PMC

RUTBC2 co-localizes with Rab9A Q66L. Upper panels, confocal micrographs of HeLa cells co-expressing Myc-RUTBC2 and GFP-Rab9A. Middle and lower panels, confocal micrographs of GFP-RUTBC2 and Myc-Rab9A Q66L. The merged micrographs are shown in the right column. Bar, 10 μm.

Endogenous RUTBC2 is stably associated with membranes in SK-N-SH cells. A, detergent extracts (100 μg) of indicated cell lines were resolved by SDS-PAGE and immunoblotted using anti-RUTBC2 antibodies. B, HEK293T cells were either mock-transfected or transfected with siRNA targeting RUTBC2 for 72 h. Shown is quantitation of the band corresponding to RUTBC2. AU, arbitrary units. C, SK-N-SH cells were fractionated into crude membranes and cytosol. Increasing volumes of each fraction were analyzed by immunoblot using RUTBC2-specific antibodies. PNS, postnuclear supernatant.

RUTBC2 interacts with Rab9. A, diagram of RUTBC2 domain structure. The RUN domain is shown in blue; the TBC domain is shown in red. The first three conserved motifs of the TBC domain are indicated at the top. Conserved residues are in bold, and the predicted catalytic arginine and glutamine are shown in red, respectively. The extended insert between motif A and motif B of RUTBC2 is compared with the same regions of human RUTBC1 and RabGAP-5 (RUTBC3) and Saccharomyces cerevisiae Gyp1p. The structures of RUTBC2-N (residues 1–449) and RUTBC2-C (450–1032) are shown. B, RUTBC2 was screened against a library of 56 human Rab GTPases. Growth after streaking on selective medium indicates an interaction between RUTBC2 and a Rab GTPase.

RUTBC2 is an effector of Rab9A. A, His-Rab2 and His-Rab9A were preloaded with GTPγS and incubated with GST or full-length GST-RUTBC2 and then collected on glutathione-Sepharose. Bound material was eluted in sample buffer and analyzed by immunoblot with anti-His antibodies. Inputs represent 5% of the added His-tagged Rab. B, His-Rab9A and His-Rab6A were preloaded with GTPγS and incubated with full-length GST-RUTBC2 and then collected on glutathione-Sepharose. Bound material was eluted in sample buffer and analyzed by immunoblot with anti-His antibodies. Inputs represent 10% of the added His-tagged Rabs. C, in vitro transcribed/translated 3×Myc-RUTBC2 was incubated with GST-Rab9A or GST-Rab9B preloaded with either GTPγS (T) or GDP (D) and analyzed as in A. D, purified GST-RUTBC2 and control proteins were incubated with untagged Rab9A loaded with [³⁵S]GTPγS and immobilized using glutathione-Sepharose. Bound Rab was detected by scintillation counting. Error bars represent S.E. from at least two independent experiments. E, His-RUTBC2-N or His-RUTBC2-C was incubated with GST or GST-Rab9A Q66L and then collected on glutathione-Sepharose. Bound material was eluted in sample buffer and analyzed by immunoblot using anti-His antibodies. Inputs represent 5% of the added His-tagged RUTBC2 constructs. F, His-Rab9A was preloaded with GTPγS and incubated with GST, full-length GST-RUTBC2, GST-RUTBC2-N, or GST-RUTBC2-C and then collected on glutathione-Sepharose. Bound material was eluted in sample buffer and analyzed by immunoblot with anti-His antibodies. Inputs represent 5% of the added His-tagged Rab.

RUTBC2 binds to, but is not a GAP for Rab9A in cells. A, HEK293T cells were transfected with 3×Myc-RUTBC2 and GFP or GFP-Rab9A for 24 h, and GFP was immunoprecipitated with GFP-binding protein-conjugated Sepharose and immunoblotted with anti-Myc antibodies (top panel) and anti-GFP antibodies (bottom panel). Input represents 2% of the lysate subjected to immunoprecipitation. B, COS-1 cells were transfected with 3×Myc-RUTBC2 for 48 h, and total cell extracts were immunoblotted with anti-CI-MPR antibodies. AU, arbitrary units. C, quantitation of CI-MPR half-life measured from pulse-chase analysis of HeLa cells transfected with 3×Myc-RUTBC2 for 48 h. Extracts were immunoprecipitated with anti-CI-MPR antibodies and exposed to a phosphor screen. D, HEK293T cells transfected with 3×Myc-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.

RUTBC2 is a GAP for Rab36 in cells. A, confocal micrographs (with identical exposure times) of HeLa cells co-expressing EGFP-Rab36 and Myc-empty vector (upper panels), 3×Myc-RUTBC2 (middle panel), or 3×Myc-RUTBC2 R829A (lower panel). Bar, 10 μm. B, enlargement of the boxed area from the corresponding middle panels of A, with brightness increased to display co-localization of EGFP-Rab36 (left) and Myc-RUTBC2 (right). Asterisks are included to facilitate identification of co-localized puncta. C, quantification of the number of Rab36 puncta and their fluorescence intensity as a measure of membrane-associated Rab36 protein. Data represent the average of two experiments (n = 40 cells). Error bars represent S.E. of two independent experiments. D, HeLa cells were co-transfected as in A, and total cell extracts were immunoblotted for RUTBC2 and Rab36 using anti-Myc and anti-GFP antibodies, respectively.

RUTBC2 TBC domain has GAP activity toward Rab36 and Rab34 in vitro. 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 MgCl₂ 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.