Human Rab32 cDNA encodes a 225 amino-acid protein of 25.0 kDa with the unusual GTP-binding sequence DIAGQE in place of DTAGQE.. We therefore adopted a different approach to the identi®ca
Trang 1Molecular cloning, bacterial expression and properties of Rab31
and Rab32
New blood platelet Rab proteins
Xiankun Bao1, Andrea E Faris2, Elliott K Jang1and Richard J Haslam1,2
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, were
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 extended to 1000
nucleotides including 630 nucleotides of coding sequence
(GenBank accession no U59878) but the 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 enriched in
Rab11A (0.85 lgámg of platelet protein)1) and 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 Rab32 was replaced by a 52-kDa immunoreactive protein Rab31 and Rab32, expressed as glutathione S-transferase (GST)-fusion proteins, did not bind [a-32P]GTP on nitrocellulose blots but did bind [35S]GTP[S] in a Mg2+-dependent manner Bind-ing of [35S]GTP[S] was optimal with 5 lMMg2+
freeand was markedly inhibited by higher Mg2+concentrations in the case of GST±Rab31 but not GST±Rab32 Both proteins displayed low steady-state GTPase activities, which were not inhibited by mutations (Rab31Q64L and Rab32Q85L) that abolish the GTPase activities of most low-Mr GTP-binding proteins
Keywords: Rab protein; GTP-binding protein; GTPase;
Mg2+; platelet
Low-MrGTP-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 granules, a-granules and lysosomes, in
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 Rab8 [3,4], as well as Rab27A [5,6] and Rab27B
[6,7] Rab6 and Rab8 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 platelet Rab proteins have been identi®ed We therefore adopted a different approach 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 the Rab and Rho families of low-MrGTP-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 of large amounts of Rab11A Here, we describe the cloning and tissue distribution of Rab31 and Rab32, their bacterial expression and some unusual biochemical properties of the recombinant proteins Brief reports of some of our ®ndings have been published in abstract form [9,10]
E X P E R I M E N T A L P R O C E D U R E S Materials
An AmpliFINDERTMRACE kit, a MarathonTM 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: glutathione S-transferase (EC 2.5.1.18).
(Received 10 July 2001, revised 16 October 2001, accepted 30 October
2001)
Trang 2[a-32P]dCTP (3000 Ciámmol)1) and [35S]guanosine
5¢-O-(c-thio)triphosphate ([35S]GTP[S], 1250 Ciámmol)1) were
from NEN and [a-32P]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, foetal bovine serum,L-glutamine, T4 DNA ligase,
Taq DNA polymerase, MMLV reverse transcriptase,
SuperscriptTM II reverse transcriptase/Taq mix and
restriction enzymes were all from Life Technologies
QIA-quickTM PCR puri®cation kits and QIAprepTM 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 were from
Amersham Pharmacia Biotech Avid AL columns were
from BioProbe International UltraLink Iodoacetyl gel for
peptide af®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 for
Molecular Biology and Technology (McMaster University,
Canada) The PEPTOOLTM program used for sequence
alignment was obtained from BioTools Inc The authors
are very grateful to P D Stahl (Washington University
School of Medicine, St Louis, MO, USA) for providing
Escherichia coli expressing GST±Rab5A [11] A sample of
recombinant Rab11A protein [12] was generously supplied
by J R Goldenring (Institute for Molecular 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 by K K Wu (University of Texas, Health Science
Center, Houston, TX, USA) Cultures of other human cell
lines were obtained from the following sources in the
Department of Pathology and 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 cells (a
basophilic leukemia cell line) from J Marshall Cell lines
were routinely grown in RPMI 1640 medium 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) was taken from
healthy human donors and centrifuged for 5 min at 140 g
to separate platelets from other blood cells To 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 Ca2+-free Tyrode's solution containing 0.35%
BSA, 5 mM Pipes, pH 6.5, 90 lgámL)1 of apyrase,
50 IUámL)1 of heparin and 20 nM iloprost Care was taken to remove any residual red and white cells during washes In some experiments, platelet 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 plateletRab cDNA sequences cDNA was synthesized from human platelet mRNA using MMLV reverse transcriptase and an RT primer (5¢-GGACTAGTGTCGACAAGCTTGAATTCT17-3¢, 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 reactions, this primer was replaced by an oligonucleotide identical except for the replacement of the 3¢G by 3¢A Two separate 5¢ primers were used to avoid degeneracy in any of the three bases at their 3¢ ends The antisense oligonucleotide was a 26-mer adaptor identical to the 5¢ half of the RT primer PCR with Taq DNA polymerase was carried out by heating the mixture at 95 °C for 2 min, followed by 40 cycles of 1 min
at 95 °C and 4 min at 68 °C, and then a ®nal 7 min at
72 °C The products were then cloned into pBluescript SK+ using the XbaI and HindIII or EcoRI restriction sites and inserts larger than 450 bp were sequenced After identi®cation of 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 AmpliFINDERTMRACE kit or a MarathonTMcDNA 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 MarathonTM
cDNA library using appropriate speci®c primers, cloned in 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)1and 107c.p.m.ámL)1) labelled with [a-32P]dCTP were prepared by PCR ampli®cation of Rab cDNA sequences encoding amino-acid residues from the 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,
Trang 3nucleotides 252±793 of human glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) cDNA (GenBank accession no
M33197) were ampli®ed from a HEL cell cDNA library
(prepared using the MarathonTMcDNA ampli®cation kit)
Hybridization was carried out at 68 °C for 1 h as described
previously [15] The same membranes were probed
succes-sively for Rab31, Rab32 and GAPDH mRNAs, with
intermediate stripping by heating at 100 °C in 0.5% SDS
for 10 min
Immunoblotting
Rabbit anti-peptide antibodies were prepared to peptide
sequences in the hypervariable C-terminal regions of
cloned Rab proteins The peptides synthesized were
(CH3CO)TIKVEKPTMQASRRC for Rab31 (Fig 1A),
(C)NEENDVDKIKLDQE(CONH2) for Rab32 (Fig 1B),
and (C)QKQMSDRRENDMS(CONH2) for 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 after 3±4 boosts with conjugated peptide in 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 rabbit immune sera, immune IgG puri®ed on Avid AL columns or antibody af®nity-puri®ed on an UltraLink Iodoacetyl column containing covalently bound peptide Bound antibody was visualized using horse-radish peroxidase-conjugated donkey anti-(rabbit IgG) Ig as the secondary antibody and ECL reagents Bacterial expression of Rab31 and Rab32
To generate an expression construct, Rab31 cDNA was ampli®ed from a platelet MarathonTMcDNA library, using
as PCR primers, 5¢-TAGGATCCGCGATACGGGAGC-TCAAAG-3¢ (P31-1) and
5¢-ATCTCGAGGATGTGGG-Fig 1 Nucleotide and deduced amino-acid sequences of Rab31 and Rab32 (A) Rab31 The nucleotide sequence shown (GenBank accession no U59877) was obtained as described under Experimental procedures An almost identical cDNA cloned at the same time from human melanocytes [30] diers 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, one obtained as described in the Experimental procedures (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 are shaded and the peptide sequences used for generating antibodies are doubly underlined The nucleotide sequences that were used for ampli®cation of cDNAs that were ligated into pGEX-4T-1 are also indicated (P31-1, P31-2, P32-1, P32-2, see Experimental procedures).
Trang 4CTCTGGCTTCT-3¢ (P31-2), containing BamHI and XhoI
restriction sites (in bold), respectively (Fig 1A) The PCR
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) and 2 min at 72 °C, for a total of 21
cycles The PCR product was puri®ed using a QIAquickTM
kit and cloned into the BamHI and XhoI sites of the
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 sequence of Rab32 and generate an
expression construct, human platelet mRNA was ®rst
isolated using a Micro-FastTrackTMkit Reverse
transcrip-tion of this platelet mRNA was carried out using a
SuperscriptTMII RT/Taq mix (30 min at 55 °C and 2 min
at 94 °C) and a primer (P32-2) containing XhoI and SalI
restriction sites (in bold) and the complement of nucleotides
700±724 of Rab32 cDNA
(5¢-AAGCTCGAGTCGAC-TTCTTCAGAGCTGAGGCACACAC-3¢) The resulting
cDNA was ampli®ed by PCR using
5¢-TGGGATCC-GGAGGAGCCGGGGACCCCGGCCTG-3¢, containing
a BamHI site, as 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 °C and
2 min at 72 °C, for 35 cycles The product was cloned into
the BamHI and XhoI sites of pGEX-4T-1 and sequenced in
both directions The GST±Rab32 fusion protein was
expressed in E coli BL21(DE3) cells, as for Rab31 In this
fusion protein, the ®rst three amino-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 mM
isopropyl thio-b-D-galactoside and the culture grown for a
further 3 h, when the bacteria were isolated by
centrifuga-tion and frozen at )70 °C until needed Bacterial pellets
(each from 50 mL of culture) were resuspended in 9.8 mL
of NaCl/Pi (pH 7.4) containing lysozyme (100 lgámL)1)
After incubation of the cells for 30 min at 0 °C, 1 mM
phenylmethanesulfonyl ¯uoride and 5 mM dithiothreitol
were added and the cells were sonicated for 10 min in a
bath sonicator Bacterial supernatant was then isolated by
centrifugation and mixed with Triton X-100 (®nal
concen-tration 0.1%), MgCl2(10 mM) and glutathione-Sepharose
4B beads After shaking the mixture for 60 min at room
temperature, the beads were isolated, washed three times
with NaCl/Pi containing 0.1% Triton X-100 and 10 mM
MgCl2and then eluted with 10 mMreduced glutathione in
50 mM Tris/HCl (pH 8.0) containing 10 mM MgCl2 The
eluted protein was concentrated by centrifugation at 4 °C
in a Centricon ®lter, diluted with Buffer A (100 mMKCl,
20 mMHepes, pH 7.5, 1 mMEDTA, 1 mMdithiothreitol)
containing 10 mMMgCl2, 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 MgCl2in the above solutions was essential to
obtain GST±Rab proteins capable of binding guanine nucleotides
Mutagenesis of Rab31 and Rab32
An attempt was made to create constitutively active forms
of these Rab proteins by mutating the glutamine residue in the DTAGQE GTP-binding motif to a leucine residue (Fig 1A,B) A dual PCR method [16] was applied to the Rab31 and Rab32 constructs in pGEX-4T-1 to give clones encoding GST±Rab31Q64L and GST±Rab32Q85L, respec-tively These proteins were expressed and puri®ed, as described for GST±Rab31 and GST±Rab32
Binding of [a-32P]GTP by GST±Rab proteins
on nitrocellulose blots After SDS/PAGE, GST±Rab proteins were electroblotted onto nitrocellulose, renatured and probed with [a-32P]GTP
by two different methods [17,18] In one, the binding buffer contained 2 lMMgCl2[17] and in the other, 10 mMMgCl2
[18]
Binding of [35S]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 [35S]GTP[S] (usually 5 lMat a speci®c radioactivity of 0.5 Ciámmol)1) and any additional MgCl2required to give a de®ned concentration of Mg2+
free
(usually 5 lM or 10 mM) The amount of MgCl2required was calculated using 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 mM KCl, 20 mM
Hepes (pH 7.5), 0.5 mMMgCl2] 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 and 8 mL of ACS and counted for35S by liquid scintillation After correction for the [35S]GTP[S] observed on ®lters from control incubations without protein, the results were expressed as pmol of [35S]GTP[S] bound per 100 pmol of protein (mean SE); this is equivalent to the percentage of protein containing bound [35S]GTP[S]
GTPase assays The GTPase activities of GST±Rab proteins were measured
by a modi®cation of the method of Kabcenell et al [19] Puri®ed protein (10±200 pmol) was incubated at 37 °C in 40±200 lL of Buffer A containing [c-32P]GTP (usually
5 lM at a speci®c radioactivity of 0.5 Ciámmol)1) and suf®cient additional MgCl2to give the required concentra-tion of Mg2+
free(usually 5 lMor 10 mM) At appropriate times (usually 180 min), triplicate 10 lL samples were mixed with 0.75 mL of 50 mM NaH2PO4 (at 0 °C) containing 10 mMEDTA and activated carbon (5% w/v)
to remove unhydrolysed [c-32P]GTP After centrifugation, 0.4 mL of each supernatant was diluted in 0.01% 4-methylumbelliferone and counted for CÏerenkov radiation Nonenzymatic release of [32P]Pi was subtracted GTPase
Trang 5activity was expressed as nmol of GTP hydrolysedámg
protein)1ámin)1
R E S U L T S
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 sequence 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 that we used,
we found that the sequences ampli®ed were restricted to
members of the Rab and Rho families of low-Mr
GTP-binding proteins The 3¢ sequences that we obtained were
extended in the 5¢ direction by 5¢-RACE In this study, two
novel Rab sequences (Rab31 and Rab32) were cloned from
platelet mRNA (Fig 1A,B) These sequences were readily
recognized as those of low-Mr GTP-binding proteins, in
that in addition to the DXXG sequence present in the
cloning primer, they encoded the GXXXXGK(S/T),
NKXD and EXSA amino-acid residues also involved in
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
prenyla-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 Kozak 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 molecular mass of 25.0 kDa (Fig 1B) In a Rab32 clone obtained later from HEL cells, we observed two nucleotide (and amino acid) changes (Fig 1B) One unusual feature was the ®nding that the WDTAGQE sequence 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 sequences most closely resembling Rab31 and Rab32 were identi®ed byBLASTsearches of the NCBI nonredundant database [24] and were aligned with Rab31 and Rab32 by usingPEPTOOLTMwith some manual adjustments (Fig 2) The results 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 almost identical to our Rab31 was cloned from human melanocytes and named Rab22B [25] The coding sequence of 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 PEPTOOLTMprogramme; minor manual adjustments were made to the alignment of the N- and C-terminal amino-acid residues Consensus sequences are shown white on black The individual percent 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 marked 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 those of related Rab proteins containing an isoleucine substitution (.) 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 sapiens; Mm, Mus musculus; Np, Nicotiana plumbaginifolia; Dd, Dictyostelium discoideum.
Trang 6obtained by ourselves (Fig 1A) The next most similar
Rab-related protein with 49% identity was Rhn1 from
tobacco [26], which is related to Rab5A (Fig 2A) A
comparison of human Rab32 with more recently identi®ed
Rab proteins demonstrated 84% identity with a mouse
protein predicted from a RIKEN cDNA clone, which is
probably a murine form of Rab32 (Fig 2B) In addition,
66% identity was observed between human Rab32 and
human Rab38 [27], which is the human ortholog of an
uncharacterized Rab protein 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], were also
related to Rab32 (Fig 2B) This group of Rab32-related
proteins are characterized by the presence of the
WDIAGQE sequence, as well as a high overall similarity,
suggesting that they form a discrete subfamily of Rab
proteins (see Discussion)
Expression of Rab31 and Rab32
Northern blots demonstrated that human tissues and
cultured cells expressed a 4.4-kb Rab31 mRNA 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 HEL cells, and
to a lesser extent K562 cells expressed Rab31 mRNA, whereas Jurkat cells did not In contrast, 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 procedures and Fig 1A,B) These antibodies gave strong signals that were blocked by the peptides to which they were prepared As shown in Fig 4, all these antibodies detected proteins in platelets with molecular masses similar to 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 not 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 standard amounts of 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 Northern blot (lanes 1±8, 2 lg of polyA+ RNA per lane) was obtained from Clontech (lane 1, heart; lane 2, brain; lane 3, placenta; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 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 blotted onto a nylon membrane, as described under Experi-mental procedures (lane 9, HEL cells; lane 10, K562 cells; lane 11, Jurkat cells) These two membranes were probed successively with
32 P-labelled DNA (300±400 nucleotides) syn-thesized by PCR ampli®cation of sequences from Rab31, Rab32 and GAPDH (see Experimental procedures) Autoradiographs are shown The positions of RNA standards are indicated on the left.
Trang 7at the same time These results showed that human platelets
contained, per mg of total platelet protein, 0.85 0.13 lg
ofRab11A,0.11 0.02 lgofRab32and0.005 0.001 lg
of Rab31 (mean values SE from four, six and four
determinations, respectively, using platelets from different
donors) The subcellular distributions of these Rab proteins
were studied by differential centrifugation using a simple
method shown to yield one particulate fraction 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 Rab31 or Rab32, and only
a small 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 protein 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 amounts of Rab31, Rab32 and Rab11A in
human platelets and related human cell lines Protein (30 lg per lane)
from human platelets (lane 1), HEL cells (lane 2), MEG-01 cells (lane
3) and KU812 cells (lane 4) was subjected to SDS/PAGE,
electro-blotted onto Immobilon-P and probed for Rab31, Rab32 and
Rab11A, using rabbit sera containing polyclonal antibodies raised
against speci®c Rab peptides (see Experimental procedures)
Immu-noreactive proteins were detected by ECL The positions of prestained
protein standards are shown 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 membranes (lane 3), and from the platelet supernatant fraction (lane 4) was subjected to SDS/PAGE and electroblotted onto Immobilon-P Rab31 was detected using a 1 : 100 dilution of rabbit anti-peptide serum, Rab32 with rabbit anity-puri®ed immune IgG and Rab11A with rabbit immune IgG anity-puri®ed
on Avid AL anity gel In each case, bound antibody was visualized
by ECL The positions of prestained protein standards are shown on the right.
Fig 6 Immunoblot of rat platelet and tissue
proteins using anti-Rab32 Ig Samples
containing 20 lg of protein were analysed
by SDS/PAGE and electroblotted onto
Immobilon-P Immunoreactive proteins were
detected using anity-puri®ed antibody to
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.
Trang 8(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 samples of human
platelet protein (Fig 6) We conclude that the 52 kDa
protein may 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 these proteins, GST±Rab31Q64Land GST±Rab32Q85L, 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 the recombinant Rab31 and Rab32 proteins bound GTP, we
®rst used [a-32P]GTP to probe nitrocellulose blots of the proteins, using two different Mg2+concentrations [17,18], but no binding of [a-32P]GTP was detected (e.g Fig 7B) To con®rm that the methods were working, samples of platelet protein and of GST-Rab5A were included and bound [a-32P]GTP (Fig 7B) We conclude that GST±Rab31 and
Fig 7 Puri®cation and properties of wild-type and mutant Rab31 and
Rab32 expressed as 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 ; lane 5, 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 proteins; (B) an
[a- 32 P]GTP overlay [17] of a nitrocellulose blot of a gel containing
40 lg of platelet protein, 0.5 lg of GST and 0.5 lg of GST±Rab
proteins, except for GST±Rab5A (0.1 lg); (C) an immunoblot of a gel
containing 20 lg of platelet protein and 0.1 lg 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 pmol of protein in 0.2 mL of Buer A) was incubated at 37 °C with 5 l M [ 35 S]GTP[S] in the presence of
5 l M Mg 2+
free (d) or 10 m M Mg 2+
free (j) [ 35 S]GTP[S]-binding by the proteins was measured at the indicated times, as described under Experimental procedures Values are means SE from three deter-minations.
Trang 9GST±Rab32 (and the mutant proteins) were unable to
renature after binding to nitrocellulose
The speci®city of our antibodies was studied in
experi-ments with the 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)
[35S]GTP[S] binding by GST±Rab31 and GST±Rab32 Several studies have shown that Mg2+can have a critical in¯uence on GTP or GTP[S] binding by decreasing the off-rates for both GDP and GTP/GTP[S] [31,32] Figure 8 shows that the time-course of binding of [35S]GTP[S] to GST±Rab31 and GST±Rab32 depended on the Mg2+
concentration in the medium With both GST±Rab31 (Fig 8A) and GST±Rab32 (Fig 8B), binding of [35S]GTP[S] (5 lM) reached a maximum within 30 min at
37 °C when 5 lM Mg2+
free was present, whereas with
10 mMMg2+
freethe binding of [35S]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 [35S]GTP[S] at either Mg2+ concentra-tion Studies on the effects of different buffered Mg2+
concentrations on binding of [35S]GTP[S] in 120 min incubations also showed this major difference between the two proteins (Fig 9) Binding of [35S]GTP[S] to GST± Rab31 reached a sharp maximum with 5 lMMg2+and then declined as the Mg2+
freeincreased, reaching the low level seen in Fig 8A with 10 mM Mg2+
free GST±Rab32 was much less sensitive than GST±Rab31 to the ability of
Mg2+ concentrations above 5 lM to inhibit [35S]GTP[S] binding These effects of Mg2+ions on [35S]GTP[S] binding,
as seen in several different experiments, are summarized in Table 1, which shows that the ratio of [35S]GTP[S] binding with 10 mMand 5 lMMg2+
freeafter 3 h of incubation was 0.19 with GST±Rab31 and 0.98 with GST±Rab32, a highly signi®cant difference (2P < 0.001) Table 1 also shows the effects of the Rab31Q64L and Rab32Q85L mutations on [35S]GTP[S] binding by the fusion proteins Less binding was seen with 5 lMMg2+
freein both cases and with 10 mM
Mg2+
free in the case of GST±Rab32Q85L 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 [35S]GTP[S] to GST±Rab31 was only observed with 5 lM
Mg2+free, the Kdvalues for [35S]GTP[S] dissociation from both GST±Rab31 and GST±Rab32 were determined at this
Mg2+concentration and gave values of 0.82 0.10 lM
and 1.7 0.3 lM, respectively (means range from two determinations)
Fig 9 Dependence of GTP[S] binding by GST±Rab31 and GST±
Rab32 on the concentration of Mg 2+
free Puri®ed GST±Rab31 (A) or GST±Rab32 (B) (in each case 50 pmol in 0.1 mL of Buer A) was
incubated for 120 min at 37 °C with 5 l M [ 35 S]GTP[S] and the
indi-cated concentrations of Mg 2+
free (buered 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±Rab proteins were expressed and isolated as described under Experimental procedures Binding of [ 35 S]GTP[S] was determined in 180-min incubations at 37 °C in the presence 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 mol 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 for each protein preparation for which both values were obtained; mean 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)
Trang 10GTPase activities of GST±Rab31, GST±Rab32 and mutants
As expected, the steady-state GTPase activities of these Rab
proteinswerelow, butastheywerelinearwithtimeforupto4
h at 37 °C with 5 lM[c-32P]GTP as substrate, whether 5 lM
Mg2+
freeor 10 mMMg2+
freewas 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 experiments with recombinant GST
preparedsimilarlyshowednegligiblecontaminatingbacterial
GTPase activity Vmax and apparent Km values for the
GTPase activity of GST±Rab31 obtained in the presence of
10 mM Mg2+
free were 0.964 0.272 nmolámg
pro-tein)1ámin)1and 14.8 2.1 lM, respectively (means SE,
four preparations), whereas those for GST±Rab32 were
1.80 0.35 nmolámg protein)1ámin)1 and 6.7 0.9 lM,
respectively (means SE, three preparations) These
re-sults suggest kcatvalues for the GTPase activities of GST±
Rab31 and GST±Rab32 of 0.046 min)1and 0.092 min)1,
respectively, though it is unlikely that all the recombinant
Rab protein was catalytically active Assays with 10 mM
Mg2+
freeand 5 lM [c-32P]GTP (Table 2) showed that the
GST±Rab31Q64L and GST±Rab32Q85L mutants did not
exhibit the expected decreases in GTPase activities, though
diminished activities were often observed after prolonged
incubation, with 5 lMMg2+
free Reproducible kinetic con-stants were not obtained, probably because of the instability
of these mutant proteins in the longer incubations required
to measure GTPase activities at high GTP concentrations
D I S C U S S I O N
Relationships of Rab31 and of Rab32 to other Rab
proteins
After the initial cloning of Rab31 in 1996 [9], aBLASTsearch
showed that the most closely related Rab protein, with 71%
identity, was canine Rab22 [33] Because the accepted
guideline for the use of the next available Rab number was,
at that time, an identity less than 85% [34], we named the
new protein Rab31 rather than Rab22B However, a
protein cloned 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 sequences considered characteristic of the Rab 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 are highly conserved only in the members of putative subfamilies of Rab proteins (RabSF1±SF4) It has been proposed that such sequences may convey effector speci®city, whereas the Switch I and II domains primarily convey sensitivity to the binding of GTP [8] There is support for this view from the crystal structure
of the complex of Rab3A/GTP/Mg2+ with the effector domain of rabphilin-3A, which shows that elements of RabSF1, RabSF3 and RabSF4 form a complementarity-determining region that binds a structural element of rabphilin-3A [36] Particular importance has been attached
to RabSF4 (in the C-terminal hypervariable region) in identi®cation of subfamilies of Rab proteins [8] However, the RabSF4 regions 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 the RabSF1-SF4 criteria for de®ning Rab protein subfamilies [8], Rab32 and Rab38 resemble each other more closely than do Rab31 and Rab22A Finally, Rab37 is 74% identical to Rab26 [37] Thus, it is certainly possible that Rab31 and Rab22A act through distinct effectors and we are unable to support the 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 of these proteins (84%) was less than usual for orthologous Rab proteins Thus, human and mouse Rab11A are 100% identical and human and mouse Rab5A 97% identical The main sequence differences between human Rab32 and the related mouse protein 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-malian Rab proteins identi®ed to date, and it is also present
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 parentheses are shown The ratio 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 for which both values were obtained; mean ratios SE are given.
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) ±