Báo cáo Y học: Molecular cloning, bacterial expression and properties of Rab31 and Rab32 docx

Molecular cloning, bacterial expression and properties of Rab31 and Rab32 New blood platelet Rab proteins Xiankun Bao 1 , Andrea E. Faris 2 , Elliott K. Jang 1 and Richard J. Haslam 1,2 Departments of 1 Pathology and Molecular Medicine and 2 Biochemistry, McMaster University, Hamilton, Ontario, Canada GTP-binding proteins of the Rab family were cloned from human platelets using RT-PCR. Clones corresponding to two novel Rab proteins, Rab31 and Rab32, and to Rab11A, which had not been detected in platelets previously, w ere isolated. The coding sequence of Rab31 (GenBank acces- sion no. U59877) corresponded to a 194 amino-acid protein of 21.6 kDa. The Rab32 sequence was exten ded to 1000 nucleotides including 630 nucleotides of coding sequence (GenBank accession no. U59878) but t he 5¢ coding sequence was only completed later by others (GenBank accession no. U71127). Human Rab32 cDNA encodes a 225 amino-acid protein of 25.0 kDa with the unusual GTP- binding sequence DIAGQE in place of DTAGQE. North- ern blots for Rab31 and Rab32 identi®ed 4.4 kb and 1.35 kb mRNA species, respectively, in some human tissues and in human erythroleukemia (HEL) cells. Rabbit polyclonal anti-peptide antibodies to Rab31, Rab32 and Rab11A detected platelet proteins of 22 kDa, 28 kDa and 26 kDa, respectively. Human platelets were highly enriche d in Rab11A ( 0.85 lgámg of platelet protein )1 ) a nd contained substantial amounts of Rab32 (0.11 lgámg protein )1 ). Little Rab31 was present (0.005 lgámg protein )1 ). All three Rab proteins were found in both granule and membrane frac- tions from platelets. In rat platelets, the 28-kDa R ab32 was replaced by a 52-kDa immunoreactive p rotein. Rab31 and Rab32, expressed as glutathione S-transferase (GST)-fusion proteins, did not bind [a- 32 P]GTP on nitrocellulose blots but did bind [ 35 S]GTP[S] in a Mg 2+ -dependent manner. Bind- ing of [ 35 S]GTP[S] was optimal with 5 l M Mg 2+ free and was markedly inhibited by higher Mg 2+ concentrations in the case of GST±Rab31 but not GST±Rab32. Both proteins displayed low steady-state GTPase activities, w hich were not inhibited by mutations (Rab31 Q64L and Rab32 Q85L ) that abolish the GTPase activities of most low-M r GTP- binding proteins. Keywords: Rab protein; GTP-binding protein; GTPase; Mg 2+ ; p latelet. Low-M r GTP-binding proteins of the Rab subfamily play important roles in vesicle and granule targeting [1]. Because blood platelets secrete the contents of three distinct granule types, namely dense g ranules, a-granules and lysosomes, i n response to physiological stimuli [2], the identities and subcellular locations of platelet Rab proteins are of considerable interest. Immunoblotting experiments using antibodies to known Rab proteins have demonstrated the presence in human platelets of Rab1, Rab3B, Rab4, Rab5, Rab6 and R ab8 [3,4], a s well as Rab27A [5,6] and Rab27B [6,7]. Rab6 and R ab8 were detected on a-granules [3], although evidence has been presented that Rab4 regulates a-granule secretion [ 4]. Over 50 different Rab proteins have now been identi®ed, some of which are highly tissue- or cell- speci®c [1,8]. Consequently, it is not certain that all the major p latelet Rab proteins have been identi®ed. We therefore adopted a different ap proach to the identi®cation of Rab proteins in human platelets and cloned sequences from platelet mRNA by RT-PCR, using a degenerate oligonucleotide corresponding to the conserved protein sequence WDTAGQE, found in members of t he Rab and Rho families of low-M r GTP-binding proteins. Antibodies to unique C-terminal peptide sequences were then prepared and used to con®rm the presence of the proteins in platelets. By these methods, we identi®ed two previously unknown Rab proteins, Rab31 and Rab32, in human platelets and also demonstrated the presence in these platelets o f large amounts of Rab11A. Here, we d escribe the cloning and tissue distributio n o f R ab31 and Rab32, their bacterial expression and s ome unusual b iochemical properties of t he recombinant p roteins. Brief reports of some of our ®ndings have been published in abstract form [9,10]. EXPERIMENTAL PROCEDURES Materials An AmpliFINDER TM RACE kit, a Marathon TM cDNA ampli®cation kit and a human multiple tissue Northern blot were obtained from Clontech Laboratories Inc. Correspondence to R. J. Haslam, Department of Pathology and Molecular Medicine, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5. Fax: + 905 777 7856, Tel.: + 905 525 9140 Ext. 22475, E-mail: haslamr@mcmaster.ca Abbreviations: ACS, aqueous counting scintillant; ECL, enhanced chemiluminescence; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GTP[S], guanosine 5¢-O-(c-thio)triphosphate; GST, glutathione S-transferase. Enzyme: g lutathione S-transferase ( EC 2.5.1.18). (Received 10 July 2001, revised 16 October 2001, accepted 30 October 2001) Eur. J. Biochem. 269, 259±271 (2002) Ó FEBS 2002 [a- 32 P]dCTP (3000 Ciámmol )1 )and[ 35 S]guanosine 5¢-O- (c-thio)triphosphate ([ 35 S]GTP[S], 1250 Ciámmol )1 )were from NEN and [a- 32 P]GTP (> 3000 Ciámmol )1 )was from ICN Pharmaceuticals. Immobilon-P membrane for blotting proteins, HAWP ®lters (0.45 lm, 25 mm) and Centricon YM-10 ®lters were from Millipore. RPMI 1640 medium, f oetal bovine serum, L -glutamine, T4 D NA ligase, Taq DNA polymerase, MMLV r everse transcriptase, Superscript TM II reverse transcriptase/Taq mix and restriction enzymes were all from Life Technologies. QIA- quick TM PCR puri®cation kits and QIAprep TM plasmid DNA puri®cation kits were from Qiagen. pBluescript SK+ DNA was from Stratagene. pGEX-4T-1 DNA, glutathione-Sepharose 4B, aqueous counting scintillant (ACS), secondary antibody for immunoblotting and enhanced chemiluminescence (ECL) reagents w ere f rom Amersham Pharmacia Biotech. Avid A L c olumns were from BioProbe International. UltraLink Iodoacetyl gel for peptide a f®nity-puri®cation of antibodies was from Pierce. Darco G60 activated carbon was from Fisher Scienti®c. Oligonucleotides and peptides were synthesized and DNA sequenced in the Central Facility of the Institute f or Molecular B iology and Technology (McMaster University, Canada). The PEPTOOL TM program used f or sequence alignment w as obtained from B ioTools Inc. The authors are very grateful to P. D. Stahl (Washington University School of Medicine, St Louis, MO, USA) f or providing Escherichia coli expressing GST±Rab5A [11]. A sample of recombinant Rab11A protein [12] was generously supplied by J. R. Goldenring (Institute for M olecular Medicine and Genetics, Medical College of Georgia, Augusta, GA, USA). Iloprost was a gift from Schering AG. Human cell lines MEG-01 cells (a megakaryoblastic leukemia cell line) were donated b y K . K. Wu ( University of Texas, H ealth Science Center, Houston, TX, USA). Cultures of other human cell lines were obtained from the following sources in the Department of Pathology a nd Molecular Medicine at McMaster University, Hamilton, Canada: HEL cells (an erythroleukemia cell line) from B. J. Clarke, K562 cells (a multipotential haematopoietic cell line) and Jurkat cells (a T-cell line) from K. Rosenthal, and KU812 ce lls (a basophilic leukemia cell line) from J. Marshall. Cell lines were routinely grown in RPMI 1640 m edium supplemented with antibiotics and 10% foetal bovine serum (heated at 56 °C for 30 min). L -Glutamine (0.03%, w/v) was added into the medium for MEG-01 cells only. Isolation of human platelets and their subcellular fractionation Platelets were isolated by a modi®cation of the method of Mustard et al. [13]. Blood (100 mL) w as taken from healthy human donors and centrifuged for 5 min at 140 g to separate platelets from o ther blood cells. T o minimize contamination by other cells, only the top one third of the platelet-rich plasma was collected when mRNA was prepared. The platelets were isolated by centrifugation for 5 min at 1160 g and the pellet was washed three times in 10 mL of Ca 2+ -free Tyrode's solution containin g 0.35% BSA, 5 m M Pipes, pH 6.5, 90 lgámL )1 of apyrase, 50 IU ámL )1 of heparin and 20 n M iloprost. Care was taken to remove any residual red and white cells during washes. In s ome experiments, platele t cytosol and partic- ulate fractions enriched in either granules or membranes were prepared by differential centrifugation of platelet sonicates [14]. Isolation of mRNA and total RNA Micro-FastTrack mRNA Isolation Kits (Invitrogen) were used to extract mRNA from platelets and HEL cells, whereas TRIzol Reagent (Life Technologies) was used to isolate total RNA from HEL, K562 and Jurkat cells, according to the manufacturer's protocol. Cloning of platelet Rab cDNA sequences cDNA was synthesized from human platelet mRNA using MMLV reverse transcriptase and an RT primer (5¢-GGACTAGTGTCGACAAGCTTGAATTCT 17 -3¢, 43-mer) consisting of oligo-dT with four added restriction sites (SpeI, SalI, HindIII and EcoRI, shown in bold). The RT reaction mixture was then added to a PCR cocktail. The sense oligonucleotide used for PCR ampli®cation was a 128-fold degenerate oligonucleotide encoding the amino- acid sequence, WDTAGQE, with BamHI and XbaI restriction sites (in bold) at the 5¢ end (5¢-CGGGATCCTCT- AGATGGGA(T/C)AC(A/G)GC(A/T/C/G)GG(A/T/C/G) CA(A/G)GAG-3¢, 35-mer). In some r eactions, this primer was replaced by an oligonucleotide identical except for the replacement o f t he 3 ¢Gby3¢A. Two separate 5 ¢ primers were used to avoid degeneracy in any of the three bases at their 3¢ ends. The antisense oligonucleotide w as a 26-mer adaptor identical to the 5 ¢ half of the R T primer. PCR w ith Taq DNA polymerase was carried out by heating the mixtur e a t 9 5 °C for 2 min, followed by 4 0 cycle s of 1 min at 9 5 °C and 4 min at 68 °C, and t hen a ®nal 7 min a t 72 °C. The products were then cloned into pBluescript SK+ using the XbaIandHindIII or EcoRI restriction sites and inserts larger than 450 bp were sequenced. After identi®cation o f novel Rab cDNAs, the 5¢ nucleotide sequences were obtained by 5 ¢-RACE, using antisense primers based on 5¢ sequences of partial clones of Rab proteins and either an AmpliFINDER TM RACE kit or a Marathon TM cDNA ampli®cation kit. PCR products were then cloned into pBluescript SK+. After a complete (Rab31) or nearly complete (Rab32) sequence was assem- bled, the ORF was reampli®ed from a platelet Marathon TM cDNA library using a ppropriate sp eci®c primers, cloned i n pBluescript SK+ and resequenced in both directions to verify the assembled sequence. Northern blotting analysis Total RNA from HEL, K562 and Jurkat cells was electrophoresed on a 1% agarose/formaldehyde gel, blotted on to nylon membrane and cross-linked with UV light. Probes ( 50 ngámL )1 and 10 7 c.p.m.ámL )1 ) labelled with [a- 32 P]dCTP were prepared by PCR ampli®cation of Rab cDNA sequences encoding amino-acid residues f rom t he C-terminal halves of the proteins ( and 3¢-untranslated sequence), as follows: Rab31, nucleotides 366±788; Rab32, nucleotides 546±868 (see Fig. 1A,B). For a control probe, 260 X. Bao et al. (Eur. J. Biochem. 269) Ó FEBS 2002 nucleotides 252±793 of human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA (GenBank accession no. M33197) were ampli®ed f rom a HEL cell cDNA library (prepared using the Marathon TM cDNA ampli®cation kit). Hybridization was carried out at 68 °C f or 1 h as described previously [15]. The same membranes w ere probed succes- sively for Rab31, Rab32 and GAPDH mRNAs, with intermediate stripping by heating at 100 °Cin0.5%SDS for 10 min. Immunoblotting Rabbit anti-peptide antibodies were prepared to peptide sequences in the hypervariable C-terminal regions of cloned R ab proteins. T he peptides synthesized were (CH 3 CO)TIKVEKP TMQASRRC for Rab31 (Fig. 1 A), (C)NEENDVDKIKLDQE(CONH 2 ) for Rab32 (Fig. 1B), and (C)QKQMSDRRENDMS(CONH 2 ) f or Rab11A (amino-acid residues 178±190). These peptides were coupled to keyhole limpet haemocyanin via their endogenous (Rab31) or added (Rab32, Rab11A) cysteine residues, using 4-(N-maleimidomethyl) cyclohexane-1-carboxylate. Rabbits were immunized by intradermal injection of the conjugated peptides (0.5±1.0 mg) with Freund's complete adjuvent. Sera with adequate titres were obtained a fter 3±4 boosts with conjugated peptide i n incomplete adjuvent. Protein for immunoblotting was analysed by SDS/PAGE, using 13% acrylamide in the separating gel, and then transferred electrophoretically to Immobilon-P. Immunoreactive pro- teins were detected using the rab bit immune sera, immune IgG puri®ed on Avid AL columns or antibody af®nity- puri®ed on an UltraLink I odoacetyl column containing covalently bound peptide. Bound antibody was visualized using horse-radish peroxidase-conjugated donkey anti- (rabbit IgG) Ig as t he se condary antibody and ECL reagents. Bacterial expression of Rab31 and Rab32 To generate an expression construct, Rab31 cDNA was ampli®ed from a platelet Marathon TM cDNA library, using as PCR primers, 5¢-TAGGATCCGCGATACGGGAGC- TCAAAG-3¢ (P31-1) and 5 ¢-ATCTCGAGGATGTGGG- Fig. 1. Nucleotide and deduced amino-acid s equences of Rab31 and Rab32. (A) Rab31. The nu cleotide sequence shown ( Ge nBank a cc ession no. U59877) was obtained as described under Experimental procedures. An almost identical cDNA cloned at the same time from human melanocytes [30] diers by two bases and one amino acid in the open reading frame (see box). (B) Rab32. The nucleotide sequence shown is derived from two clones, o ne obtained as describ ed i n the Experimental proc edures (GenBank accession no. U59878) and a second, which completed the 5¢ end of the open reading frame, obtained later from GenBank (U71127). Sequence variants found in Rab32 cDNA from HEL cells are boxed. For both Rab31 and Rab32, the conserved amino-acid sequences involved in binding GDP/GTP are shown white on black, the glutamine residues mutated in this study a re shade d and the pe ptide sequences used for generating a ntib odies are doubly un derlined. The nucleotide se qu ences that were u sed f o r ampli®cation of cD NAs that were ligated into pG EX -4T-1 are also indicated (P31-1, P31-2, P32-1, P32-2, see E xperim ental procedure s). Ó FEBS 2002 Rab proteins from platelets (Eur. J. Biochem. 269) 261 CTCTGGCTTCT-3¢ (P31-2), containing BamHI and Xh oI restriction sites (in bold), respectively (Fig. 1A). The PC R reaction conditions (with Taq DNA polymerase) were 1 min at 95 °C, 2 min at 60 °C (®rst cycle) or 2 min at 65 °C (remaining cycles) a nd 2 min at 72 °C, for a tot al o f 21 cycles. T he PCR p roduct was puri®ed using a QIAquick TM kit and cloned into the BamHI and XhoIsitesofthe pGEX-4T-1 expression vector. The sequence of the insert was veri®ed in both directions and competent E. coli BL21 (DE3) cells transformed. In the expressed GST±Rab31 fusion protein, the initiating methionine of Rab31 was replaced by GS residues (from the BamHI restriction site). To verify the coding seq uence of Rab32 and generate an expression construct, human platelet mRNA was ®rst isolated using a Micro-FastTrack TM kit. Reverse trans crip- tion of this platelet mRNA was carried out using a Superscript TM II RT/Taq mix (30 min at 55 °Cand2min at 94 °C) and a primer (P32-2) containing XhoIandSalI restriction sites (in bold) and the complement of nucleotides 700±724 of Rab32 cDNA (5¢-AAGCTCGAGTCGAC- TTCTTCAGAGCTGAGGCACACAC-3¢). The resulting cDNA was a mpli®ed by PCR using 5¢-TGGGATCC- GGAGGAGCCGGGGACCCCGGCCTG-3¢, containing a BamHI si te, a s the 5¢ primer (P32-1) and P32-2 as the 3¢ primer (Fig. 1B). The PCR reaction conditions (with Taq DNA polymerase) were 1 min at 94 °C, 2 min at 60 °Cand 2 min at 72 °C, for 3 5 cycles. The p roduct was c loned into the BamHI a nd XhoI sites of pGEX-4T-1 a nd sequenced in both directions. Th e GST±Rab32 fusion protein was expressed in E. coli BL21(DE3) cells, as for Rab31. In this fusion pro tein, the ®rst three a mino-acid residues of Rab32 (MAG) were replaced by GS residues. To express GST-fusion proteins and GST itself, 4 mL of Luria±Bertani medium (containing 100 lg of ampicillin per mL) was inoculated with E. coli BL21(DE3) cells contain- ing the appropriate pGEX-4T-1 construct and grown overnight at 37 °C. This culture was used to seed a larger culture (200±500 mL), which was grown at 37 °C until D  0.5. The fusion protein was then induced with 0.1 m M isopropyl t hio-b- D -galactoside and the culture grown for a further 3 h, when the b acteria were isolated by centrifuga- tionandfrozenat)70 °C until needed. B acterial pellets (each from 50 m L of culture) were resuspended in 9.8 mL of NaCl/P i (pH 7.4) containing lysozyme (100 lgámL )1 ). After incubation of the cells for 30 min at 0 °C, 1 m M phenylmethanesulfonyl ¯uoride and 5 m M dithiothreitol were ad ded and the cells were sonicated f or 10 min in a bath sonicator. Bacterial supernatant was then isolated by centrifugation and mixed with Triton X-100 (®nal concen- tration 0.1%), MgCl 2 (10 m M ) and glutathione-Sepharose 4B beads. After s haking the mixture for 60 min at room temperature, the beads were isolated, washed three times with NaC l/P i containing 0.1% Triton X-100 and 10 m M MgCl 2 andthenelutedwith10m M reduced glutathione in 50 m M Tris/HCl (pH 8.0) containing 10 m M MgCl 2 .The eluted protein was concentrated by centrifugation at 4 °C in a Centricon ®lter, diluted with Buffer A (100 m M KCl, 20 m M Hepes, pH 7.5, 1 m M EDTA, 1 m M dithiothreitol) containing 10 m M MgCl 2 , and reconcentrated. GST-fusion proteins were stable in this solution for 1±3 weeks at 4 °C. Protein concentrations were determined by the Lowry method using Sigma protein standard diluted in Buffer A. Inclusion of MgCl 2 in the above solutions was essential to obtain GST±Rab proteins capable o f binding guanine nucleotides. Mutagenesis of Rab31 and Rab32 An attempt was made to create constitutively active forms of these R ab proteins by mutating the g lutamine re sidue in the DTAGQE GTP-binding motif to a leucine r esidue (Fig. 1A,B). A dual PCR method [16] was applied to the Rab31 and Rab32 c onstructs in pGEX-4T-1 t o g ive clones encoding GST±Rab31 Q64L and GST±Rab32 Q85L ,respec- tively. These proteins were expressed and puri®ed, as described for GST±Rab31 and GST±Rab32. Binding of [a- 32 P]GTP by GST±Rab proteins on nitrocellulose blots After SDS/PAGE, GST±Rab proteins were electroblotted onto nitrocellulose, renatured and p robed w ith [a- 32 P]GTP by two different methods [17,18]. I n one, t he binding buffer contained 2 l M MgCl 2 [17] and in t he other, 10 m M MgCl 2 [18]. Binding of [ 35 S]GTP[S] by GST±Rab proteins A modi®cation of the method of Kabcenell et al. [19] was used. Puri®ed protein (10±200 pmol) was incubated at 37 °C in Buffer A containing [ 35 S]GTP[S] (usually 5 l M at a speci®c radioactivity of 0.5 Ciámmol )1 ) and any additional MgCl 2 required to g ive a de®ned concentration of Mg 2+ free (usually 5 l M or 10 m M ). The amount of MgCl 2 required was calcu lated u sing a computer version of the programme described by Fabiato & Fabiato [20] and the binding constants used by the same authors. At speci®c times, triplicate 10 lL samples of the incubation mixture were diluted into 100 lL of wash buffer [100 m M KCl, 20 m M Hepes (pH 7.5), 0.5 m M MgCl 2 ] and immediately applied to HAWP ®lters in a Millipore vacuum ®ltration unit. After three washes with 2 mL of wash buffer, the ®lters were placed in vials with 0 .5 mL of water a nd 8 mL of A CS and counted for 35 S by liquid scintillation. After correction for the [ 35 S]GTP[S] observed on ®lters from control incubations without protein, t he results w ere e xpressed as p mol of [ 35 S]GTP[S] bound per 100 pmol of protein ( mean  SE); this is equivalent to the percentage of protein containing bound [ 35 S]GTP[S]. GTPase assays The GTPase activities of GST±Rab proteins were measured by a modi®cation o f the method of Kabcenell et al.[19]. Puri®ed protein (10±200 pmol) was incubated at 37 °Cin 40±200 lL o f Buffer A containing [c- 32 P]GTP (usually 5 l M at a speci®c radioactivity of 0.5 Ciámmol )1 )and suf®cient additional MgCl 2 to give the required concentra- tion of Mg 2+ free (usually 5 l M or 10 m M ). At appropriate times (usually 180 min), triplicate 10 lLsampleswere mixed with 0.75 mL of 50 m M NaH 2 PO 4 (at 0 °C) containing 10 m M EDTA and activated carbon (5% w/v) to remove unhydrolysed [c- 32 P]GTP. After centrifugation, 0.4 m L of each supernatant was diluted in 0 .01% 4- methylumbelliferone and counted for C Ï erenkov radiation. Nonenzymatic release of [ 32 P]P i was subtracted. GTPase 262 X. Bao et al. (Eur. J. Biochem. 269) Ó FEBS 2002 activity was expressed as nmol of GTP hydrolysedámg protein )1 ámin )1 . RESULTS Cloning of platelet Rab proteins We used degenerate sense primers corresponding to the conserved sequence WDTAGQE and 3¢-RACE to amplify Rab-related sequences from platelet cDNA. Previously, this consensus se quence had been successfully used to clone the 5¢ ends of Rab sequences from mouse kidney [21]. Although the deoxynucleotides corresponding to the speci®c trypto- phan residue were at the 5¢ end of the primers t hat we used, we found that the s equences ampli®ed were restricted to members o f t he Rab and Rho families o f l ow-M r GTP- binding proteins. The 3¢ sequences that we obtained were extended i n the 5¢ direction by 5¢-RACE. In this study, two novel Rab s equences (Rab31 and Rab32) were cloned from platelet mRNA (Fig. 1A,B). These sequences were readily recognized as those of low-M r GTP-binding proteins, in that in addition to the D XXG sequence present in the cloning primer, they encoded the GXXXXGK(S/T), NKXD and EXSA amino-acid residues also i nvolved i n binding GDP or GTP [22] (Figs 1A,B and 2). In addition, the glycine residue present in the Switch I region and the two C-terminal cysteine residues found in both Rab31 and Rab32 are characteristic of Rab proteins [22]. The coding sequence of Rab31 that we obtained (Gen- Bank accession no. U59877) corresponded to a 194-residue protein with a nominal molecular mass (ignoring p renyla- tion) of 21.6 kDa. Of two adjacent potential translation initiation codons (nucleotides 58±60 and 61±63 in Fig. 1A) only the second is surrounded by a plausible K ozak consensus sequence [23]. A 5¢ in-frame stop codon (nucle- otides 28±30 in Fig. 1A), precludes translation of a larger protein. The Rab32 sequence obtained by ourselves (Gen- Bank accession no. U59878) was incomplete but a later sequence submitted by Seabra and colleagues (GenBank accession no. U71127) completed a plausible coding sequence with an additional 15 amino acids at the N-terminus, though no 5¢ in-frame stop codon was found. This sequence of Rab32 encodes a 225-residue protein with a nominal m olecular mass of 25.0 kDa (Fig. 1B). In a Rab32 clone o btained later from HEL cells, we observed two nucleotide (and amino acid) changes (Fig. 1 B). One unusual feature was the ®nding that the W DTAGQE s equence typical of Rab proteins was replaced by WDIAGQE in Rab32 (Fig. 1B). In addition to Rab31 and Rab32, several clones encoding human Rab11A were isolated from human platelets. For unknown reasons, clones corresponding to the Rab proteins previously detected in platelets by immunob- lotting [3,4,6] were not obtained. The Rab protein s equences most closely resembling Rab31 and Rab32 were identi®ed by BLAST searches of the NCBI nonredundant database [24] and were aligned with Rab31 and Rab32 by using PEPTOOL TM with some manual adjustments (Fig. 2). The re sults initially showed that human Rab31 was most closely related to canine Rab22 with which it shared 71% amino-acid identity. In a simultaneous study, a protein a lmost identical to our Rab31 w as cloned from human melanocytes and named Rab22B [25]. The coding sequence o f the latter differed by two nucleotides and one amino-acid residue from that Fig. 2. Alignment of the deduced amino-acid sequences of Rab31 and Rab32 with those of closely related Rab proteins. Multiple sequence alignments were carried out using the PEPTOOL TM programme; m inor manual adjustments were made t o the alignment of the N- and C- terminal amino-acid residues. Consensus sequences are shown white on black. T he individual perc ent identities of proteins related to Rab31 and Rab32 are shown on the right (% ID). Conserved residues that participate in the binding of guanine nucleotide [22] are mar ked with asterisks. The Switch I and Switch II regions (from Rab3A [54]) are also indicated. (A) The deduced amino-acid sequence of Rab31/22B (Fig. 1A) is aligned with those of human Rab22A (XM_009454), human Rab5A (U18420) and tobacco Rhn1 (P31583). (B) The deduced amino-acid sequence of Rab32 (Fig. 1B) is aligned with th ose of related Rab proteins containing an isoleucine su bstitution (.) in the PM3 GTP-binding motif [22]. These proteins are a mouse Rab32- like protein (NM_026405), human Rab38 (AF235022 [27]), Rab7L1 (D84488) [29] which is the human ortholog of rat Rab29 [30] and Dictyostelium RabE (AF116859). Hs, Homo sa piens;Mm,Mus m usculus;Np,Nicotiana p lumbaginifolia;Dd,Dictyostelium discoideum. Ó FEBS 2002 Rab proteins from platelets (Eur. J. Biochem. 269) 263 obtained b y ours elves (Fig. 1A). The next most similar Rab-related p rotein with 49% identity w as Rhn1 from tobacco [26], which is related to Rab5A (Fig. 2A). A comparison of human Rab32 with m ore recently identi®ed Rab proteins d emonstrated 84% i dentity with a mouse protein p redicted from a RIKEN cDNA clone, which is probably a murine form of Rab32 (Fig. 2B). In addition, 66% identity was observed b etween human Rab32 and human Rab38 [27], which is the human ortholog of an uncharacterized Rab p rotein previously cloned from rat alveolar type II cells (GenBank accession no. M94043). RabE from Dictyostelium [28] and human Rab7L1 [29], apparently the human ortholog of rat Rab29 [30], w ere also related t o R ab32 (Fig. 2B). T his g roup of Rab32-related proteins are c haracterized by the presence of t he WDIAGQE seque nce, as well as a high overall similarity, suggesting that they form a discrete subfamily o f Rab proteins (see Discussion). Expression of Rab31 and Rab32 Northern blots demonstrated that human tissues and cultured cells expressed a 4.4-kb Rab31 mRN A and a 1.35-kb Rab32 mRNA, though the distribution of these two mRNAs was very different (Fig. 3). Rab31 mRNA was expressed most strongly in placenta and brain and to a lesser extent in heart and lung, but no signal was detected from liver, skeletal muscle, kidney and pancreas. H EL cells, and to a lesser extent K 562 cells expressed Rab3 1 mRNA, whereas Jurkat c ells did not. In c ontrast, the 1.35-kb Rab32 mRNA was expressed in most of the human tissues examined, but particularly in heart, liver and kidney, and was also found in HEL and K562 cells (Fig. 3). A 2.0-kb Rab32 mRNA was also detected in some preparations of RNA from HEL cells. To demonstrate the presence of Rab31, Rab32 and Rab11A proteins in platelets, rabbit antibodies were gen- erated to unique peptides from the variable C -terminal regions of the proteins ( see Experimental p rocedures and Fig. 1A,B). These antibodies gave strong signals that were blocked by the peptides to which they were prepared. As shown i n Fig. 4, a ll these a ntibodies detected proteins in platelets with m olecular m asses s imilar t o or slightly higher than those predicted from their cDNA sequences (Rab31, 22 kDa; Rab32, 28 kDa; Rab11A, 26 kDa; Fig. 4). Pre- sumably, the higher values re¯ect geranylgeranylation of the proteins. For unknown reasons, HEL cells did n ot contain detectable amounts of Rab31 and Rab32, using immuno- blotting techniques. Rab31 protein was found in both MEG-01 cells and KU812 cells, whereas Rab32 was not. Rab11A was found in all cells tested, but appeared to be present in particularly large amounts in platelets (Fig. 4). To determine the amounts of these Rab proteins in platelets, immunoblots of 10±20 lg of platelet protein were compared with those of s tandard amounts o f the recombinant Rab proteins (0.5±20 ng), which were subjected to SDS/PAGE Fig. 3. Expression of Rab31 and Rab32 mRNA in human tissues and cell lines. A human multiple tissue N orthern blot ( lanes 1±8, 2 lg of p olyA+ RNA per lane) was obtained from Clontech (lane 1, heart; lane 2, brain; lane 3, placenta; l ane 4, lung; lane 5, liver; l ane 6, s keletal muscle; l ane 7, kidney; lane 8, pancreas). In addition, total RNA (12±15 lg per lane) was extracted from three human cell lines, electrophoresed on a 1% agarose-formaldehyde gel and blo tted onto a nylon membrane, as described under Experi- mental procedures (lane 9, HEL cells; lane 10, K562 ce lls; lane 11, Jurkat cells). These two membranes were probed successively with 32 P-labelled DNA (300±400 nucleotides) s yn- thesized by PCR a m pli®cation of sequences from Rab31, Rab32 and GAPDH (see Experimental procedures). Autoradiographs are shown. T he positions of RNA standards are indicated on t he left. 264 X. Bao et al. (Eur. J. Biochem. 269) Ó FEBS 2002 at the same time. These results showed that human platelets contained, per mg of total platelet protein, 0.85  0.13 lg of Rab11A, 0.11  0.02 lg of Rab32 and 0.005  0.001 lg of Rab31 ( mean valu es  SE from f our, s ix and four determinations, respectively, using platelets from different donors). The subcellular distributions of these R ab proteins were studied by differential centrifugation using a simple method shown to y ield one particulate f raction enriched in granules and mitochondria and another enriched in both plasma and intracellular membranes [14]. The results (Fig. 5) showed that Rab31, Rab32 and Rab11A were equally enriched in the granule/mitochondrion and membrane fractions and that no R ab31 or R ab32, and only a s mall amount of Rab11A, was present in the supernatant (cytosol) fraction. Rab31 and Rab11A were detected in rat platelets in amounts similar to those observed in human platelets, when the same antibodies were used. Far smaller amounts of Rab11A were observed in p rotein from rat kidney, liver, heart, lung and brain, con®rming that platelets have an exceptionally high Rab11A content (not shown). In contrast, no 28 kDa immunoreactive species corresponding to human Rab32 was found in rat platelets or in any rat tissue examined Fig. 4. Comparison of the a mounts of Rab31, Rab32 and Rab11A in human platelets and related human cell lines. Protein (30 lgperlane) from human platelets (lane 1), HEL cells (lane 2), MEG-01 cells (lane 3) and KU812 cells (lane 4) was subjected to SDS/PAG E, electro- blotted onto Immobilon-P and probed for Rab31, Rab32 and Rab11A, u sing rabbit ser a containing polyclonal antibodies raised against speci®c Rab peptides (se e Experimental proced ures). Immu- noreactive proteins were detected by ECL. The positions of prestained protein standards are s hown on the right. Fig. 5. Subcellular distributions of Rab proteins in human platelets. Protein (30 lg) from platelet lysate (lane 1 ), from platelet fractions enriched in granules (lane 2) or mem branes (la ne 3), a nd f rom the platelet sup ernata nt f ract ion ( lane 4) was subjected to SDS/PAGE and electroblotted onto Immobilon-P. Rab31 was detected using a 1 : 100 dilution of rabb it anti-p eptide se rum, Rab32 w ith r abbit a nity- puri®ed immune I gG and Rab11A with rabbit i mmune I gG puri®ed on Avid AL anity gel. In each case, b ound antibody was vi sualized by ECL. T he positions of prestained p rotein standards are s hown on the right. Fig. 6. Immunoblot of rat platelet and tissue proteins usi ng anti-Rab32 I g. Samples containing 20 lg of p rotein were analys ed by SDS/PAGE and e lectroblo tted onto Immobilon-P. Immunoreactive proteins were detected using anity-puri®ed antibody t o Rab32 and visualized by ECL. Lane 1, human platelets; lane 2, rat platelets; lane 3, rat aorta; lane 4, rat heart; lane 5 , rat kidney. Ó FEBS 2002 Rab proteins from platelets (Eur. J. Biochem. 269) 265 (Fig. 6). Instead, an equivalent amount of an immunoreac- tive protein of 52 kDa was observed in rat platelets and a much smaller amount was detected in rat heart. A very weak 52-kDa signal was also observed in s amples of human platelet protein (Fig. 6). We conclude that the 52 kDa protein m ay be a long form of Rab32 (see Discussion). Bacterial expression of Rab31 and Rab32 GST±Rab31, GST±Rab32 and the potentially GTPase- de®cient mutants of t hese proteins, GST±Rab31 Q64L and GST±R ab32 Q85L , were cloned and expressed as described in Experimental procedures. GST±Rab5A was expressed using bacteria provided by P. Stahl. The puri®ed fusion proteins (and GST itself) were almost homogeneous (Fig. 7A) and suitable for experimental studies. To determine whether t he recombinant Rab31 and Rab32 proteins bound GTP, we ®rst u sed [a- 32 P]GTP t o probe nitrocellulose blots of the proteins, using two different Mg 2+ concentrations [17,18], but no binding of [a- 32 P]GTP was detected (e.g. Fig. 7B). To con®rm t hat the methods were working, samples of p latelet protein and of GST-Rab5A were included and bound [a- 32 P]GTP (Fig. 7B). We conclude that GST±Rab31 and Fig. 7. Puri®cation and properties o f wild-type and mutant Rab31 and Rab32 expressed a s GST±fusion proteins. Samples of platelet particu- late fraction protein and of puri®ed GST and GST±Rab proteins were subjected to SDS/PAGE as follows: lane 1, platelet protein; lane 2, GST; lane 3, GST±Rab31; lane 4, GST±Rab31 Q64L ;lane5,GST± Rab32; lane 6, GST±Rab32 Q85L ; lane 7 , GST±Rab5A. Gels were processed as follows: (A) a Coomassie Blue-stained gel showing 10 lg of platelet protein and 0.5 lg of GST and GST±Rab p roteins; (B) an [a- 32 P]GTP overlay [17] of a nitrocellulose blot of a gel containing 40 lg of platelet protein, 0.5 lgofGSTand0.5lgofGST±Rab proteins, except for GST±Rab5A (0.1 lg); (C) an immunoblot of a gel containing 20 lgofplateletproteinand0.1lg of GST and GST±Rab proteins, using a 1 : 1000 dilution of anti-Rab31 antiserum; (D) a similar immunoblot using a 1 : 1000 dilution of anti-Rab32 antiserum. The positions of prestained protein standards are shown on the right. Fig. 8. Kinetics of GTP[S] binding by GST±Rab31 and GST±Rab32 at low and high Mg 2+ free concentrations. Puri®ed GST±Rab31 (A) or GST±Rab32 (B) (in each case 200 pm ol of prote in in 0.2 m L of Bu er A) was i ncub ated at 37 °Cwith5l M [ 35 S]GTP[S] in the presence of 5 l M Mg 2+ free (d)or10m M Mg 2+ free (j). [ 35 S]GTP[S]-binding by the proteins was measured at the indicated tim es, as described under Experimental procedures. Values are means  SE from three d eter- minations. 266 X. Bao et al. (Eur. J. Biochem. 269) Ó FEBS 2002 GST±Rab32 (and t he mutant proteins) were unable to renature after binding to nitrocellulose. The speci®city of our antibodies was studied in experi- ments with t he GST-fusion proteins . Antibody to Rab31 detected only Rab31 and not GST, GST±Rab32 or GST±Rab5A (Fig. 7C). Similarly, antibody to Rab32 detected only Rab32 (and minor proteolytic fragments) (Fig. 7D). [ 35 S]GTP[S] binding by GST±Rab31 and GST±Rab32 Several studies have shown that Mg 2+ can have a critical in¯uence o n GTP or GTP[S] binding by decreasing the o ff- rates for both GDP and GTP/GTP[S] [31,32]. Figure 8 shows that the time-course of binding of [ 35 S]GTP[S] to GST±Rab31 and GST±Rab32 depended on the Mg 2+ concentration in t he medium. W ith both GST±Rab31 (Fig. 8 A) and GST±Rab32 (Fig. 8B), binding of [ 35 S]GTP[S] (5 l M ) reached a maximum within 30 min at 37 °Cwhen5l M Mg 2+ free was present, whereas with 10 m M Mg 2+ free the binding of [ 35 S]GTP[S] did not reach this maximum in the case of GST±Rab31 and required 1 ± 2 h incubation with GST±Rab32. In control experiments, GST did not bind [ 35 S]GTP[S] at either Mg 2+ concentra- tion. Studies on the e ffects o f d ifferent buffered Mg 2+ concentrations on binding of [ 35 S]GTP[S] in 120 min incubations also showed this major difference b etween the two proteins (Fig. 9). Binding of [ 35 S]GTP[S] to GST± Rab31 reached a sharp maximum with  5 l M Mg 2+ and then declined as the Mg 2+ free increased, reaching the low level s een in Fig. 8A with 10 m M Mg 2+ free . GST±Rab32 was much less sensitive than GST±Rab31 to the ability of Mg 2+ concentrations above 5 l M to inhibit [ 35 S]GTP[S] binding. These effects of Mg 2+ ions on [ 35 S]GTP[S] binding, as seen in several different experiments, are summarized in Table 1, w hich shows that t he ratio o f [ 35 S]GTP[S] binding with 10 m M and 5 l M Mg 2+ free after 3 h of incubation was 0.19 with GST±Rab31 a nd 0.98 with GST±Rab32, a highly signi®cant difference (2P < 0.001). Table 1 also shows the effects of the Rab31 Q64L and Rab32 Q85L mutations on [ 35 S]GTP[S] binding by the f usion p roteins. Less binding was seen w ith 5 l M Mg 2+ free in both cases and with 1 0 m M Mg 2+ free in the case of GST±Rab32 Q85L . This re¯ects a progressive loss of stability of these proteins under incubation conditions in which GDP could dissociate relatively rapidly. Because optimal equilibrium binding of [ 35 S]GTP[S] to GST±Rab31 was only observed with 5 l M Mg 2+ free ,theK d values for [ 35 S]GTP[S] dissociation from both GST±Rab31 and GST±Rab32 were determined at this Mg 2+ concentration and gave v alues o f 0.82  0.10 l M and 1.7  0.3 l M , r espectively (means  range from two determinations). Fig. 9. Dependence of GTP[S] binding by GST±Rab31 and GST± Rab32 on t he concentration o f Mg 2+ free . Pur i®ed GST±Rab31 (A) o r GST±Rab32(B)(ineachcase50pmolin0.1mLofBuerA)was incubatedfor120minat37°Cwith5l M [ 35 S]GTP[S] and t he indi- cated concentrations of Mg 2+ free (buered by 1 m M EDTA). [ 35 S]GTP[S]-binding by the proteins was then measured as described under Experimental procedures. Values are means  SE from three determinations. Table 1. [ 35 S]GTP[S]-binding by puri®ed GST±Rab proteins and mutants. GST±Rabproteinswereexpressedandisolatedasdescribedunder Experimental procedures. Binding of [ 35 S]GTP[S] was determined in 180-min incubations at 37 °C in the p resen ce of 5 l M [ 35 S]GTP[S] and the indicated concentrations of Mg 2+ free , and is expressed as mol of [ 35 S]GTP[S] bound per 100 m ol of protein. Mean values  SE from the numbers of separate protein preparations indicated in parentheses are shown. The ratio of [ 35 S]GTP[S] binding at 10 m M Mg 2+ free to that at 5 l M Mg 2+ free was calculated f or each protein preparation f or which both values w ere obtained; me an ratios  SE are given. Protein [ 35 S]GTP[S] binding (mol [ 35 S]GTP[S] per 100 mol of protein) Ratio of [ 35 S]GTP[S] binding (10 m M Mg 2+ free /5 l M Mg 2+ free ) 5 l M Mg 2+ free 10 m M Mg 2+ free GST-Rab31 35.3  2.8 (8) 7.0  0.7 (6) 0.19  0.01 (6) GST-Rab31 Q64L 18.3  2.6 (4) 9.7  1.8 (4) 0.55  0.12 (4) GST-Rab32 26.5  2.7 (9) 25.4  1.7 (8) 0.98  0.10 (8) GST-Rab32 Q85L 11.2  4.2 (4) 5.2  1.1 (4) 0.69  0.26 (4) Ó FEBS 2002 Rab proteins from platelets (Eur. J. Biochem. 269) 267 GTPase activities of GST±Rab31, GST±Rab32 and mutants As expected, the steady-state GTPase activities of these Rab proteins were low, but as they were linear with time for up to 4 hat37 °Cwith5 l M [c- 32 P]GTP as substrate, whether 5 l M Mg 2+ free or 10 m M Mg 2+ free was used, valid measurements could be obtained (Table 2). Under both of these conditions, the GTPase activity of GST±Rab32 was signi®cantly higher than that of GST±Rab31 (2P < 0.05, unpaired t-test) (Table 2). Control e xperiments with recombinant GST prepared similarly showed negligible contaminating bacterial GTPase activity. V max and a pparent K m values f or the GTPase activity of GST±Rab31 obtained i n the presence of 10 m M Mg 2+ free were 0.964  0.272 nmolámg pro- tein )1 ámin )1 and 14.8  2.1 l M , respectively ( means  SE, four preparations), wher eas those for GST±Rab32 were 1.80  0.35 n molámg protein )1 ámi n )1 and 6.7  0.9 l M , respectively (means  SE, three p reparations). These re- sults suggest k cat values for the GTPase activities of GST± Rab31 and GST±Rab32 of 0.046 min )1 and 0.092 min )1 , respectively, though it is unlikely that all the recombinant Rab prot ein w as c atalytica lly a ctive. A ssays w ith 10 m M Mg 2+ free and 5 l M [c- 32 P]GTP (Table 2) showed that the GST±R ab31 Q64L and GST±Rab32 Q85L mutants did no t exhibit the expected decreases in GTPase activities, though diminished activities were often observed after prolonged incubation, with 5 l M Mg 2+ free . Reproducible kinetic con- stants were not obtained, probably because of the instability of these mutan t proteins i n the longer incubations required to measure GTPase activities at high GTP concentrations. DISCUSSION Relationships of Rab31 and of Rab32 to other Rab proteins After the initial cloning of Rab31 in 1996 [9], a BLAST search showed that the most closely related Rab protein, with 71% identity, was canine Rab22 [33]. Because the accepted guideline for the u se of the next available R ab number w as, at that time, an identity less than 85% [34], we named the new p rotein Rab31 r ather than Rab22B. However, a protein c loned from human melanocytes that is almost identical to Rab31 has been named Rab22B [25]. There have been at least two attempts to de®ne criteria that permit classi®cation of Rab proteins into subfamilies on the basis of their primary amino -acid sequences [8,35]. In addition to ®ve short s equences considered characteristic of the R ab family as a whole (RabF1-F5), which include one within the Switch I region (RabF1) and two within the Switch II region (RabF3 and RabF4), three [35] or four [8] sequences have been identi®ed that a re highly conserved only in the members of putative subfamilies of Rab proteins (RabSF1±SF4). It has been proposed that such sequences may c onvey effector speci®city, whereas the Switch I and II domains primarily convey sensitivity t o the binding of GTP [8]. There is support f or this view from the crystal structure of the complex of Rab3A/GTP/Mg 2+ with the effector domain of r abphilin-3A, which shows that elements o f RabSF1, RabSF3 and RabSF4 form a complementarity- determining region t hat binds a structural element of rabphilin-3A [36]. Particular importance has been attached to RabSF4 (in the C-terminal hypervariable region) in identi®cation o f subfamilies of Rab proteins [8]. However, the RabSF4 r egions of Rab31 and Rab22A (amino-acid residues 168±180) contain only one residue out of 13 in common (8%), compared with 58% in Rab subfamilies as a whole [ 8]. Moreover, based on t he RabSF1-SF4 criteria for de®ning Rab protein subfamilies [8], Rab32 and Rab38 resemble each other more closely than do Rab31 and Rab22A. F inally, Rab37 is 74% i dentical to Rab26 [37]. Thus, it is certainly possible that Rab31 and Rab22A act through distinct e ffectors and we are unable to support t he suggestion [8] that Rab proteins with > 70% identity should be assigned the same number. The amino-acid sequence of human Rab32 was most similar to that predicted for a recently cloned mouse Rab protein (Fig. 2B). However, the percent identity o f these proteins (84%) was less than usual for orthologous Rab proteins. Thus, human and mouse Rab11A are 100% identical and human and mouse R ab5A 97% identical. The main sequence differences between human Rab32 and the related mouse p rotein are in the N- and C-terminal regions and it is unlikely that our antibody to the human protein would recognize this mouse protein. This raises the possi- bility that a mouse protein that is more closely related to human Rab32 remains to be identi®ed. Rab32 contains amino-acid sequences that are shared with only a small number of other Rab proteins. Most conspicuously, the threonine in the WDTAGQE sequence found in almost all Rab proteins was replaced by isoleucine. This WDIAGQE sequence is also found in the above mouse protein, Rab38 [27] and Rab7L1/29 [29], amongst mam- malianRabproteinsidenti®edtodate,anditisalsopresent Table 2. GTPase activities of puri®ed GST±Rab proteins and mutants. GST-Rab proteins were expressed and isolated as described in Experimental procedures. GTPase activities were determined in 180±240 min incubations at 37 °C in the presence of 5 l M [c- 32 P]GTP and the indicated concentrations of Mg 2+ free , and are expressed as nmol of GTP hydrolysedámg protein )1 ámin )1 . Mean values  SE from the numbers of separate protein preparations indicated in p arentheses are sho wn. The r atio of the GTPase activity at 10 m M Mg 2+ free to that at 5 l M Mg 2+ free was calculated for each protein preparation f or which b oth values w ere obtained; mean ratios  S E are g iven. Protein GTPase activity (nmolámg protein )1 ámin )1 ) Ratio of GTPase activities (10 m M Mg 2+ free /5 l M Mg 2+ free ) 5 l M Mg 2+ free 10 m M Mg 2+ free GST-Rab31 0.205  0.057 (8) 0.195  0.044 (9) 1.15  0.13 (8) GST-Rab31 Q64L ± 0.262  0.078 (6) ± GST-Rab32 0.335  0.071 (8) 0.498  0.088 (8) 1.72  0.29 (8) GST-Rab32 Q85L ± 0.515  0.151 (6) ± 268 X. Bao et al. (Eur. J. Biochem. 269) Ó FEBS 2002 [...]... glucose transporter (GLUT4) and could therefore play a role in the fusion of these vesicles with the plasma membrane [45] Binding of guanine nucleotides and hydrolysis of GTP by GST Rab31 and GST Rab32 Many studies have shown that bacterially expressed Rab proteins, devoid of post-translational modi®cations, contain bound GDP and retain their native GTPase activities after exchange of GDP for GTP [11,19,31,32,46±49]... naturally replaced by leucine Our results showed no decreases in the GTPase activities of GST±Rab31Q64L and GST±Rab32Q85L relative to the unmutated proteins, suggesting that Rab31 and Rab32 fall into the same category as Rab11A and may not be constitutively activated by this mutation These observations emphasize the importance of establishing the biochemical properties of mutant Rab GTPases before they are... [51] Binding of [35S]GTP[S] by the native proteins provides a more critical 270 X Bao et al (Eur J Biochem 269) test and was found to be highly dependent on the Mg2+ concentration Not only was Mg2+ required for binding of [35S]GTP[S] by both GST Rab31 and GST Rab32 but Mg2+free concentrations above 5 lM markedly inhibited the rate and ®nal extent of [35S]GTP[S] binding by GST Rab31 With GST Rab32, high... GTPase activity, as found for Rab3A at high Mg2+ concentrations [31] With GST± Rab31, the same GTPase activities were seen with 5 lM and 10 mM Mg2+free This could be coincidental, with GTP hydrolysis rate-limiting at 5 lM Mg2+free and GDP-GTP exchange rate-limiting at 10 mM Mg2+free In the present study, GST Rab32 consistently exhibited a higher GTPase activity than GST Rab31 and GTP hydrolysis could... inhibitory These observations can most easily be explained by an ability of the higher Mg2+ concentrations to inhibit the dissociation of GDP bound to the Rab proteins, as previously found for Rab3A [31] and Rab5A [32] Thus, with 10 mM Mg2+free, the ko€ for GDP release from GST± Rab31 may be much lower than the ko€ for GDP release from GST Rab32 The GTPase activities of Rab proteins are very low in... Dictyostelium discoideum [28] Rab28, which is more distantly related to other Rab proteins, contains isoleucine in the sequence, WDIGGQT [38] Two other unusual structural features are shared by Rab32, the related mouse protein, Rab38 and Rab7L1/29, namely replacement of the glycine residue that usually precedes the guanine nucleotide-binding NKXD motif by alanine and replacement of a conserved phenylalanine... tag (EST) includes the predicted transcription start site and TATA box of the 28-kDa Rab32 In rat platelets and heart, the 28-kDa Rab32 appeared to be completely replaced by a protein corresponding to Rab32L Although we cannot yet exclude the possibility that the antibody to Rab32 detects an unrelated 52-kDa protein, an N-terminal extension of a Rab protein is not without Rab proteins from platelets... granule and membrane fractions, and only Rab11A was also present in the cytosol This observation may be explained by the ®nding that Rab11A is a preferential target for GDP dissociation inhibitor [41] The very high content of Rab11A in platelets (0.85 lg per mg of platelet protein) is consistent with a major role for this protein Although studies in a variety of cells have indicated that Rab11A is closely... replaced by isoleucine in Rab32 makes contacts with other residues and water molecules involved in Mg2+ and nucleotide binding [39] Expression of Rab31, Rab32 and Rab11A in platelets Use of antibodies directed against C-terminal amino-acid sequences speci®c to each Rab protein studied demonstrated the presence in human platelets of proteins with molecular masses corresponding closely to those predicted... largely in the cytosol fraction, the other Rab proteins were predominantly particulate [3,4] Rab6 and Rab8 were associated with a-granules [3] and other Rab proteins (Rab4, Rab5, Rab27A and Rab27B) have been found in fractions containing a-granules, as well as in membrane fractions [4,7] No Rab proteins have yet been clearly associated with the platelet dense granules In the present study, Rab31, Rab32 and . Antibody to Rab31 detected only Rab31 and not GST, GST Rab32 or GST±Rab5A (Fig. 7C). Similarly, antibody to Rab32 detected only Rab32 (and minor proteolytic. donkey anti- (rabbit IgG) Ig as t he se condary antibody and ECL reagents. Bacterial expression of Rab31 and Rab32 To generate an expression construct, Rab31