Analysis of Rab GTPase and GTPase‐Activating Protein Function at Primary Cilia

C H A P T E R

T W E N T Y- S I X

Analysis of Rab GTPase and GTPase-Activating Protein Function at Primary Cilia Shin-ichiro Yoshimura, Alexander K. Haas, and Francis A. Barr Contents 1. Introduction 2. Methods 2.1. Cloning of RabGAPs and Rabs 2.2. Expression of Rabs and RabGAPs in hTERT-RPE1 cells 2.3. In vitro RabGAP assays 2.4. Identification and analysis of Rab8a-specific effector proteins References

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Abstract Primary cilia are sensory structures on the cell surface whose formation requires tight integration of microtubules and polarized membrane trafficking events. Rabs are GTP-binding proteins of the Ras superfamily that control directed membrane trafficking by regulating membrane–membrane and membrane–cytoskeleton interactions. This chapter describes cell biological and biochemical methods for the analysis of Rab function and Rab GTPaseactivating proteins during primary cilia formation.

1. Introduction Primary cilia are sensory structures on the surface of mammalian cells involved in morphogen signaling during development, sensing liquid flow in the kidney, mechanosensation, sight, and smell (Badano et al., 2006; Eggenschwiler and Anderson, 2006; Satir and Christensen, 2007) Mutations that affect primary cilia cause a number of diseases, including neural tube defect, polycystic disease, retinal degeneration, and cancers (Badano et al., 2006; University of Liverpool, Cancer Research Centre, Liverpool, United Kingdom Methods in Enzymology, Volume 439 ISSN 0076-6879, DOI: 10.1016/S0076-6879(07)00426-0

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2008 Elsevier Inc. All rights reserved.

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Michaud and Yoder, 2006; Zariwala et al., 2007) The primary cilium consists of a ninefold array of doublet microtubules closely wrapped by the plasma membrane. Unlike motile cilia, it lacks a central pair of doublet microtubules and is therefore referred to as a 9þ0 axoneme. Cilium formation in mammalian cells and tissues has been described in detail by electron microscopy and can be divided into a number of discrete stages (Sorokin, 1968). First, the basal body moves to the cell surface; following this docking event, microtubules are nucleated from the basal body toward the plasma membrane, and cilium formation is then completed by coordinated microtubule and plasma membrane extension (Sorokin, 1962, 1968). It is therefore an interesting system in which to study the underlying principles of how microtubule and membrane dynamics cooperate to generate specific cellular structures. This chapter focuses on the role of Rab proteins in the formation of primary cilia. Rab proteins are GTPases of the Ras superfamily that recruit specific effector protein to membrane surfaces when in the active GTP-bound form. These effector complexes are thought to define the identity of the specific membrane compartment due to their involvement in membrane–membrane recognition events and link membranes to the cytoskeleton (Gillingham and Munro, 2003; Munro, 2002). Both these classes of events are often referred to as tethering and are known to be important in vesicle-mediated trafficking. The best understood examples are the role of Rab5 and its effector protein EEA1 in endosome fusion (Christoforidis et al., 1999) and Rab1 with its effector protein p115 in ER to Golgi transport vesicle fusion with the Golgi (Allan et al., 2000) The guanine nucleotide-binding state of Rabs, and hence their specific activation and inactivation, is under the control of additional factors called GDP/GTP exchange factors (GEFs) and GTPase-activating proteins (GAPs). This chapter focuses on the 40 RabGAP proteins encoded in the human genome, each of which is thought to act on a specific Rab. To identify Rabs implicated in specific cellular processes, this chapter describes the application to primary cilia using three powerful screening methods: (i) RabGAP overexpression screening to specifically inactivate endogenous Rabs, (ii) Rab localization in human telomerase-immortalized retinal-pigmented epithelial (hTERT-RPE1) cells, and (iii) biochemical GAP assay with all Rab proteins. By combining these methods, Rabs that act at or localize to primary cilia could be identified, and their role in primary cilium function further characterized. This resulted in the identification of three Rab proteins, Rab8a, Rab17, and Rab23, that function in primary ciliogenesis (Yoshimura et al., 2007). In the case of Rab8a, its role in ciliogenesis was confirmed independently by the identification of Rabin8, a GEF for Rab8, as a binding partner for the core complex of Bardet–Biedl syndrome proteins known to be mutated in a number of ciliopathies (Nachury et al., 2007). The details of these three screening methods and their application are described next. In addition, variations on these methods are also useful to

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pinpoint Rab function in other membrane transport pathways (Fuchs et al., 2007; Haas et al., 2007).

2. Methods 2.1. Cloning of RabGAPs and Rabs RabGAP proteins are defined by the conserved TBC (Tre2/Bub2/Cdc16) domain containing conserved residues required for catalyzing GTP hydrolysis. By searching the nucleotide, protein, and EST databases held at the National Center for Biotechnology Information with a TBC domain consensus built using yeast proteins, 40 genes encoded TBC domaincontaining proteins were identified in humans. Of these, cDNA for 38 RabGAPs were obtained by polymerase chain reaction (PCR) methods using KOD DNA polymerase (Toyobo, Novagen) or pfu DNA polymerase (Stratagene) from human fetus, liver, and testis Marathon-Ready cDNA (Clontech Laboratories, Inc.) according to the manufacturer’s standard protocol (Fuchs et al., 2007; Haas et al., 2005, 2007; Yoshimura et al., 2007). TBC1D12 could not be amplified using standard PCR conditions because of its guanosine/cytosine-rich content; however, it could be amplified by PCR with KOD in the presence of 5% (vol/vol) dimethyl sulfoxide (DMSO). The cloning of Rabs is performed with the same protocol as RabGAPs, except for Rab12. The Rab12 gene has also a guanosine/ cytosine region, and 7.5% (vol/vol) DMSO with Taq DNA polymerase (New England Biolabs.) is used to amplify it successfully, whereas PCR by KOD and pfu polymerase with or without DMSO is not successful. The recent crystal structure of a Rab, together with a TBC domain, reveals that these two residues are important for the nucleotide hydrolysis reaction (Pan et al., 2006). The glutamine is important for positioning the nucleophilic water molecule required for GTP hydrolysis, whereas the arginine stabilizes the developing charge on the b- and g-phosphates of the bound GTP. Accordingly, catalytically inactive RabGAP mutants are created by site-directed mutagenesis of either one of these residues to alanine using the Quickchange method and pfu polymerase (Stratagene). Similarly, the conserved glutamine residues of the Rabs are mutated to alanine to generate hydrolysis-defective mutants. All the primers for cloning and mutagenesis are described in Tables 26.1 and 26.2 (in order to view these tables, please refer to the URL: http://books. elsevier.com/companions/9780123743114); the restriction sites used for cloning are shown as uppercase letters. The wild-type and catalytically inactive mutant RabGAP and Rab cDNA are then subcloned into pCRII TOPO (Invitrogen) prior to transfer into pEGFP-C2 (Clontech) and pcDNA3.1/Myc (Invitrogen) for mammalian cell expression or into

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pQE32 (Qiagen) and pFAT2 (a His-GST-tagging vector modified from pGAT2 to have a polylinker compatible with pQE32).

2.2. Expression of Rabs and RabGAPs in hTERT-RPE1 cells 2.2.1. Cell culture and primary cilium formation The hTERT-RPE1 cell line can be used for the analysis of primary cilium formation. The hTERT-RPE1 cell line can be obtained from Clontech Laboratories or the American Type Culture Collection (ATCC). Cells are grown at 37 and 5% CO2 in a 1:1 mixture of dimethyl ether and Ham’s F12 medium containing 10% calf serum, 2.5 mM L-glutamine, and 1.2 g/liter sodium bicarbonate. When these cells are cultured after serum withdrawal, they rapidly form primary cilia with 24 to 48 h. To do this, cells are seeded at 0.5 to 1.0  105 cells per well of a six-well plate and grown for 24 h. The growth medium is then removed and the cells are washed in phosphate-buffered saline (PBS) and replaced with growth medium lacking serum. Under these conditions, primary cilium formation is around 60% after 24 h and, after 48 h, has reached a maximum of around 80%. Other cell lines, for example, mouse NIH/3T3 cells and Madine–Darby canine kidney cells, also have primary cilia and can be used for similar experiments with only a slight variation of the methods described later. 2.2.2. RabGAP overexpression screening on primary cilium formation To test the effects of RabGAPs on cilium formation, 0.5 mg of the pEGFP-C2 vector encoding the 38 RabGAPs is transfected by the Fugene 6 transfection regent (Roche) to 0.5 to 1.0  105 of hTERT-RPE1 cells on coverslips in separate wells of six-well plates. After 24 h the cells are washed three times with PBS and replaced with new culture medium as described earlier, except without calf serum. The cells are cultured for another 48 h to induce primary cilia formation. At this time point, the cells are treated on ice for 1 h to depolymerize cytoplasmic microtubules. The cells are fixed with ice-cold methanol for 4 min and then washed three times with PBS. The fixed cells are used for indirect immunofluorescence analysis with mouse antiacetylated tubulin antibody (6–11B-1; Sigma-Aldrich) as the primary antibody and Cy3conjugated donkey antimouse secondary antibody ( Jackson ImmunoResearch Laboratories). The cells are observed by fluorescence microscopy [Axioskop 2 with a 63 Plan Apochromat oil-immersion objective of NA 1.4, standard filter sets (Carl Zeiss MicroImaging, Inc.), and a 1300  1030 pixel-cooled, charge-coupled device camera (Model CCD-1300-Y; Princeton Instruments)]. RabGAP expression is monitored by EGFP fluorescence, and primary cilium formation is judged by Cy3-acetylated tubulin signals.

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For the analysis, 100 RabGAP expressing cells are counted and plotted the number of ciliated cells to the graph. With seven RabGAP proteins, TBC1D3, TBC1D12, RUTBC1, RUTBC2, USP6, AK074305, and KIAA0882 reduce cell viability and increase levels of apoptosis; therefore, we exclude these proteins from the analysis of cilium formation. Using this method, we have shown that TBC1D7, EVI5-like, and XM_037557 cause significant reduction on primary cilia formation, from 80 to 5–40% (Yoshimura et al., 2007). 2.2.3. Rab localization and function in primary cilium in hTERT-RPE1 cells To identify which Rab proteins localize to primary cilia, hTERT-RPE1 cells are transfected with the pEGFP-tagged Rab constructs described earlier and the same method is used for RabGAPs, except the primary cilium is left induced or induced for 72 h. Of all the Rabs tested, only Rab8a shows cilium localization after serum starvation (Fig. 26.1) (Yoshimura et al., 2007). Notably, Rab8a shows primary cilium localization but not Rab8b (Yoshimura et al., 2007). To examine the subcellular localization of endogenous Rab8a, we use the affinity-purified rabbit anti-Rab8a antibody, a kind gift of Johan Pera¨nen (Institute of Biotechnology, University of Helsinki, Helsinki, Finland). This antibody is also used to check the depletion efficiency of Rab8a protein by siRNA. For siRNA experiments, 2  104 of hTERT-RPE1 cells is plated per well of a six-well plate. The target sequences are as follows: control, CGUACGCGGAAUACUUCGA; Rab8a, CAGGAACGGUUUCGGA CGA, GAAUUAAACUGCAGAUAUG, GAACAAGUGUGAUGUG AAU. All siRNA duplexes are obtained from Dharmacon Inc. Cells are transfected with 3 ml 20 mM of the Rab8a siRNA duplex using 3 ml of the Oligofectamine transfection reagent (Invitrogen) mixed with 100 ml Optimem (Invitrogen). After 15 min of incubation, this mixture is added to the cells, which are then grown for 48 h in normal medium with 10% calf serum. After 48 h, the cells are washed three times with PBS, and fresh culture medium without serum is added and then transfected with the same duplex once again. After culture for a further 48 h, the cells are fixed and then costained with the acetylated tubulin and Rab8a antibodies. The length of primary cilia in EGFP-Rab8a expression cells or Rab8adepleted cells indicated by acetylated tubulin and EGFP signals is measured and calculated with the measurement tools in either Metavue (Molecular Devices, MDS Inc.) or Photoshop (Adobe Systems Inc.). The length of cilia is measured in at least 100 independent cells, and the significance is judged by a Students t test (Yoshimura et al., 2007).

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A

EGFP-Rab8a

Acetylated tubulin

B

EGFP-Rab8b

Acetylated tubulin

Figure 26.1 Rab8a and Rab8b localization to primary cilium. hTERT-RPE1 cells were transfected with EGFP-Rab8a and Rab8b, cultured for 24 h, and then serum starved for 72 h to induce primary cilia. Cells were fixed and then observed by immunofluorescence microscopy. (A) EGFP-Rab8a localized in primary cilia indicated by acetylated tubulin staining, whereas (B) EGFP-Rab8b did not.

2.3. In vitro RabGAP assays 2.3.1. Recombinant protein preparation of Rab and RabGAP proteins from bacteria All cDNA of Rab are subcloned into the pFAT2 vector to generate hexahistidine (6xHis)–GST-tagged fusion proteins when expressed in bacteria. It is important to note that maltose-binding protein (MBP) fusions, while yielding high levels of expression, do not to give rise to Rabs with normal GTP-binding and hydrolysis activities. For this reason, we recommend using either GST or His-tagged Rab proteins. The vector is

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transformed to Escherichia coli BL 21(DE3 lysogen) containing the pRIL plasmid containing rare tRNAs for arginine, isoleucine, and leucine. Bacteria are plated on LB agar with 100 mg/ml of ampicillin to select pFAT2 and 34 mg/ml of chloramphenicol to select pRIL. The plates are kept at 37 for 12 h. Single colonies are then picked from each plate and transferred between 500 ml and 4 liters of LB liquid medium depending on the Rab with 100 mg/ ml of ampicillin and 34 mg/ml of chloramphenicol and cultured at 3 to reach an OD600 of between 0.5 and 0.6 in a shaking incubator (Infors AG). The cultured is then transferred to an actively cooled 18 incubator and cultured for 1 h, and then protein expression is induced by the addition of 0.25 mM isopropyl-b-D-thiogalactoside (IPTG). The liquid cultures are grown for another 12 to 14 h at 18 . With most Rab proteins, except Rab8a, Rab8b, Rab10, and Rab13, an alternative quick expression protocol can be used. A single colony is picked and transferred into LB liquid medium with 100 mg/ ml of ampicillin and 34 mg/ml of chloramphenicol and cultured at 37 to reach an OD600 of 0.5 to 0.6. Expression is then induced by adding 0.5 mM of IPTG and culturing for 3 h at 37 . Finally, bacteria are recovered by centrifugation for protein preparation. For RabGAP proteins, TBC1D7, EVI5-like, and XM_037557 expression and purification from bacteria are tested as either MBP or His-tagged fusion proteins. While the yield of MBP fusion protein is high with each of these three RabGAP proteins, they do not show significant GAP activity with any Rab protein tested. Although MBP fusions of RabGAP-5, RN-tre, and TBC1D20 do show specific activity toward discrete target Rabs (Haas et al., 2005, 2007), we therefore do not recommend use of the MBP tag for RabGAP expression. With the 6xHis-tagged proteins it is difficult to obtain a high yield from bacteria, as highly specifically active proteins are obtained. TBC1D7, EVI5-like, and XM_037557 in pQE32 fuse the 6xHis tag and cleavage site of TEV protease to the N terminus of RabGAP proteins. These constructs are transformed into the E. coli JM109 (pRIL) strain and plated into LB agarose medium with 100 mg/ml of ampicillin and 34 mg/ml of chloramphenicol. The plates are kept overnight (12 h) to form colonies. A single colony is picked and transferred into 4 liters of LB liquid medium with 100 mg/ml of ampicillin and 34 mg/ml of chloramphenicol and then cultured at 37 to reach an OD600 of 0.5 to 0.6. Bacteria are subsequently cultured at 18 for 12 to 14 h, without any induction of protein expression by IPTG. The bacterial pellet is recovered by centrifugation, and the pellet is processed to the following protein purification step immediately. Rabs and RabGAPs are purified on 1-ml NTA-agarose columns (Qiagen) as described previously (Fuchs et al., 2005). The peak fractions of eluted protein from NTA-agarose are dialyzed overnight in Tris-buffered saline (TBS: 50 mM Tris-HCl, pH 7.4, 150 mM NaCl) with 2 mM dithiothreitol (DTT). The protein concentration is measured using a standard Bradford protein assay kit (Bio-Rad).

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With these methods, between 1 and 10 mg of Rab protein and 0.75 to 3 mg of the RabGAP proteins can be obtained. Rab proteins and RabGAP proteins should be aliquoted and frozen in liquid nitrogen or dry ice and stored at 80 . An example of EVI5 purification is shown (Fig. 26.2A).

A

Fraction CL UB W

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2

3

4

5

6

7

8

9

10

MW 212 158 116 97.2

EVI5-like

66.4 55.6 42.7 34.6 B

180

GTP hydrolysis (pmol/h)

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RabGAP: EVI5-like

80 60 40 20 0 Rab23

Rab6a

Rab-like5

Figure 26.2 Purification profile of EVI5-like and measurement of EVI5-like GAP activity. Bacteria from a 4-liter culture of the JM109 (pRIL) strain transformed with pQE32 EVI5-like were homogenized, and a cleared lysate was prepared by centrifugation (A, CL).The cleared lysate was bound to 1 ml of NTA-agarose beads for 60 min and separated into NTA bead-bound and unbound fractions (A, UB). NTA-agarose beads were then washed with 20 mM imidazole (A,W), and the bound protein was eluted by 250 mM of imidazole. Ten 1-ml fractions were collected, and 10 ml from each fraction was loaded onto a 10% SDS-PAGE gel stained by Coomassie brilliant blue (A, fractions 1^10.) The proteins in fractions 2 and 3 were pooled, dialyzed, and used for biochemical GAP assays. (B) GAP assays were performed as described. EVI5-like shows specific activity with Rab23, but not with Rab6a and Rab-like 5.

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2.3.2. Biochemical RabGAP assay with purified proteins To measure GAP-stimulated GTP-hydrolysis, 100 pmol of Rab protein is mixed on ice with 10 ml of 10 assay buffer (500 mM HEPES-NaOH, pH 6.8, 10 mM DTT, 2 mg/ml bovine serum albumin), 10 ml of 10 mM EDTA (pH 8.0), 5 ml of 1 mM Mg2þ-GTP, and 2 ml of [g-32P]GTP (GE Healthcare PB10244: 10 mCi/ml; 5000 Ci/mmol or Perkin-Elmer NEG004Z250UC: 10 mCi/ml; 6000 Ci/mmol) and adjusted to 100 ml by dH2O. GTP and [g-32P]GTP should not be freeze–thawed. The GTPloading reaction is incubated for 15 min at 30 and is then kept briefly on ice for use in the following GAP reaction. For GAP assays, two 5-ml aliquots from the GTP-loading reaction are taken and immediately added to 795 ml of ice-cold 5% (wt/vol)-activated charcoal slurry in 50 mM NaH2PO4 and left on ice as the t ¼ 0 background values. The Rab loading mix is then split into two equal halves. In one half, 0.5 to 10 pmol of GAP is added. This is the GAP reaction. In the other half, the equal volume of TBS is added; this reaction is used to calculate basal or GAP-independent GTP hydrolysis. Reactions are then incubated at 30 , taking 5-ml samples in duplicate at each time point (5, 10, 15, 45, and 60 min). The 5-ml aliquots are added immediately to 795 ml of ice-cold 5% (wt/vol)-activated charcoal slurry in 50 mM NaH2PO4, left for 1 h on ice, and then centrifuged at 16000 gav in a bench-top microcentrifuge (Eppendorf 5417C with 30 space rotor and aerosol-resistant lid) to pellet the charcoal. The supernatant is carefully removed with a pipette, and 400-ml aliquots are scintillation counted in 4 ml of Ultima Gold scintillation liquid (Perkin-Elmer). A 2.5-ml aliquot of the assay mix is also scintillation counted to allow calculation of the specific activity of the reaction. 2.3.3. Calculation of GAP activity To calculate the number of counts per picomole of GTP in the reaction mixture, the activity measured in 2.5 ml of assay mix is multiplied by 40 to obtain the total activity for the full assay volume of 100 ml. This value is then divided by the total of 5000 pmol GTP in the reaction to obtain the specific activity in cpm/pmol of GTP. The measured values of the 5-ml aliquots taken from each reaction in duplicate at each time point are then averaged and then multiplied by 2, as only 400 ml out of the 800-ml charcoal mix is measured by scintillation counting. Because 5 ml of the 100-ml reaction mix is measured, these values are then multiplied by 20. To calculate the amount of GTP hydrolyzed in picomoles, these values are then divided by the specific activity per picomole of GTP (cpm/pmol). The t ¼ 0 values are then subtracted to eliminate the background of the reaction. The amount of GTP hydrolyzed by Rabs alone (-GAP) is then subtracted from the stimulated reactions (þGAP). Via this calculation the stimulation of GTP hydrolysis by all Rabs upon addition of the same GAP can be compared.

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An example of EVI5-like activity toward Rab23, Rab6a, and Rabl5 is plotted in Fig. 26.2B. In biochemical RabGAP assays, EVI5-like, TBC1D7, and XM_037557 showed activity with Rab23, Rab17, and Rab8a, respectively, and these Rabs were therefore implicated in cilium formation (Yoshimura et al., 2007).

2.4. Identification and analysis of Rab8a-specific effector proteins 2.4.1. Identification of cenexin/ODF2 variant 3 as Rab8a-specific effector protein Cenexin was originally identified as a centriolar protein (Lange and Gull, 1995) and as a component of the outer dense fibers (ODF) of rat sperm (Brohmann et al., 1997). These data suggest that cenexin/ODF2 has a function as a centriole/centrosome and microtubule-binding protein. Importantly, it was also reported that cenexin/ODF2 was involved in primary cilium formation in mouse F9 cells (Ishikawa et al., 2005). To identify effector proteins for Rab8a protein, yeast two-hybrid screening is performed using the Matchmaker system with a human testis cDNA library (Clontech) as described before (Fuchs et al., 2005). With this approach, 13 truncated cenexin clones derived from the third splice variant of this gene are obtained. A full-length clone is reexamined by yeast twohybrid analysis, and cenexin 3 interacts with Rab8a, but not with Rab8b (Yoshimura et al., 2007). To assess the subcellular localization of cenexin 3 proteins, cenexin 3 is subcloned into the pcDNA3.1 flag to introduce the flag tag to the N terminus of cenexin 3, and the mixture of 50 ng of this plasmid and 1 mg pBluescript-II is transfected with hTERT-RPE1 cells and analysis the localization by costaining by the rabbit anti-flag antibody (F7425; Sigma-Aldrich) and acetylated tubulin or g-tubulin antibody (GTU88; Sigma-Aldrich). When cenexin is transfected with normal plasmid amounts (0.5–1 mg), it aggregates and causes abnormal microtubule bundling (Nakagawa et al., 2001). The method described earlier retains high transfection efficiency, but results in much lower expression per cell, and thus avoids these cenexin aggregation artifacts. 2.4.2. Analysis of the Rab8a–cenexin interaction For binding assays, recombinant purified cenexin 3 protein is required; however, the full length of cenexin 3 could not be obtained in soluble form from bacteria. A C-terminal domain of cenexin 3 (amino acids 397– 657) encoded in pQE32 is obtained successfully as a soluble form by the same methods as RabGAP protein preparation. For binding assays, 10 mg of GST-Rab proteins (Rab1b, Rab8a, Rab8b, Rab10, Rab13, and Rab35) is bound to 25 ml of packed glutathione-Sepharose (GE Healthcare) in 1 ml

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total volume of PBS for 60 min at 4 . The beads are first washed three times in 500 ml with nucleotide exchange buffer [20 mM HEPES-NaOH, pH 7.5, 100 mM NaOAc, 10 mM EDTA, and 0.1% (vol/vol) NP-40], followed by two washes in 500 ml nucleotide loading buffer [NL100: 20 mM HEPES-NaOH, pH 7.5, 100 mM NaOAc, 0.1 mM MgCl2, and 0.1% (vol/ vol) NP-40]. The beads are then resuspended in 200 ml NL100, and 20 ml of 100 mM GTP and 10 mg of cenexin 3 (amino acids 397–657) are added. Binding is then allowed to proceed for 60 min at 4 , rotating to mix. The beads are then washed three times with 500 ml NL100, and bound proteins are eluted by the addition of elution buffer [20 mM HEPES-NaOH, pH 7.5, 200 mM NaCl, 20 mM EDTA, and 0.1% (vol/vol) NP-40], rotating at 4 for 15 min. Beads are pelleted by centrifugation at 2000 g for 1 min, and the supernatant is transferred to a fresh tube. To remove contaminating Rabs, 50 ml of packed glutathione-Sepharose is added to the eluate and incubated for 10 min at 4 with mixing. The beads are pelleted by centrifugation at 2000 g for 1 min, and the supernatant is transferred to a fresh tube. This procedure is repeated three times. Eluted proteins are then precipitated using trichloracetic acid and analyzed on 12% minigels stained with Coomassie brilliant blue.

REFERENCES Allan, B. B., Moyer, B. D., and Balch, W. E. (2000). Rab1 recruitment of p115 into a cisSNARE complex: Programming budding COPII vesicles for fusion. Science 289(5478), 444–448. Badano, J. L., Mitsuma, N., Beales, P. L., and Katsanis, N. (2006). The ciliopathies: An emerging class of human genetic disorders. Annu. Rev. Genomics Hum. Genet. 7, 125–148. Brohmann, H., Pinnecke, S., and Hoyer-Fender, S. (1997). Identification and characterization of new cDNAs encoding outer dense fiber proteins of rat sperm. J. Biol. Chem. 272 (15), 10327–10332. Christoforidis, S., McBride, H. M., Burgoyne, R. D., and Zerial, M. (1999). The Rab5 effector EEA1 is a core component of endosome docking. Nature 397(6720), 621–625. Eggenschwiler, J. T., and Anderson, K. V. (2006). Cilia and developmental signaling. Annu. Rev. Cell Dev. Biol. 23, 345–373. Fuchs, E., Haas, A. K., Spooner, R. A., Yoshimura, S., Lord, J. M., and Barr, F. A. (2007). Specific Rab GTPase-activating proteins define the Shiga toxin and epidermal growth factor uptake pathways. J. Cell Biol. 177(6), 1133–1143. Fuchs, E., Short, B., and Barr, F. A. (2005). Assay and properties of rab6 interaction with dynein-dynactin complexes. Methods Enzymol. 403, 607–618. Gillingham, A. K., and Munro, S. (2003). Long coiled-coil proteins and membrane traffic. Biochim. Biophys. Acta 1641(2–3), 71–85. Haas, A. K., Fuchs, E., Kopajtich, R., and Barr, F. A. (2005). A GTPase-activating protein controls Rab5 function in endocytic trafficking. Nat. Cell Biol. 7(9), 887–893. Haas, A. K., Yoshimura, S., Stephens, D. J., Preisinger, C., Fuchs, E., and Barr, F. A. (2007). Analysis of GTPase-activating proteins: Rab1 and Rab43 are key Rabs required to maintain a functional Golgi complex in human cells. J. Cell Sci. 120(Pt 17), 2997–3010.

364

Shin-ichiro Yoshimura et al.

Ishikawa, H., Kubo, A., Tsukita, S., and Tsukita, S. (2005). Odf2-deficient mother centrioles lack distal/subdistal appendages and the ability to generate primary cilia. Nat. Cell Biol. 7(5), 517–524. Lange, B. M., and Gull, K. (1995). A molecular marker for centriole maturation in the mammalian cell cycle. J. Cell Biol. 130(4), 919–927. Michaud, E. J., and Yoder, B. K. (2006). The primary cilium in cell signaling and cancer. Cancer Res. 66(13), 6463–6467. Munro, S. (2002). Organelle identity and the targeting of peripheral membrane proteins. Curr. Opin. Cell Biol. 14(4), 506–514. Nachury, M. V., Loktev, A. V., Zhang, Q., Westlake, C. J., Pera¨nen, J., Merdes, A., Slusarski, D. C., Scheller, R. H., Bazan, J. F., Sheffield, V. C., and Jackson, P. K. (2007). A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis. Cell 129(6), 1201–1213. Nakagawa, Y., Yamane, Y., Okanoue, T., Tsukita, S., and Tsukita, S. (2001). Outer dense fiber 2 is a widespread centrosome scaffold component preferentially associated with mother centrioles: Its identification from isolated centrosomes. Mol. Biol. Cell 12(6), 1687–1697. Pan, X., Eathiraj, S., Munson, M., and Lambright, D. G. (2006). TBC-domain GAPs for Rab GTPases accelerate GTP hydrolysis by a dual-finger mechanism. Nature 442(7100), 303–306. Satir, P., and Christensen, S. T. (2007). Overview of structure and function of mammalian cilia. Annu. Rev. Physiol. 69, 377–400. Sorokin, S. (1962). Centrioles and the formation of rudimentary cilia by fibroblasts and smooth muscle cells. J. Cell Biol. 15, 363–377. Sorokin, S. P. (1968). Reconstructions of centriole formation and ciliogenesis in mammalian lungs. J. Cell Sci. 3(2), 207–230. Yoshimura, S., Egerer, J., Fuchs, E., Haas, A. K., and Barr, F. A. (2007). Functional dissection of Rab GTPases involved in primary cilium formation. J. Cell Biol. 178(3), 363–369. Zariwala, M. A., Knowles, M. R., and Omran, H. (2007). Genetic defects in ciliary structure and function. Annu. Rev. Physiol. 69, 423–450.