[36] R a b PROTEINS AND GENE FAMILY IN ANIMALS 387

4. Mix the EDC-treated KLH (step 2) and peptide solution (step 3), and incubate with agitation for 3 hr at room temperature.

5. Dialyze overnight against several changes of PBS. 6. For immunization of rabbits, use 200/~g of coupled peptide and

follow the procedure as described above.

Glutaraldehyde Coupling

I. Prepare a 2 ml solution of peptide and KLH (final concentrations, 3 and 7 mg/ml, respectively) in PBS. Perform the following steps in a fume hood.

2. Add 3.2/A of a 12.5% (v/v) aqueous glutaraldehyde solution [stock solution 25%, electron microscopy (EM) grade I, Sigma, St. Louis, MO) store at - 80°].

3. Stir for 5 min at room temperature on a magnetic stirrer. 4. Repeat steps 2 and 3 five times. 5. Follow steps 5 and 6 as described under EDC Coupling (above).

The protocols for immunization, preparation of serum, partial purifi- cation of IgG fraction, affinity purification, and calculation of antibody titer are the same as described above.

Acknowledgments

We are thankful to members of the Gallwitz laboratory for discussion and to K. Larson- Becker for secretarial assistance. This work was supported in part by grants to D.G. from the Deutsche Forschungsgemeinschaft and the Bundesministerium far Forschung und Technologic.

[36] R a b P r o t e i n s a n d G e n e F a m i l y in A n i m a l s

By A R M A N D TAVITIAN a n d A H M E D ZAHRAOUI

Introduction

The acronym "rab" was first used in 1987 when four additional mem- bers of the ras gene family were isolated from a rat brain cDNA library by means of synthetic oligonucleotide probes) It was readily observed that these mammalian tab genes constituted a distinct branch of the ras super- family, more closely related to two genes discovered in Saccharomyces

1 N. Touchot, P. Chardin, and A. Tavitian, Proc. Natl. Acad. Sci. USA 84, 8210 (1987).

Copyright © 1992 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 219 All riots of reproduction in any form reserved.

388 IDENTIFICATION OF TRANSPORT INTERMEDIATES [36]

cerevisiae: the YPT gene found serendipitously in 1983 by Gallwitz et aL 2 and the then newly characterized SEC4 gene involved in yeast secretion)

rab genes have been the focus of intense research and, indeed, this branch of research has grown. Additional members have been character- ized by several groups: the YPT cDNA of mouse, capable of complement- ing that of yeast, confirmed that rabl (rabIA) was the homolog of the yeast YPT gene? G proteins purified from bovine brain permitted the design of oligonucleotide that revealed additional members related to rab3 (denoted smg25A, smg25B, and smg25C). 5 The search for human counterparts of the rat rab cDNAs in human cDNA libraries revealed the same four genes (rabl, rab2, rab3, rab4) and additionally rab3B, rab5, and rab6. 6 BRL-ras, isolated from a rat liver cell line cDNA library, 7 is referred to as rab7. Another clone, very close to rablA, was isolated from a rat brain cDNA library and denoted rablB, s More recently additional genes, rab8, rab9, rablO, and rabll, were characterized in canine cells. 9 The fission yeast Schizosaccharomyces pombe has three genes, YPT1, YPT2, and Ryh, for which the human counterpart is rab6? °

At present, there are some 16 members, characterized in mammals, that pertain to the rab branch of the family. This figure may represent only a fraction of the total number of the existing Rab proteins, even though different approaches have often led to the rediscovery of previously known members. One may foretell that there are, perhaps, as many as 30 different genes pertaining to the rab family.

The small G proteins of the Rab branch have molecular weights of 22K-27K. They are well conserved throughout the phylogeny and espe- cially between mammalian species. For instance, the percentage of identity between the human RablA and the YPT protein is 75%. The rat and the human RablA proteins are identical. There is only one conservative change between the human and rat Rab3A proteins. The four domains of the Ras proteins that were shown to be involved in the binding of GTP/ GDP and which correspond to some seven or eight amino acid residues around positions 15, 60, 115, and 145 are highly conserved. Some varia-

2 D. Gallwitz, C. Donath, and C. Sander, Nature (London) 306, 704 (1983). 3 A. Salminen and P. J. Noviek, Ce1149, 527 (1987). 4 H. Haubruck, R. Prange, C. Vorgias, and D. Gallwitz, EMBO J. 8, 1427 (1989). 5 y. Matsui, A. Kikuchi, J. Kondo, T. Hishida, Y. Teranishi, and Y. Takai, J. Biol. Chem.

263, 11071 (1988). 6 A. Zahraoui, N. Touchot, P. Chardin, and A. Tavitian, J. Biol. Chem. 264, 12394 (1989). 7 C. Bucci, R. Frunzio, L. Chiarotti, A. L. Brown, M. M. Rechier, and C. B. Bruni, Nucleic

Acids Res. 16, 9979 (1988). s E. Vielh, N. Touchot, A. Zaharoui, and A. Tavifian, Nucelic Acids Res. 17, 1770 (1989). 9 p. Chavrier, R. G. Parton, H. H. Hare'i, K. Simons, and M. Zerial, Ce1162, 317 (1990).

1o L. Hengst, T. Lehmeier, and D. Galiwitz, EMBO J. 9, 1949 (1990).

[36] RAa PROTEINS AND GENE FAMILY IN ANIMALS 389

tions are observed, however, in region 10-17, where the glycines in posi- tion 12 and sometimes 13 of Ras are replaced by other conservative (or even nonconservative for Rab6) amino acids.

Two additional sequences corresponding to residues 35-40 (known as the effector region in Ras) and 51- 85 are highly conserved among all the Rab proteins. In particular, region 35-40 points to different effectors from the Ras. There are probably different effectors for the Rab proteins; it is remarkable that the YPT2 protein, whose sequence in the effector domain is identical to the corresponding SEC4 sequence, can complement the SEC4 mutations and cannot complement the YPT l mutations.

Isolation of Rab cDNAs

There are several well-defined procedures for the isolation of a cDNA insert coding for a protein of interest. In recent years different techniques have been developed to isolate Rab eDNAs encoding 23K-27K Ras-like small GTP-binding proteins2 ,4,9 The preferred technique utilizes eDNA libraries made in ~ t l 0 cloning vector. Large number of plaques can be screened with different probes. In our laboratory, an improved oligonu- cleotide strategy has been applied to detect a number of Rab sequences in cDNA libraries. This strategy was based on the use of degenerate oligonu- cleotide probes coding for the DTAGQE amino acid sequence in positions 57-62 strictly conserved (except in Rap/D-Ras3 proteins) in Ras and Ras-related proteins identified so far. Moreover, this sequence is not found in the heterotrimeric G proteins and in other nucleotide-binding proteins. The degenerate oligonucleotide was used to screen a 2gt I 0 rat brain eDNA library (for details see Ref. 1). Four cDNAs encoding four Rab proteins were isolated. The same method, utilizing a degenerate oligonueleotide based on the conserved sequence WDTAGQE in Rab, Rho, yeast YPT 1, and SEC4 proteins, had led to the identification of new Rab proteins.l~ Other techniques, such as the screening of eDNA libraries at low stringency with known Rab cDNA probes, also led to the isolation of additional Rab proteins. 6

Construction of Rab/pET-3c Expression Vector

Methods for expressing large amount of protein from cloned eDNA coding region introduced in Escherichia coli have been described and have proved valuable for the biochemical and functional analysis of proteins.

L1 p. Chavrier, M. Vingron, C. Sander, K. Simons, and M. Zerial, Mol. Cell. Biol. 10, 6578 (1990).

390 IDENTIFICATION OF TRANSPORT INTERMEDIATES [36]

Because most of the Rab proteins are not expressed efficiently in ptac vector, 12 we used the expression vector pET-3c. This system utilizes a bacteriophage T7 RNA polymerase/promoter system.13 The pET-3c vector allows cloning of target DNAs at sites where they will be selectively and actively transcribed by T7 RNA polymerase in vitro and in E. coli cells. Transcription is controlled by the strong ~plO promoter for T7 RNA polymerase, pET-3c is a derivative ofpBR322 that carries sequentially the bacteriophage T7 gene qblO promoter, the ribosome-binding site, a transla- tion start (ATG), and a BamHI cloning site. The nucleotide sequence upstream from the gene 10 initiation codon contains a NdeI site that includes the ATG. Thus, the NdeI site is unique and can be useful for joining coding regions to the ATG initiator codon without making any change in the coding sequence, pET-3c was used for production of intact, native Rab proteins. In fact, any ATG start codon could be joined at the NdeI site (Fig. 1).

In Vitro Direct Mutagenesis

Site-directed mutagenesis to introduce an NdeI site upstream from the coding region is performed on single-stranded MI 3 templates with oligonu- cleotides synthesized on an automatic DNA synthesizer.

Step 1: Kinasing Oligonucleotide for Mutagenesis

Mix in an Eppendorf tube:

Oligonucleotide primer (100 pmol), 10/tl 10 × kinase buffer (500 mM Tris-HC 1, pH 8.0, 100 m M MgC12), 2/tl Dithiothreitol (DTT; 100 raM), 1/zl ATP (10 raM), 2/zl Distilled water, 4/zl Polynucleotide kinase, 5 units

Incubate for 30 rain at 37 °, 10 min at 70 °, and then store at - 2 0 °.

Step 2: Annealing the Template, the Mutagenic Kinased Oligonucleotide, and the Universal M13 Sequencing Primer

Because the universal primer lies at the 5' side of the mutagenic primer, it does not need to be kinased.

12 j. Tucker, G. Sczakiel, J. Feuerstein, J. John, R. S. Goody, and A. Wittinghofer, EMBO J. 5, 1351 (1986).

~3 A. H. Rosenberg, B. N. Lade, D. S. Chui, S. W. Lin, J. J. Dunn and F. W. Studier, GeneS6, 125 (1987).

[36] Rab PROTEINS AND GENE FAMILY IN ANIMALS 391

Stop ATG Codon

Site-directed ~utagenesis to create ~del site

I Prepare double.stranded [ mutagenized M13/Rab cDNA and isolate the I Ndel.BamHl fragment I

containing the $coding region

Stop Codon

N ~ BamH]

Ndel, BamHl

ctor

~ ~ x ' , . ~ i g a t i o n /

C A T ~ V St°p

pEr-$c/Rab eDNA 7 ) E;(oression olasrnid

Fro. I. Scheme for the construction of rab/pET-3c expression vector, pET-3c expression vector carries the bacteriophage T7 ~bl 0 promoter (Ptb 10), a Shine-Delgarno (SD) sequence, and 11 amino acids of the T7 bacteriophage major eapsid protein inserted between NdeI and BamHl restriction sites. The NdeI site (CATATG) is located at the translation site and used to construct the rab/pET-3e plasmid. The Rab eDNA in MI3 is represented by a box; A, noncoding sequence; CR, coding region; B, Y-noncoding sequence.

392 I D E N T I F I C A T I O N O F T R A N S P O R T I N T E R M E D I A T E S [36]

Mix in an Eppendorf tube:

Kinased mutagenic oligonucleotide (10 pmol), 2/zl M13 universal primer (10 pmol), l/zl M 13 template (1/zg), 1/zl 10× TM buffer (100 m M Tris-HC1, pH 8.0, 100 mMMgC12, 1/zl Distilled water, 5/zl

Heat to 80 ° and let cool to room temperature.

Step 3: Extension-Ligation

Add the following compounds to the tube:

10 × TM buffer 1/zl dNTPs (5 mM), 1/tl ATP (5 raM), 1/A DTT (100 raM), 1 ~ul Distilled water, 4/11

Add 10 units of T4 DNA ligase and 2 units of DNA polymerase Klenow fragment (cloned) (Amersham, Arlington Heights, IL). Then incubate overnight at 14 ° .

Ligation mixture (0.5 to 1.0/11) is used to transform E. coli strain JM 105. M 13 plaques are transferred to nitrocellulose filters and hybridized to 32P-labeled oligonucleotide. Hybridization is carried out at a tempera- ture corresponding to Tm= 3GC + 2AT, in 5 SSPE, 5X Denhardt's solu- tion, 0.1% (v/v) sodium dodecyl sulfate (SDS) for 4 hr. Filters are washed in 2X SSC (1X SSC: 0.15 M NaC1, 0.015 M sodium citrate), 0.1% (w/v) SDS for 15 min at room temperature, and 20 min at Tm+ 2*. Single- stranded DNA is prepared from positive plaques and sequenced to confirm the mutagenesis.

Expression of Rab Protein in Escherichia coli

1. After the introduction of the NdeI site upstream the rab coding sequence, prepare the corresponding double-stranded M 13 vector, and cut with NdeI- BamHI.

2. Clone the NdeI-BamHI insert containing the complete tab coding sequence, including the ATG codon, into the bacteriophage T7 promoter expression plasmid pET-3c.

3. Transform E. coli strain BL21(DE3) lysS and select for ampicillin- resistant (50/zg/ml) and chloramphenicol-rcsistant (12.5/~g/ml) transfor- mants. This strain is a lysogen bearing the bacteriophage T7 polymerase gene under the control of the lacUV5 promoter. It also bears the plasmid

[36] Rab PROTEINS AND GENE FAMILY IN ANIMALS 393

plysS carrying the bacteriophage T7 lysozyme gene. T7 lysozyme is a specific inhibitor of T7 RNA polymerase. The presence of plysS in a host that carries the inducible gene for T7 RNA polymerase increases the tolerance for toxic target plasmids) 4

4. Identify the correct recombinant plasmid either by minipreparation, analysis or by hydridization with the appropriate probe.

5. Inoculate LB medium containing 100 and 12.5 #g/ml of ampicillin and chloramphenicol, respectively, and incubate overnight at 37 ° to obtain a saturated culture.

6. Dilute the saturated culture 1 : 50 in LB medium containing 50/~g/ ml ampicillin and incubate for 1.5 hr at 37 °. Remove 1 ml of the culture. Induce the remaining culture by adding isopropylthio-fl-D-galactoside (IPTG) to a final concentration of 0.4 mM. Continue the incubation of both cultures at 37" for 1 hr.

7. Centrifuge in an Eppendorf microfuge at 12,000 g for 3 min at 4 °. Resuspend each pellet in 100 #1 of SDS gel-loading buffer. Extracts from induced and uninduced cultures are boiled 5 min and 15 #1 is subjected to a 15% (w/v) SDS-polyacrylamide gel. Clones expressing the Rab proteins are identified by Coomassie blue staining and GTP binding (see below). Amounts of produced Rab proteins are estimated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). They might attain as much as 5 - 10% of the total E. coli proteins.

Purification of Rab Proteins

Cell Culture

1. Inoculate 250 ml of an overnight culture ofE. coli DE3 (BL21)plysS carrying the rab/pET-3c recombinant plasmid in LB medium (with 50 pg of ampicillin/ml) in 5 liters of the same medium. Incubate for 1.5 hr at 37 ° .

2. Induce the production of the Rab proteins with 0.4 m M IPTG for 5 hr at 37 °. The cells are harvested by centrifugation and washed once with buffer A (see below) plus 100 m M NaC 1 to give - 30 g of cell paste, which is stored at - 7 0 ° .

Isolation Procedure

The solubilization and purification procedures of Rab proteins are carried out according to the method previously described.~2 All the purifi- cation steps are carried out at 4 ° .

14 B. A. Moffatt and F. W. Studier, Cell 49, 221 (1987).

394 IDENTIFICATION OF TRANSPORT INTERMEDIATES [ 3 6 ]

1. Frozen bacteria (30 g) are resuspended in 150 ml of buffer A [50 m M Tris-HC1, pH 7.8, 1 m M sodium azide, 0.5 m M DTT, 0.1 m M phenylmethylsulfonyl fluoride (PMSF)].

2. Add 300 pl of 0.5 M ethylenediaminetetraacetic acid (EDTA) and 60 mg of lysozyme. Homogenize the bacterial suspension. Incubate at 4 ° for 30 min. Add 850/tl of 5% (v/v) sodium deoxycholate and 20 mg of DNase. After a further 30-min incubation at 4 °, the suspension (less viscous) is centrifuged at 25,000 rpm for 30 min in an SW28 rotor (Beck- man, Palo Alto, CA).

3. QAE-Sepharose column: The supernatant (160 ml) is applied to a column of QAE-Sepharose fast flow (2.5 × 25 cm) (Pharmacia-LKB Bio- technology, Inc., Piscataway, NJ) equilibrated with 5 vol of buffer B (buffer A plus 10 m M MgC12). The column is washed with the same buffer until the OD280 m ~ 0.100 and eluted with an 800-ml linear gradient from 0 to 0.4 M NaC1 in buffer B. The flow rate is 25 ml/hr. Fractions of 5 ml are collected. Fractions containing Rab proteins (as detected by SDS-PAGE and GTP binding) are pooled.

4. Ammonium sulfate precipitation: Precipitate the pooled fraction to 60% saturation with ammonium sulfate. After 30 min, the precipitate is recovered by centrifugation, dissolved in 3 ml buffer B, and clarified again by centrifugation.

5. Load the dissolved precipitate on two consecutive columns of AcA 54 (IBF, France) (2.5 × 200 cm) equilibrated with buffer C (buffer B + 0.1 m M G D P and 200 m M NaC1). The column is eluted with buffer C at a flow rate of 20 ml/hr. Fractions (2.5 ml) are collected. The peak of GTP-binding activity is located and fractions containing Rab proteins relatively free from contaminants are pooled and brought to 70% satura- tion with ammonium sulfate. The precipitate is resuspended in 3 ml of buffer B and dialyzed against the same buffer at 4 ° overnight. Human Rab proteins with a purity exceeding 90-95% are obtained. Protein concentra- tion is determined by the method of Bradford I~ using bovine serum albu- min for calibration. [The remaining impurities can be removed by an additional purification step on a Mono Q column (Pharmacia-LKB Bio- technology, Inc.)

GTP-Binding Assay

Guanine nucleotide-binding proteins are a class of enzymes that per- form diverse functions by the same general principles, namely a conforma- tional change between an active GTP state and an inactive GDP state, which is triggered by the intrinsic GTPase activity.

15 M. M. Bradford, Anal. Biochem. 72, 248 (1976).

[36] Rab PROTEINS AND GENE FAMILY IN ANIMALS 395

pKM-tacI pET-3C

<

30K •

21.5K •

FIG. 2. GTP binding of human Rab proteins. Total proteins from induced and nonin- duced E. coli carrying rab expression vectors as indicated in the figure were solubilized by boiling for 5 rain in SDS sample buffer, separated on SDS-PAGE, and transferred to nitrocel- lulose. The filter was probed with [o#2P]GTP. NI, Noninduced bacteria transformed with pKM.tacI (rab5) or with pET-3c (rab4).

The nucleotide-binding capacity of Rab protein can be analyzed by Western blot. This method has been shown to allow the renaturation of some GTP-binding proteins. ~6 Under the conditions described below, the ability of Rab proteins to bind GTP after transfer onto nitrocellulose filters is highly variable (Fig. 2). No quantitative conclusions on the respective amounts of different proteins can be drawn by this method.

1. Rab proteins are separated on 15% (w/v) polyacrylamide-SDS gels. Gels are incubated for 30 min at 4 ° in phosphate-buffered saline (PBS: 50 m M Na2HPO4/KH2PO4, pH 7.2, O. 12 MNaCI) . The proteins are then electrophoretically transferred to nitrocellulose paper (BA85, 0.45 #m; Schleicher & SchueU, Keene, NH) in 25 m M Tris/0.15 m M glycine and 20% methanol as transfer buffer.

2. The transfer filter is washed for 1 hr in buffer A [ 10 m M Tris-HC 1, pH 7.8, 0.15 M NaC1, 5% (w/v) bovine serum albumin (BSA), and 0.05% (v/v) Tween 20].

3. Incubate the filter for 30 min at room temperature in l0 ml of binding buffer [20 m M Tris-HCl, pH 7.8, l0 m M MgCl2, 2 m M D T T ,

16 E. Lapetina and B. Reep, Proc. Natl. Acad. Sci. USA 84, 2261 (1987).

396 IDENTIFICATION OF TRANSPORT INTERMEDIATES [36]

0.1% (v/v) Nonidet P-40, and 0.3% (w/v) BSA] containing 30/iCi of [a-32p]GTP (3000 Ci/mmol; Amersham).

4. Wash the filter briefly (twice, 3 min each) in 100 ml of buffer [10 mM Tris-HCl, pH 7.8, 0.15 M NaC1, 0.5% (v/v) BSA, 0.5% (v/v) Triton X-100, and 0.2% (v/v) SDS]. The filter is dried and autoradio- graphed (Fig. 2).

The same procedure could be used to analyze the binding of other nucleotides and to study the competition of guanine nucleotide binding with that of other nucleotides, in which case a 10-fold excess of nonlabeled nucleotide is present in the incubation solution. In our laboratory, we have performed the competition for GTP binding as follows: 1 gg of purified Rab protein was incubated for 10 min in 50 mM Tris buffer, pH 7.5, containing 1 mMDTT, 2 mMEDTA, 1 gA/[a-32p]GTP (10 Ci/mmol)in the presence or absence of 100 gM nonlabeled GTP, GDP, GTP~,S, ATP, CTP, TTP, and UTP. Samples were passed through nitrocellulose filters (BA85, 0.45/~m; Schleicher & Schuell), washed three times with 3 ml of ice-cold buffer (20 mM Tris-HCl, pH 8, 5 mM MgC12, 10 mM NH4C1, 0.5 mM DTT, and 0.1 M KC1), and dried. The radioactivity bound to the filter was determined by liquid scintillation counting. Both techniques showed that Rab proteins specifically bind GTP and GDP.

GTPase Assay

Rab proteins, like all the Ras and Ras-related proteins, hydrolyze GTP very slowly. 6 We used the purified Rab proteins to test their capacities to hydrolyze GTP. Equal amounts of active Rab proteins, as determined by their GTP binding, were incubated with radioactive GTP. The GTP hy- drolysis can be measured.

1. Rab proteins (I gM) are incubated for 15 rain at 37 ° in a total volume of 100/zl in a solution containing 50 mM Tris-HC1, pH 7.8, 2 mM EDTA, 1 mM DTT, 10 gM [a-32p]GTP (2000 cpm/pmol). Under these conditions (EDTA > Mg 2+) GDP/GTP exchange is promoted.

2. GTPase activity is initiated by adding MgC12 to a final concentra- tion of 10 mM. Samples of 5 gl are withdrawn from the incubation mix- ture at different times, mixed with 5/A of a solution consisting of 0.2% (v/v) SDS, 5 mM EDTA, 50 mM GDP, and 50 mM GTP at 4*. Samples are then heated at 70* for 2 min to dissociate protein-bound nucleotides and l-gl aliquots are spotted on polyethyleneimine-cellulose thin-layer chromatography plates. They are developed in 0.6 M sodium phosphate buffer, pH 3.4, for 25 min, dried, and autoradiographed. GTP-bound hy- drolysis is followed here by the generation of GDP. The GTP and GDP

[36] Rab PROTEINS AND GENE FAMILY IN ANIMALS 397

B A t t

C

1

0 15 30 60 120 0 15 30 60 120 0 15 30 60 120

FIG. 3. GTP hydrolysis of human Rab proteins. Bacterially produced, purified proteins (1 gM final concentration) were incubated at 37* for 10 rain with 10/tM [a32P]GTP in the presence of 2 mM EDTA. The GTPase activity was started by addition of MgC12 (10 mM final concentration). Aliquots were withdrawn from the incubation mixture at the indicated times (in minutes) and analyzed by thin-layer chromatography. A, Rabl; B, Rab2; C, Rab3A; D, Rab4; E, Rab5; F, Rab6.

spots are excised and radioactivity is measured by liquid scintillation counting (Fig. 3).

The GTPase activity can also be measured by release of [32p]p i. In this case GTP hydrolytic activity is detected by preequih'brating Rab proteins with [7-J2p]GTP, incubating the mixture at 37 °, and then quantitating the decrease in radiolabeled Rab-GTP complex by nitrocellulose filtration? 7

t7 C. Leupold, R. S. Goody, and A. Wittinghofer, Eur. J. Biochem. 124, 237 (1983).