab9A. Using the GST binding assay described above, in vitro translated RUTBC2 was bound much more efficiently by GST-Rab9A when loaded with GTP␥S as compared with GDP (Fig. 2C). These results also suggest that RUTBC2 prefers to bind Rab9A despite Rab9B interaction observed in the two-hybrid screen. Thus, RUTBC2 is a bona fide effector of Rab9A.
We next investigated the location of the Rab9A binding site in RUTBC2. Using purified proteins, full-length GST-RUTBC2 bound to Rab9A at levels comparable with the positive control for Rab9A binding: the GST-tagged, C-terminal 110 amino acids of GCC185 protein (21) (Fig. 2D). 5-fold less Rab9A bound to the negative control, the GST-tagged C-terminal 123 amino acids of Golgin245 (Fig. 2D). Rab9A bound poorly to the TBC domain alone (His-tagged RUTBC2-C terminus or RUTBC2-C) but very well to His-tagged RUTBC2-N represent-ing the N-terminal half; only background binding was detected for GST-Rab9A binding to RUTBC2-C (Fig. 2E). His-Rab9A bound comparably to RUTBC2 full-length and RUTBC2-N constructs (Fig. 2F). This suggests that the N-terminal half is available for binding in the context of the full-length protein.
An earlier two-hybrid screen for GAP-Rab interactions failed to detect Rab9A binding to RUTBC2 (26). This was very likely due to the use of constructs lacking non-TBC domain sequences. Taken together, these data demonstrate that Rab9A binds directly to RUTBC2 in its N-terminal half.
RUTBC2 Interacts with RAB9A in Cells-RAB9A regulates the recycling of mannose 6-phosphate receptors (MPRs) from late endosomes to the trans Golgi network (10 -12). To test whether Rab9A and RUTBC2 interact in cells, HEK293T cells were transfected with 3ϫMyc-RUTBC2 and GFP or GFP-Rab9A. Using GFP-binding protein-conjugated Sepharose to precipitate these proteins, we detected the presence of 3ϫMyc-RUTBC2 in immunoprecipitates of GFP-Rab9A but not GFP (Fig. 3A). Thus, RUTBC2 can interact with Rab9A in living cells.
We further characterized this interaction by investigating the effects of exogenously expressed RUTBC2 on MPR recycling. When Rab9A function is disturbed in cells by overexpression of a dominant negative mutant Rab9A (12) or by depletion of its effectors (18,20,27), MPRs are missorted to the lysosome. We reasoned that if RUTBC2 had GAP activity on Rab9A, the phenotype of RUTBC2 overexpression should be similar to that of a GDP-preferring Rab9A mutant or Rab9A depletion.
When RUTBC2 was overexpressed in COS-1 cells, MPR levels at steady state were decreased by ϳ40% (Fig. 3B). This observation led us to investigate the turnover rate of MPR in HeLa cells overexpressing RUTBC2, and we found it to be essentially unchanged (Fig. 3C). This suggested that although some amount of MPRs was being missorted to lysosomes, there was likely a decrease in MPR transcription or translation. In a functional test of MPR trafficking, we measured the amount of hexosaminidase activity secreted into the medium by cells transfected with RUTBC2. Hexosaminidase is usually sorted to the lysosome, but when MPR levels are deficient in the trans Golgi network due to missorting events, the hydrolase is secreted. In 293T cells transfected with RUTBC2, slightly higher hexosaminidase activity was detected in the medium than in control cells (Fig. 3D). Cells transfected with a catalytically inactive protein, RUTBC2 R829A, showed a similar effect. Taken together, perturbation of MPR trafficking is likely due to titration of Rab9A by the overexpression of RUTBC2 rather than the GAP activity of this protein.
Further proof that Rab9A interacts with RUTBC2 in HeLa cells comes from experiments in which expression of the activated, GTP-locked form of Rab9A, Rab9A Q66L, led to increased localization of RUTBC2 to membrane compartments that harbor Rab9A (Fig. 4). In cells transfected with wild type Rab9A, very little co-localization between Rab9A and RUTBC2 was observed (ϳ10%). In contrast, 78% of RUTBC2-positive structures were also positive for Rab9A Q66L (n ϭ 700). Thus, RUTBC2 binds Rab9A in a variety of cell types.
RUTBC2 Has Narrow Substrate Specificity-Identification of the Rab substrates of RUTBC2 should provide important insight regarding its cellular function and the significance of the

RUTBC2, a Rab9A Effector and GAP for Rab36
Rab9A-RUTBC2 interaction. 32 different mammalian Rab GTPases were screened under single turnover conditions as substrates for RUTBC2 using purified, His-tagged RUTBC2-C that contains the catalytic TBC domain. Fig. 5 summarizes these results by comparing observed second order rate constants for GAP-catalyzed hydrolysis with the rate constants for intrinsic hydrolysis by each Rab protein. The TBC domain of RUTBC2 had the highest activity against Rab36 and Rab34, whereas no activity was detected against Rab9A or Rab2. There  Extracts were immunoprecipitated with anti-CI-MPR antibodies and exposed to a phosphor screen. D, HEK293T cells transfected with 3ϫMyc-RUTBC2 wild type or R829A for 24 h were assayed for secreted and intracellular hexosaminidase activity. Error bars in panels B-D represent S.E. from at least two independent experiments.
was also activity toward Rab32, which is a substrate for the closely related RUTBC1 (28). Both Rab36 and Rab34 are reported to be localized to the Golgi (29,30), but little else is known about their precise roles. Additionally, His-Rab2 was tested as a substrate for full-length RUTBC2; no enhancement of GTP hydrolysis rate was detected in reactions containing up to equimolar amounts of Rab2 and RUTBC2 proteins (data not shown).
RUTBC2 Is Enriched in Neural Cells-Analysis of different cell lines revealed that RUTBC2 is present in different amounts in HEK293T, Vero, and SK-N-SH cells (Fig. 6A). SK-N-SH cells are a human neuroblastoma cell line that might be similar to the mouse Neuro2a neuroblastoma cells used by Shimizu and coworkers (9) in earlier RUTBC2 studies. RUTBC2 was highly expressed in these cells as compared with kidney cells, consistent with that study. RUTBC2 could be efficiently depleted by siRNA treatment of 293T cells (Fig. 6B). Quantitation revealed approximately ϳ90% depletion. Fractionation of SK-N-SH cells showed that most RUTBC2 was cytosolic. Nevertheless, a small amount of RUTBC2 stably associated with membranes (Fig. 6C). Unfortunately, the antibody could not be used for immunofluorescence microscopy to determine the specific membranes with which endogenous RUTBC2 associates.
RUTBC2 Acts on Rab36 in Cells-To test whether Rab34 or Rab36 are substrates for RUTBC2 in cells, GFP-tagged Rabs were co-expressed with RUTBC2 or a catalytically inactive RUTBC2 R829A mutant protein. As described earlier, most RUTBC2 is cytosolic (Fig. 6C). To remove the cytosolic pool and permit detection of RUTBC2 on membranes, cells were frozen in liquid nitrogen to break membranes and washed to deplete cytosol prior to fixation.
When expressed alone, GFP-tagged Rab36 protein localized to ϳ125 punctate structures per cell (Fig. 7A, top panel). These did not contain late endosomal MPRs or Rab9A, lysosomal LAMP-1, early endosomal EEA1, early or recycling endosomal transferrin, or the Golgi protein, GM130 (not shown). We conclude that Rab36 is present in a distinct class of endosomal structures.   32 purified, mammalian Rab GTPases were preloaded with GTP for 1 h at room temperature and then desalted to remove free nucleotide. Rabs were diluted with MDCC-PBP and mixed with MgCl 2 containing varying concentrations of purified His-RUTBC2-C to start the reaction. Phosphate release was monitored continuously by microplate fluorometer (see "Experimental Procedures"). Catalytic efficiency (k cat /K m ) relative to the intrinsic rate constant (k intr ) for GTP hydrolysis was determined. Plots represent the mean from duplicate wells. The Rab isoforms used in the screen were the ubiquitously expressed isoforms; Rab9A was assayed. Error bars represent S.E. from at least two independent experiments. RUTBC2 protein was detected on the same cytosolic puncta that were labeled with GFP-Rab36 (Fig. 7A, middle panels shown enlarged in Fig. 7B). However, when catalytically active, wild type RUTBC2 protein was present, less Rab36 was seen on those structures as determined by quantification of the fluorescence intensity and the number of Rab36-positive puncta in each cell (Fig. 7, A and C). The level of Rab36 on vesicles did not change in cells in which similar levels of RUTBC2-R829A protein were expressed (Fig. 7, A and C). Importantly, immunoblot analysis of transfected cells showed that under all conditions tested, the cells expressed comparable amounts of Rab36 and either wild type or mutant RUTBC2 proteins (Fig. 7D).
Analogous experiments carried out with GFP-Rab34 failed to reveal any change in localization upon expression of RUTBC2 protein (not shown). Together, these data demonstrate that RUTBC2 is a GAP for Rab36 in cells and suggest a model where RUTBC2 in some way links Rab9A function to Rab36-controlled pathways.

DISCUSSION
Here we have shown that a predicted RabGAP protein, RUTBC2, is a novel Rab9A effector. RUTBC2 does not display GAP activity on Rab9A but instead has GAP activity for Rab34 and Rab36 in vitro and acts on Rab36 in cells. RUTBC2 is present in many cell types but is enriched in cells of neural origin. A small pool of RUTBC2 also stably associates with membranes, which appear to represent a distinct class of endosome in HeLa cells. Although Rab9B appeared to interact with RUTBC2 in a two-hybrid screen, only purified Rab9A bound directly to RUTBC2. Binding to either Rab would be consistent with the observation that at least in some cell types, murine RUTBC2 localizes to the trans Golgi network (9) or to endosomes (this study). Rab9B is localized to the Golgi (19), whereas Rab9A regulates the transport of MPRs from late endosomes to the trans Golgi network. It is not yet clear precisely where Rab9A and RUTBC2 interact.
It is interesting to contrast the substrate specificity of RUTBC2 with that of RUTBC1 (28). RUTBC1 shows GAP activity toward Rab32 and Rab33B in vitro but only toward Rab32 in cells. RUTBC2 also displays GAP activity toward Rab32 but much less than RUTBC1 and shows no activity toward Rab33B. Because most of the sequence divergence between the two proteins occurs in the TBC domain between the A and B motifs, it would be interesting to compare structural models of the two proteins to see whether this region may fold back and contact the Rab substrates. The co-crystal of Gyp1p and Rab33B suggests that this region does not contact the Rab (21), but in this region, the RUTBC proteins are almost five times longer. Currently, there are no known direct links between Rab9Amediated trafficking and either Rab36-regulated or Rab34-regulated events. However, both Rab36 and Rab34 are thought to influence the localization of lysosomes (29,30). GTP-restricted Rab34 or Rab36 causes LAMP1-positive lysosomes (and not MPR-positive structures) to cluster in the perinuclear area (29,30,31). This phenomenon is mediated by Rab-interacting lysosomal protein (RILP), an effector of both Rab34 and Rab36 as well as Rab7, a Rab known to play a role in lysosomal biogenesis. A mutation in the switch I region of Rab34 (K82Q) that blocks association with RILP fails to redistribute lysosomes (29). Little else is known about Rab36 other than the fact that it is deleted in many cases of malignant rhabdoid tumors (32).
Rab34 has also been suggested to play an additional role in constitutive secretion. It binds to hmunc-13, a diacylglycerolbinding protein (33). Depletion of Rab34 and hmunc-13 from HeLa cells blocks temperature-sensitive vesicular stomatitis virus glycoprotein (VSV-G) arrival at the plasma membrane (31,34). Brefeldin A treatment revealed that blocked VSV-G returned to the endoplasmic reticulum, suggesting that Rab34 depletion causes a block in intra-Golgi transport rather than exit from the trans Golgi network (31). A previous screen for the ability of overexpressed RabGAPs to block VSV-G secretion in multiple cell types showed that RUTBC2 had no effect on this process (35). This suggests that RUTBC2 may only have activity on Rab36 in living cells, consistent with the data presented herein.
RUTBC2 has also been shown to interact with Nurr1, an orphan nuclear receptor required for the development of dopaminergic neurons in the mouse brain (36). Co-expression of RUTBC2 and Nurr1 increased transcription of endogenous tyrosine hydroxylase, part of the dopamine synthesis pathway (36). Moreover, suppression of RUTBC2 by siRNA led to decreased cell division and decreased expression of the dopamine transporter, a Nurr1 target gene (36). This has led to speculation that RUTBC2 may be important in the pathogenesis of Parkinson disease, which is influenced by Nurr1 (37).
There is currently no known connection between Nurr1 and Rab36. One attractive, but highly speculative, possibility might be that Rab36 is required for proper trafficking of the dopamine transporter. Further work will be needed to clarify the link between Rab9A, RUTBC2, Rab36, and Nurr1 and may enhance our understanding of the etiology of Parkinson disease.