a 1759 (2006) 281–295www.elsevier.com/locate/bbaexp
Biochimica et Biophysica Act
The Rab11-FIP1/RCP gene codes for multiple protein transcriptsrelated to the plasma membrane recycling system
Min Jin a, James R. Goldenring a,b,⁎
a Departments of Surgery, Vanderbilt University School of Medicine, Vanderbilt-Ingram Cancer Center and the Nashville VA Medical Center, Nashville,TN 37232, USA
b Cell and Developmental Biology, Vanderbilt University School of Medicine, Vanderbilt-Ingram Cancer Center and the Nashville VA Medical Center, Nashville,TN 37232, USA
Received 19 January 2006; received in revised form 16 May 2006; accepted 8 June 2006Available online 27 June 2006
Rab11a, Rab11b and Rab25 comprise a subfamily of Rabproteins that belong to the Ras-related small GTPase proteinsuperfamily [1,2]. Rab11a and Rab11b are ubiquitouslyexpressed in most tissues [1,3,4], while Rab25 is only expressedin epithelial cells [5]. A number of studies have shown that
Abbreviations: Rab11-FIP, Rab11-family interacting protein; RCP, Rabcoupling protein; MTC panel, multi-tissue cDNAs panel; TfnR, transferrinreceptor; TF, transferrin⁎ Corresponding author. Vanderbilt University School of Medicine, Depart-
ment of Surgery, Epithelial Biology Program, 4160AMRB III, 465 21st AvenueSouth, Nashville, TN 37232-2733, USA. Tel.: +1 615 936 3726; fax: +1 615 3431355.
Rab11 proteins are involved in endocytosis and plasmamembrane recycling in non-polarized and polarized cells[3,6–13]. Rab11a, Rab11b and Rab25 appear to play differentroles in protein trafficking. Expression of the dominant negativeGDP-bound form of Rab11a inhibits acid sequestration ingastric glands and inhibits the apical recycling and basolateral toapical transcytosis of polymeric IgA receptor in MDCK cells[10,14]. Rab11b resides in an apical pericentriolar regiondistinct from Rab11a in MDCK cells [15]. Rab11b is also apotent inhibitor of Ca2+-regulated exocytosis in neurons andneuroendocrine cells [16]. Rab25 co-localizes with Rab11a inthe recycling endosomes, but inhibits apical recycling inMDCK cells [9].
Rab11 interacting proteins play important roles in thephysiological functions of the Rab11 family [17–22]. The
282 M. Jin, J.R. Goldenring / Biochimica et Biophysica Acta 1759 (2006) 281–295
Rab11-family interacting proteins (Rab11-FIP) include Rab11-FIP1, Rab11-FIP2, Rab11-FIP3, Rab11-FIP4 as well as Rabcoupling protein (RCP) and pp75/Rip11 (Rab11-FIP5). All ofthe Rab11-FIP proteins described to date are associated with theplasma membrane recycling system containing Rab11a, andinteract with Rab11 family members through an amphipathicalpha-helical motif in their carboxyl-termini Rab11 bindingdomain [18,23]. Overexpression of the carboxyl termini ofRab11-FIP proteins generally inhibits plasma membranerecycling [17–19]. Overexpression of the carboxyl terminalregion of Rab11-FIP1 or RCP elicits tubulation of the recyclingsystem and inhibition of recycling trafficking [17,19,20]. Inaddition to their interaction with Rab11a, Rab11-FIP proteinsform homodimers and heterodimers in vitro [21]. Rab11-FIP2uniquely interacts with another Rab11-interacting protein,Myosin Vb [22]. All of these results indicate that complexmulti-protein interactions among Rab11-interacting proteinsmay regulate the function of the plasma membrane recyclingsystem.
Currently Rab11 interacting proteins have been categorizedinto three classes based on their structures. The class I Rab11-FIPs (pp75/Rip11/Rab11-FIP5, RCP and Rab11-FIP2) share aC2 domain at their amino-termini, are membrane bound, andlocalize to recycling endosomes [21,23–25]. The class IIRab11-FIPs (Rab11-FIP3/Eferin/arfophilin and Rab11-FIP4)contain two EF-hands and a proline rich region, and arelocalized to recycling endosomes, the trans-Golgi network andthe centrosome [26–29]. The class III Rab11-FIPs has only onemember, rabbit Rab11-FIP1, which lacks any amino terminalstructural domains [23].
The presence of multiple Rab11-FIP members in the samecell indicates the complexity of influences that regulatetrafficking through the Rab11a-containing recycling systems[22,23]. We now report the identification of eight differenttranscripts coded from the Rab11-FIP1 gene on humanchromosome 8. These transcripts include both the humanhomolog of rabbit Rab11-FIP1 (designated Rab11-FIP1A) aswell as RCP (now designated Rab11-FIP1C/RCP). They alsoinclude two previously unrecognized isoforms with Rab11-binding capacity, designated Rab11-FIP1B and Rab11-FIP1D.We have further identified four other transcripts, designated asRab11-FIP1E-H, that code for proteins that lack the Rab11binding domain. Our results demonstrate tissue and cell specificexpression of Rab11-FIP1 isoform transcripts. Rab11-FIP1
Table 1Primer pairs for PCR amplification of Rab11-FIP1 members
Sense primer, anti-sense primer and the predicted length of each DNA product for i
family proteins expressing the Rab11 binding domain associatewith the plasma membrane trafficking system in HeLa cells.However, individual Rab11-FIP1 members may define differ-entiable domains within the recycling system. The resultssuggest that individual Rab11-FIP1 proteins may be responsiblefor a continuum of trafficking decisions within the Rab11a-containing plasma membrane recycling system.
2. Materials and methods
2.1. Cloning of Rab11-FIP1 gene transcripts
Using PCR with primers made from Rab11-FIP1 sequences and the Celeradatabase, eight alternatively spliced Rab11-FIP1 gene transcripts were identifiedfrom the HCA-7 colon cell line and human gastric cDNAs. Rab11-FIP1A andRab11-FIP1F were amplified with 5′-GACGTTCTCCCCTGGTGAATGAC(sense) and 5′-TTACATCTTTCCTGCTTTTTTGCCAACCTGAGT (anti-sense) primers. Rab11-FIP1B, Rab11-FIP1C/RCP and Rab11-FIP1D wereamplified with 5′-GTTATCGTCACCATGTCCCTAATG (sense) and 5′-TTA-CATCTTTCCTGCTTTTTTGCCAACCTGAGT (anti-sense) primers. Rab11-FIP1E, Rab11-FIP1G and Rab11-FIP1H were amplified with the same anti-sense primer 5′-CTAACGAACACACCAGCTTCTCAGAG, but differentsense primers. The sense primers were 5′-GACGTTCTCCCCTGGTGAAT-GAC for Rab11-FIP1E, 5′-AGCTCTGGCCAGGCATCTGTC for Rab11-FIP1G, and 5′-ACACTGGATCCTATCTTTGTCTAG for Rab11-FIP1H. Eachsplice variant was first amplified using BD Advantage 2 polymerase (ClontechLaboratories, Inc., Mountain View, CA) and cloned into pTOPO vector, thencloned into pEGFP-C and pAD GAL4 vectors. All transcript sequences wereconfirmed by direct sequencing (Vanderbilt DNA Sequencing Core).
2.2. Distribution of Rab11-FIP1 gene transcripts in human tissuecDNA panels
To distinguish individual splice variants, sense and anti-sense primer pairsspecific for each variant were designed based on each transcript sequence andthe human Celera database (Table 1) to amplify transcript sequences across atleast one exon–intron boundary.
PCR reactions using BD Advantage 2 polymerase, human multi-tissuecDNAs (MTC) panels (Clontech Laboratories, Inc., Mountain View, CA)and the specific primer pairs were utilized to evaluate the expressionpatterns of these splice variants in various human tissues. The extensiontime for each PCR was based on the predicted length of the DNA beingamplified (Table 1).
2.3. Protein alignment
Open reading frame of each Rab11-FIP1 gene transcript was deduced, andmultiple protein sequences were aligned using CLUSTAL W program on theBiology WorkBench at http://workbench.sdsc.edu (CLUSTAL W: Julie D.Thompson, Desmond G. Higgins and Toby J. Gibson, modified).
Yeast two-hybrid assays were conducted following the standard Clontechprotocol for the Matchmaker two-hybrid system (Clontech Laboratories, Inc.,Mountain View, CA). Each splice variant of the Rab11-FIP1 family was clonedinto pAD GAL4 vector, and the plasmids were confirmed by automatedsequencing. The Rab11a wild type in the pLexAvector was a gift from Dr. MaryW. McCaffrey. Each Rab11-FIP1 splice variant and Rab11a were cotransfectedinto L40 yeast strain, and allowed to grow for 3 to 5 days at 30 °C. A positiveresult of yeast two-hybrid was determined by blue color within 4 h usingincubation using X-GAL substrate as a β-galactosidase assay.
2.5. Cell culture, transfection and immunocytochemistry
HeLa cells were cultured on glass coverslips in RPMI mediumsupplemented with 10% fetal bovine serum and penicillin–streptomycin (50I.U./ml and 50 μg/ml). HeLa cells were transiently transfected for 24h witheach pEGFP construct using Effectene Transfection Reagent (QIAGEN,Valencia, CA). Cells were washes three times, then fixed for 15 min at 4 °C in4% paraformaldehyde and washed 3 times with PBS.
Staining of the fixed cells was conducted as follows. Cells were blocked for20min in the blocking buffer (10mg/ml donkey serum, 0.3% Triton X-100,0.15M NaCl in PBS), then incubated with appropriate primary antibodies for 2hat room temperature, followed with appropriate fluorescently conjugatedsecondary antibodies (Alexa 488, Cy3, Cy5, Molecular Probes, Eugene, OR)for 1h at room temperature. Coverslips were mounted onto slides using ProlongAntifade (Molecular Probes, Eugene, OR). HeLa cells were observed using aZeiss Axiphot equipped with a SPOT digital imaging system.
2.6. Flow cytometry based transferrin uptake and recycling assays
HeLa cells were transiently transfected with pEGFP-Rab11-FIP1 splicevariants for 24 h using Effectene Transfection Reagent (QIAGEN, Valencia,CA), and serum starved for 30min. For immunocytochemistry, cells were loadedwith Alexa 568-conjugated transferrin (Molecular Probes, Eugene, OR), thenfixed and stained with anti-TfnR antibody (CD 71, SIGMA, St. Louis, MS).
Flow cytometry was used to quantify the effects of EGFP-Rab11-FIP1A,EGFP-Rab11-FIP1B, EGFP-Rab11-FIP1C/RCP, EGFP-Rab11-FIP1D, EGFP-Rab11-FIP1E/F and EGFP-Rab11-FIP1H on Alexa 633-conjugated transferrin(Molecular Probes, Eugene, OR) uptake and recycling. Empty EGFP vector wasincluded in the experiment as control. For uptake assays, transiently transfectedHeLa cells were serum starved and incubated for 30 min with Alexa 633-conjugated transferrin at 4 °C to allow binding, followed by incubation at 37 °Cfor 5, 10 and 20 min for internalization. For recycling assays, followingincubation with Alexa 633-conjugated transferrin for 30 min at 4 °C, the cellswere incubated for 20 min at 37 °C in the presence of Alexa 633-conjugatedtransferrin. The medium was then replaced by regular serum-containing mediumand chased for 5, 10, and 20 min at 37 °C. The cells were washed, trypsinizedand resuspended in single cell resuspension. The fluorescence intensity of cell-associated Alexa 633-conjugated transferrin was measured by flow cytometryusing a BD LSRII flow cytometer (BD Biosciences, San Jose, CA) and theaverage fluorescent intensity of the cell population (5000 to 10,000 cells) wasrecorded for each time point. The intensity of Alexa 633-conjugated transferrinwas gated according to the EGFP fluorescence intensity. In cells transfected withempty EGFP vector, EGFP-Rab11-FIP1A, EGFP-Rab11-FIP1B, EGFP-Rab11-FIPC/RCP, EGFP-Rab11-FIP1D, and EGFP-Rab11-FIP1E/F and EGFP-Rab11-FIP1H, the EGFP fluorescence demonstrated three gating categories, whichwere no EGFP (non-transfected), moderate EGFP and high EGFP fluorescenceintensity. High EGFP fluorescent cells showed impaired cellular function due tothe toxicity caused by highly overexpressed EGFP and were excluded from thestudy. Therefore, moderate EGFP fluorescent cells were studied as healthytransfected cells for EGFP-Rab11-FIP1A-D, E/F and H. No EGFP fluorescent(non-transfected) cells served as the internal control for each sample. Therelative fluorescence data were normalized to the mean fluorescence of theinternal control cells, and the retention of transferrin fluorescence data amongcells expressing EGFP-Rab11-FIP1A-D, E/F and H, the empty EGFP vector andnon-transfected control cells were compared.
2.7. Antibody development
Polyclonal anti-Rab11-FIP1B antibody was produced in rabbits againstpeptide (HRDQGRRKTQWYK) spanning amino acids 115–127 of Rab11-FIP1B and Rab11-FIP1C/RCP. Polyclonal anti-Rab11-FIP1C antibody wasproduced against peptide (RAVKPRLHPVK) spanning amino acids 536–546 ofRab11-FIP1C/RCP (COVANCE, Denver, PA). The sera were affinity purifiedusing UltraLink Immobilization Kit (PIERCE, Rockford, IL), and eluted first in100mM glycine (pH 2.5) for antibodies that are bound by acid-sensitiveinteractions, and then second in 100 mM sodium phosphate (pH 11.5) forantibodies that are bound by base-sensitive interactions. For peptide competi-tion, 5μg/ml of the antigen peptide was incubated with the appropriate antibodyfor 1h before applying in immunocytochemistry and western blotting.
2.8. Western blotting
Confluent cells were harvested freshly, and resuspended in modified RIPAbuffer (0.1% sodium deoxycholate, 0.01% SDS, 1% NP-40, and 20 mMmagnesium acetate) supplemented with protease inhibitors, then lysed byrotating at 4 °C for 30min. Cell lysates were then centrifuged at 100,000×g for1h at 4 °C, and the protein concentration of the supernatant was quantified usingthe BCA method (PIERCE, Rockford, IL). SDS-PAGE, transferring, blockingand antibody probing were performed according to standard western blottingprotocols. Anti-GFP antibody (ab290, Abcam Inc., Cambridge, MA) was usedat a 1:2500 dilution for 1 h at room temperature or overnight at 4 °C. Polyclonalanti-Rab11-FIP1B antibody was used at 1:100 dilution, and incubated overnightat 4 °C. Polyclonal anti-Rab11-FIP1C antibody was used at 1:200 dilution, andincubated for 1 h at room temperature. Following incubation with HRP-conjugated anti-rabbit secondary antibody (Jackson ImmunoResearch Labora-tories, Inc., West Grove, PA), binding was visualized with SuperSignal WestPico Chemiluminescent Substrate (PIERCE, Rockford, IL).
3. Results
3.1. Identification of a family of transcripts from the genecoding for Rab11-Family Interacting Protein 1
A comparison of the deduced protein sequences of rabbitRab11-FIP1 and human Rab Coupling Protein (RCP) revealed ahigh degree of sequence identity in the carboxyl terminal half ofboth proteins. Further examination of human EST's andgenomic sequence indicated that only one gene could accountfor transcripts coding for both proteins. Given these insights wesought to establish the range of alternate splice products fromthe Rab11-FIP1 gene on human chromosome 8. Guided byhuman EST sequences and the derived exons for Rab11-FIP1and RCP in the Celera database, we designed PCR primers toresolve splice variants from the gene initiated from two apparentalternative 5′ initiation sites and two alternative 3′ splice variantpatterns. We have identified eight alternatively spliced Rab11-FIP1 gene transcripts from the HCA-7 colon cell line andhuman gastric cDNAs. Four of the transcripts, which we havedesignated Rab11-FIP1A, Rab11-FIP1B, Rab11-FIP1C/RCP,and Rab11-FIP1D, contained the exon coding for the carboxylterminal Rab11 binding domain (Fig. 1A). The genomic map ofRab11-FIP1B, the largest family member of Rab11-FIP1, isshown in Fig. 1B according to the Celera database. Rab11-FIP1C/RCP is 98% homologous to RCP (AF368294). How-ever, Rab11-FIP1C/RCP sequence is more consistent with theCelera human genome database when compared with thepreviously published RCP sequence. The Rab11-FIP1B
Fig. 1. Genomic structures of Rab11-FIP1 gene transcripts. Using PCR with primers made from RCP, Rab11-FIP1B sequences and Celera database, eight alternativelyspliced Rab11-FIP1 gene transcripts were identified from the HCA-7 colon cell line and human gastric cDNAs. (A) Schematic structures of human Rab11-FIP1A-Hgene transcripts. The arrow pairs represent each specific primer pair used to distinguish among Rab11-FIP1 gene transcripts. The exon coding for the Rab11 bindingdomain is indicated as RBD. The positions of termination codons are indicated for each transcript with an asterisk. The lengths of the transcripts are indicated on theright. (B) The genomic structure of human Rab11-FIP1B in human chromosome 8 is shown to represent Rab11-FIP1 family. The numbers on the top are the derivednumbers for the exons. The numbers on the bottom are the length of the introns that are spliced out.
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sequence contains all of the exons present in both Rab11-FIP1Aand Rab11-FIP1C/RCP. Both Rab11-FIP1B and Rab11-FIP1C/RCP contain amino terminal C2 domains. The C2 domain istruncated by an internal splicing variation in Rab11-FIP1D. TheRab11-FIP1A sequence is similar to the original Rab11-FIP1sequence cloned from the rabbit parietal cell and lacks an aminoterminal C2 domain based on an alternative 5′ initiation site.However, in addition we identified four transcripts with read-through early terminations on their 3′ ends that eliminate theexon coding for the Rab11 binding domain, which aredesignated as Rab11-FIP1E-H (Fig. 1A). In the case of theRab11-FIP1E and F transcripts, the termination occurs in thefirst codon within the intron present in the cDNA sequence.
To distinguish the splice transcripts from each other, wedesigned internal primer pairs specific for each Rab11-FIP1member, shown as arrow pairs in Fig. 1A. Table 1 shows theprimer pair for each Rab11-FIP1 transcript and the predictedlength of DNA product from PCR amplifications. Fig. 2Ademonstrates that Rab11-FIP1 gene transcripts have diverseexpression patterns in human multi-tissue cDNA (MTC) panels.Rab11-FIP1B and Rab11-FIP1F were expressed in similarpatterns in all of the tissue cDNAs. Rab11-FIP1A, Rab11-FIP1C/RCP and Rab11-FIP1D were more highly expressed incertain tissues with different patterns. In particular, Rab11-FIP1A was expressed in certain organs including the stomach,lung and pancreas as well as in spleen and leukocytes (Fig. 2A).
Rab11-FIP1E and Rab11-FIP1F were expressed in most tissues,while Rab11-FIP1G and Rab11-FIP1H showed a morerestricted pattern of expression compared to the other Rab11-FIP1 gene transcripts. HeLa cells have been used extensively asa non-polar cell model to study endocytosis and proteintrafficking, thus we also studied Rab11-FIP1 family expressionin these cells. We observed expression of all eight Rab11-FIP1gene transcripts in HeLa cell cDNAs prepared by reversetranscription from RNA (Fig. 2B).
To analyze the amino acid sequences coded by Rab11-FIP1gene transcripts, we have deduced the open reading frame ofeach Rab11-FIP1 member. Fig. 3 shows the schematicalignment of Rab11-FIP1 proteins and the predicted molecularweight of each protein (Fig. 3). We noted that the Rab11-FIP1Eand Rab11-FIP1F transcripts share the same open reading framedue to the stop codon (TGA) immediately downstream of the 3′end of Exon 4 (Fig. 1B). Thus the term Rab11-FIP1E/F wasused to designate the coding sequence for both Rab11-FIP1Eand Rab11-FIP1F transcripts.
Using Clustal W program, we aligned the protein sequencesof Rab11-FIP1 members. The alignment showed that Rab11-FIP1 proteins fall into two structural groups: (1) Rab11-FIP1B,Rab11-FIP1C/RCP, Rab11-FIP1D and Rab11-FIP1H, whichshare exons coding for their amino and carboxyl termini, and (2)Rab11-FIP1A, Rab11-FIP1E/F and Rab11-FIP1G, which sharethe same amino terminus and a conserved central region (Fig.
Fig. 2. Tissue distribution of Rab11-FIP1 gene transcripts. (A) Distribution of human Rab11-FIP1 family in various human tissues at cDNA level. Human multi-tissuecDNA (MTC) panels and the specific primer pairs (shown as arrow pairs in Fig. 1A) were used in PCR reactions. (B) Human Rab11-FIP1 gene transcripts areexpressed at the RNA level in HeLa cells. Using HeLa cDNAs (reverse transcribed from HeLa RNA) as template, PCR reactions were performed with primer pairsdesigned for each splice variant.
4). Due to the 3′ read through termination that eliminates theexon coding for the Rab11 binding domain, Rab11-FIP1G andRab11-FIP1H share the same carboxyl terminal sequence(VCPLRSWCVR) that is different from the other Rab11-FIP1members (Fig. 4).
Among the splice variants, Rab11-FIP1A, Rab11-FIP1B,Rab11-FIP1C/RCP and Rab11-FIP1D have identical carboxylterminal sequences that contain the Rab11 binding domain,which presumably allows them to bind to Rab11. In addition,
Fig. 3. Schematic alignment of Rab11-FIP1 protein open reading frames. Open reashown. Rab11-FIP1E and Rab11-FIP1F have the same coding sequence due to the srepresented as Rab11-FIP1E/F. The amino terminal C2 domain and carboxyl terminaleach protein is also listed on the right.
Rab11-FIP1B and Rab11-FIP1C/RCP both have the C2 domainat the amino-terminus, while Rab11-FIP1A and Rab11-FIP1Dlack the C2 domain. Rab11-FIP1E/F, Rab11-FIP1G and Rab11-FIP1H are novel splice variants that lack the carboxyl terminalRab11 binding domain.
A yeast two-hybrid binary assay using L40 yeast strainshowed that Rab11-FIP1A, Rab11-FIP1B, Rab11-FIP1C/RCP,and Rab11-FIP1D interact with Rab11a wild type, while Rab11-FIP1E/F, Rab11-FIP1G and Rab11-FIP1H do not interact with
ding frames of Rab11-FIP1A-H were deduced and the schematic alignment istop codon TGA immediate downstream of Exon 4, therefore the shared ORF isRab11 binding domain (RBD) are indicated. The predicted molecular weight of
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Fig. 4. Protein alignment of Rab11-FIP1 proteins using Clustal W program. Amino acid sequences of Rab11-FIP1 proteins were aligned using CLUSTALW program.The methionine (M) in the initiator position for each protein is bolded and underlined. The unique carboxyl terminal sequence shared by Rab11-FIP1G and Rab11-FIP1H is also bolded.
Rab11a. The result is consistent with the presence or absence ofthe Rab11 binding domain in Rab11-FIP1 members. Proteinoverlay (far western) of recombinant Rab11a and Rab11-FIP1C/RCP or Rab11-FIP1D confirmed their binding to Rab11a (datanot shown).
3.2. EGFP targeting of Rab11-FIP1 in HeLa cells
Open reading frames of Rab11-FIP1A, Rab11-FIP1B,Rab11-FIP1C/RCP, Rab11-FIP1D, Rab11-FIP1E/F, Rab11-FIP1G and Rab11-FIP1H were cloned into the pEGFP vectorto study the targeting and overexpression of Rab11-FIP1 geneproducts. HeLa cells were transiently transfected with EGFP-Rab11-FIP1A, Rab11-FIP1B, Rab11-FIP1C/RCP, Rab11-FIP1D, Rab11-FIP1E/F, Rab11-FIP1G or Rab11-FIP1H andimmunostained with anti-Rab11a antibody and anti-transferrinreceptor antibody (Fig. 5). Each EGFP-FIP1 protein exhibited adifferentiable pattern of distribution. EGFP-Rab11-FIP1A wasconcentrated in the perinuclear region, extending out to thelamellipodia in a diffuse tubulovesicular pattern. In contrast,EGFP-Rab11-FIP1B displayed a more complex tubular vesiclepattern compared to the more localized focal tubules formed byEGFP-Rab11-FIP1C/RCP in the peri-nuclear region. Strikingly,overexpression of EGFP-Rab11-FIP1B and EGFP-Rab11-FIP1C/RCP, which both contain the amino terminal C2 domain,significantly concentrated Rab11a in tubular cisternae. EGFP-Rab11-FIP1D concentrated at the perinuclear region, but not astightly as EGFP-Rab11-FIP1A. EGFP-Rab11-FIP1D alsoextended out to plasma membrane and lamellipodia in a veryfine tubular distribution. EGFP-Rab11-FIP1E/F had a cytoplas-mic distribution and did not co-localize with Rab11a. EGFP-Rab11-FIP1H was concentrated in the perinuclear region, but
did not co-localize with Rab11a. Due to the apparent toxicity ofEGFP-Rab11-FIP1G, we could not evaluate transient over-expression of EGFP-Rab11-FIP1G in HeLa cells. In general,consistent with their coding sequences, EGFP-Rab11-FIP1A,Rab11-FIP1B, Rab11-FIP1C/RCP and Rab11-FIP1D co-loca-lized with Rab11a at the perinuclear region, while Rab11-FIP1E/F and Rab11-FIP1H did not overlap with Rab11a. Theimmunostaining of anti-transferrin receptor (TfnR) overlappedwith the staining of anti-Rab11a in most of the HeLa cellstransfected with EGFP-Rab11-FIP1A-D, while EGFP-Rab11-FIP1H demonstrated a partial overlap with anti-TfnR at theperinuclear region.
3.3. Transferrin uptake and recycling in EGFP-Rab11-FIP1transfected HeLa cells
We studied transferrin uptake and recycling in HeLa cellstransiently expressing EGFP-Rab11-FIP1A, Rab11-FIP1B,Rab11-FIP1C/RCP, and Rab11-FIP1D that contain the Rab11binding domain. Fig. 6 shows that Alexa 568-conjugatedtransferrin was internalized into HeLa cells, and co-localizedwith EGFP-Rab11-FIP1 proteins. Together with our findingsthat EGFP-Rab11-FIP1A-D, Rab11a and TfnR co-localize witheach other (Fig. 5), these results further confirm that Rab11-FIP1A, Rab11-FIP1B, Rab11-FIP1C/RCP, and Rab11-FIP1Dreside in the plasma membrane recycling system.
Flow cytometry was used to quantify the amount of cell-associated Alexa 633-conjugated transferrin in transferrinuptake and recycling assays in HeLa cells transiently transfectedwith EGFP-Rab11-FIP1A, Rab11-FIP1B, Rab11-FIP1C/RCP,Rab11-FIP1D, Rab11-FIP1E/F, Rab11-FIP1H and empty EGFPvector, respectively. Table 2A shows the relative fluorescence of
Fig. 5. Overexpression of EGFP-Rab11-FIP1 in HeLa cells. HeLa cells were transiently transfected with EGFP-Rab11-FIP1A, Rab11-FIP1B, Rab11-FIP1C/RCP,Rab11-FIP1D, Rab11-FIP1E/F, or Rab11-FIP1H (green), and immunostained with anti-Rab11a (cy3-red) and anti-TfnR (cy5-blue) antibodies. EGFP-Rab11-FIP1proteins demonstrated differences in subcellular targeting as well as their relations to Rab11a and TfnR in HeLa cells. Scale bar, 10μm. (For interpretation of thereferences to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 6. Effects of Rab11-FIP1A-D overexpression on transferrin recycling assay. HeLa cells were transiently transfected with EGFP-Rab11-FIP1A, Rab11-FIP1B,Rab11-FIP1C/RCP or Rab11-FIP1D (green). Alexa 568-conjugated transferrin (red) was internalized into HeLa cells and co-localized with the EGFP-Rab11-FIP1A-Dproteins. Scale bar, 10μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Alexa 633-transferrin collected by a BD LSRII flow cytometerat each time point of the transferrin uptake and recycling assays.Table 2B and C show the rates of Alexa-633 transferrin uptakeand recycling in these cells. Overexpression of EGFP-Rab11-FIP1A, Rab11-FIP1B, Rab11-FIP1C/RCP, Rab11-FIP1D,Rab11-FIP1E/F, or Rab11-FIP1H did not cause significantdifferences in Alexa 633-transferring uptake and recycling,compared with either non-transfected HeLa cells or HeLa cellstransfected with empty EGFP vector.
To ensure that the EGFP-tagged proteins were expressedproperly, western blotting was performed on HeLa cellsexpressing the EGFP-FIP1 constructs. Postnuclear supernatants(25μg) of HeLa cells transfected with individual EGFP-Rab11-FIP1A-D, E/F and G were electrophoresed on SDS-PAGE, thenprobed with anti-GFP antibody (ab290). Fig. 7 shows thatEGFP-Rab11-FIP1A-H proteins were all expressed, althoughEGFP-Rab11-FIP1B, the largest spice variant, was expressed ata much lower level. One great advantage of flow cytometry is
indicated here. With flow cytometry, we were able to detect andanalyze EGFP-Rab11-FIP1B even expressed at a lower level.
EGFP-Rab11-FIP1A, EGFP-Rab11-FIP1B and EGFP-Rab11-FIP1E/F demonstrate a band that has a much higherapparent molecular mass than the predicted EGFP proteinmolecular weight, but less than a dimer. We speculate that thiscould be due to the inclusion of Exon 4 (Fig. 1) in these threesplice variants. We have observed that the constructs expressingExon 4 sequences often show aberrant mobilities in SDS-PAGEgels. Alternatively, these splice variants may be subject to as yetunidentified post-translational modifications.
3.4. Antibodies specific for Rab11-FIP1B andRab11-FIP1C/RCP
We have speculated that Rab11-FIP1 family proteins mayplay different roles in recycling system function. Rab11-FIP1Band Rab11-FIP1C/RCP share amino terminal and carboxyl
Table 2Quantification and statistic analysis of Alexa 633-transferrin uptake and recycling assays using flow cytometry
A. Relative fluorescence data generated by a BD LSRII flow cytometer (arbitrary fluorescence units, mean ± SEM)
0 min 100% 100% 100% 100% 100% 100% 100% 100%5 min 69% 70% 69% 69% 65% 64% 67% 65%10 min 54% 54% 53% 51% 51% 48% 50% 49%20 min 39% 39% 40% 38% 35% 36% 35% 35%
Alexa 633-transferrin uptake and recycling assays were performed in HeLa cells were transiently transfected with EGFP-Rab11-FIP1A, Rab11-FIP1B, Rab11-FIP1C,Rab11-FIP1D, Rab11-FIP1E/F, or Rab11-FIP1H. Quantification and statistics were performed to analyze uptake and recycling assays.
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terminal sequences including C2 and the Rab11 bindingdomains. However, Rab11-FIP1B contains a large centralprotein domain not present in Rab11-FIP1C/RCP. Thus, wedeveloped rabbit polyclonal antibodies specific for Rab11-FIP1B and Rab11-FIP1C/RCP to differentiate between some ofthe related family members. Polyclonal anti-Rab11-FIP1Bantibody was produced against a peptide (HRDQG-RRKTQWYKC) spanning the C2 domain of both Rab11-FIP1B and Rab11-FIP1C/RCP. Polyclonal anti-Rab11-FIP1C/RCP antibody was produced against a peptide sequence(RAVKPRLHPVKC) unique to Rab11-FIP1C/RCP, Rab11-FIP1D and Rab11-FIP1H spanning the splice junction betweenExons 3 and Exon 5 (Fig. 1B). Both polyclonal antibodies wereaffinity purified and tested in western blotting and immunos-taining. The anti-Rab11-FIP1B antibody was obtained from thebasic eluate from affinity purification, which only detected a
Fig. 7. Expression of EGFP-Rab11-FIP1A-H proteins in HeLa cells. Proteinsfrom HeLa cells transfected with EGFP-Rab11-FIP1A, B, C, D, E/F and H wereresolved on SDS-PAGE gels and western blots were probed with anti-GFPantibodies. All transfected cells demonstrated bands with appropriate sizes, andRab11-FIP1A, B and E/F also showed lower mobility immunoreactive species.
137kDa band consistent with Rab11-FIP1B in western blottingusing HeLa, MDCK and HCA-7 cell lysates (Fig. 8A). Incontrast, the acidic eluate from the same sera detected only a77kDa band consistent with Rab11-FIP1C/RCP (data notshown). Such specificity may have derived from the immuno-genic complexity in the tested animals. All immunoblot stainingwas inhibited by preincubation with antigen peptide (data notshown). The anti-Rab11-FIP1C antibody only recognized aband at 77kDa in HeLa, MDCK and HCA-7 cell lysates, whichwas consistent with the size of Rab11-FIP1C/RCP (Fig. 8A). Inthese cell lysates, anti-Rab11-FIP1C antibody did not detectbands consistent with either Rab11-FIP1D (68kDa) or Rab11-FIP1H (48kDa), which also have the splice junction domainalong with Rab11-FIP1C/RCP, likely due to the low expressionlevels of these proteins (Fig. 8A). Immunocytochemistry usinganti-Rab11-FIP1C antibody in HeLa cells transiently transfectedwith EGFP-Rab11-FIP1C/RCP showed that this antibodyrecognized EGFP-Rab11-FIP1C/RCP. All immunocytochemis-try staining could be blocked by preincubation of anti-Rab11-FIP1C antibody with the splice junction antigen peptide (datanot shown). Unfortunately, the anti-Rab11-FIP1B antibody didnot elicit specific immunocytochemistry staining.
3.5. Endogenous staining of Rab11-FIP1C/RCP antibody inHeLa cells
We utilized the anti-Rab11-FIP1C antibody to study theendogenous expression of Rab11-FIP1C/RCP in HeLa cells.The endogenous staining of Rab11-FIP1C/RCP co-localizedwith endogenous Rab11a in a tight perinuclear puncta (Fig. 8B).
Fig. 8. Development of rabbit polyclonal anti-Rab11-FIP1B and anti-Rab11-FIP1C/RCP antibodies. (A) Western blotting using anti-Rab11-FIP1B recognized a bandat 137 kDa, and anti-Rab11-FIP1C/RCP antibody detected a band at 77 kDa in HCA-7, HeLa and MDCK cell lysates. (B) Endogenous staining of Rab11a and Rab11-FIP1C/RCP in HeLa cells using anti-Rab11a and anti-Rab11-FIP1C/RCP antibodies. Endogenous Rab11a and Rab11-FIP1C/RCP co-localize with each other in HeLacells. Scale bar, 10μm.
Rab11-FIP1C/RCP staining was less apparent in more periph-eral Rab11a-containing vesicles.
We have hypothesized that some of the Rab11-FIP1 familymembers reside in different subcellular compartments and playdifferent roles in the plasma membrane recycling system.Therefore, we transiently transfected EGFP-Rab11-FIP1A andEGFP-Rab11-FIP1B into HeLa cells and stained with anti-Rab11-FIP1C antibody (Fig. 9). Although there was someoverlap of Rab11-FIP1C/RCP with EGFP-Rab11-FIP1A orEGFP-Rab11-FIP1B at the perinuclear region, endogenousRab11-FIP1C/RCP displayed a more focal distribution withinthe recycling system compared to either EGFP-Rab11-FIP1A orEGFP-Rab11-FIP1B. The staining for endogenous Rab11-FIP1C/RCP was consistent with the focal distribution ofEGFP-Rab11-FIP1C/RCP expression in HeLa cells (Fig. 5).We also consistently observed that EGFP-Rab11-FIP1A andEGFP-Rab11-FIP1B expressing HeLa cells showed a strongerendogenous Rab11-FIP1C/RCP staining compared to non-transfected cells, suggesting that overexpression of EGFP-Rab11-FIP1A and EGFP-Rab11-FIP1B may further concen-trate Rab11-FIP1C/RCP to a certain extent. These data supportthe hypothesis that Rab11-FIP1A, Rab11-FIP1B and Rab11-FIP1C/RCP define overlapping yet potentially differentiableregions within the plasma membrane recycling system.
Rab11a in HeLa cells transiently transfected with EGFP-Rab11a co-localized with endogenous Rab11-FIP1C/RCP.However, endogenous Rab11-FIP1C/RCP resided in a moredefined subset of Rab11a containing vesicles (Fig. 9). Over-expression of EGFP-Rab11a did not alter endogenous Rab11-FIP1C/RCP distribution (Fig. 9). In contrast, as noted in Fig. 5,overexpression of Rab11-FIP1C/RCP significantly alteredendogenous Rab11a distribution. These data are consistent
with the role of Rab11-FIP1C/RCP as part of a multi-proteincomplex associating with Rab11a. Myosin Vb interacts withboth Rab11a and Rab11-FIP2, and regulates plasma membranerecycling in HeLa cells [22,30]. Overexpression of the MyosinVb-tail domain, which lacks the motor head domain, potentlyinhibits trafficking through the recycling system and concen-trates regulators of recycling including Rab11a, Rab11-FIP2and pp75/Rip11/rab11-FIP5 [22,30]. Therefore we studied theoverexpression of EGFP-Myosin Vb-tail on the distribution ofendogenous Rab11-FIP1C/RCP in HeLa cells. EGFP-MyosinVb-tail co-localizing with endogenous Rab11-FIP1C/RCPfurther confirmed the association of Rab11-FIP1C/RCP withthe plasma membrane recycling system (Fig. 9).
4. Discussion
The plasma membrane recycling system is a dynamicorganelle comprised of tubulovesicular membrane cisternae.Present concepts of membrane trafficking suggest that earlyendosomes containing Rab5 fuse into a Rab4-containingsorting endosome compartment [31]. Proteins can thenrecycle directly out of the Rab4 compartment through a fastrecycling pathway or progress into a slower recyclingpathway through the Rab11a-containing plasma membranerecycling system [31]. In polarized cells, the Rab11a-containing recycling system is specialized for apical mem-brane recycling, while a separate recycling pathway governsthe recycling of basolateral proteins such as the transferrinreceptor [10,32]. In the cases of both the general recyclingsystem in non-polarized cells and the apical recycling systemin polarized cells, Rab11a appears to coordinate trafficking ofproteins through and out of a complex series of tubular
Fig. 9. Rab11-FIP1C/RCP localizes to a subset of recycling system membranes. Endogenous staining of Rab11-FIP1C/RCP was evaluated with anti-Rab11-FIP1C/RCP antibody in HeLa cells transiently transfected with EGFP-Rab11-FIP1A, EGFP-Rab11-FIP1B, EGFP-Rab11a or EGFP-Myosin Vb-tail. The high-resolutionpanels for each set of immunocytochemistry staining demonstrate the more restricted distribution of Rab11-FIP1C/RCP compared with EGFP-Rab11-FIP1A, EGFP-Rab11-FIP1B, or EGFP-Rab11a. Scale bar, 10μm.
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membranes [6,8–10,22,30]. An array of previous investiga-tions has indicated that Rab11a functions as the nidus for theassembly of proteins, which then regulate the processing ofcargoes through the recycling system [9,10,19,23,30,33–35].Our previous studies have noted the requirement for themotor protein myosin Vb for the progression of traffickingcargoes through the recycling system [30]. We and otherresearchers have also noted the existence of a complex familyof proteins, the Rab11-FIP proteins, that all interact withRab11a through amphipathic alpha-helical motifs in theirterminal carboxyl terminal domains [10,17,18,23,26,36,37].We have now provided evidence for the existence of anunrecognized group of Rab11-interacting proteins, which arethe product of a multiply spliced gene on human chromosome8. While the products of this gene include two previouslydescribed proteins, the human homolog of rabbit Rab11-FIP1and Rab Coupling Protein (RCP), we have now identified two
further transcripts for Rab11 interacting proteins. To create aunified nomenclature, we have designated the humanhomolog of rabbit Rab11-FIP1 as Rab11-FIP1A and thetranscript containing all of the exons in both Rab11-FIP1Aand RCP as Rab11-FIP1B. We have renamed the transcriptoriginally identified as RCP as Rab11-FIP1C/RCP and thealternative 5′ initiation site splice variant of RCP as Rab11-FIP1D.
We have previously noted the presence of multiple Rab11family interacting proteins in the membrane recycling system[23]. While all of these proteins appear to co-localize withRab11a, we and other labs have noted indications of subcellularspecializations. A portion of Rab11-FIP2 appears to lose co-localization with Rab11a after treatment of cells with taxol [23].Also, we have noted that rabbit Rab11-FIP1 associates with thecentrosomes during cell division [33]. More recent investiga-tions have supported a specific role for Rab11-FIP3 in the
delivery of membranes to the cleavage furrow during division[27,29]. The results presented here show that all of the Rab11-FIP1 family transcripts are expressed in HeLa cells. However, itis clear from the inspection of PCR-product distribution fromamplification of multi-tissue cDNA panels that some of thespecializations of Rab11-FIP1 may accrue from tissue-specificdistribution. Thus, while Rab11-FIP1B, the longest transcript,displays a ubiquitous distribution, Rab11-FIP1A has a muchmore limited distribution. Furthermore, since Rab11b andRab25 can also bind to the Rab11 binding domain of Rab11-FIP1A-D, some further complexity of sub-specialization maystem from their interactions with different members of theRab11 family of small GTPases. The existence of multiplemembers of the Rab11-FIP1 family in individual cells suggeststhat some of these proteins either work in concert in largermulti-protein complexes coordinated by Rab11, or that somefamily members may define sub-domains within the Rab11a-containing recycling system.
Because of the overlap of exons, it is difficult to developsplice variant selective antibodies for Rab11-FIP1 proteins. Wehave developed two splice variant specific antisera. Due to theimmunogenic complexity in the rabbits, the anti-Rab11-FIP1Bantibody (a basic eluate from affinity purification) only detectsRab11-FIP1B, while the acidic eluate from the same sera detectsonly Rab11-FIP1C/RCP. This antibody, which unfortunatelydoes not work in immunocytochemistry, demonstrated a 137kDa immunoreactivity in all cell lines tested, consistent with RT-PCR analysis indicating the ubiquitous expression of Rab11-FIP1B. Because the anti-Rab11-FIP1C/RCP antibody wasdeveloped against the assembled splice junction sequence inRab11-FIP1C/RCP that is not present in Rab11-FIP1B, thisantibody only recognizes Rab11-FIP1C/RCP at the predictedmolecular weight of 77 kDa in all cell lines tested, but notRab11-FIP1A or Rab11-FIP1B. We do note that this antibodyshould detect Rab11-FIP1D and Rab11-FIP1H, but we have notobserved bands of the appropriate molecular weight in HeLacells, likely because of the extremely low abundance of the twoproteins. This Rab11-FIP1C/RCP specific antibody has allowedus to compare the distribution of transiently expressed EGFP-Rab11-FIP1B with endogenous Rab11-FIP1C/RCP. While bothEGFP-Rab11-FIP1B and endogenous Rab11-FIP1C/RCPshowed overlapping distributions with Rab11a, the pattern forRab11-FIP1C/RCP was more restricted to a centrally locatedfocus of staining. These results suggest that there must beregional specialization for some of Rab11-FIP1 proteins withinthe recycling system. This specialization could account forassembly of unique multi-protein complexes or handoffsbetween modified assemblies during the process of traffickingthrough the recycling system. The presence of multiple Rab11-FIP complexes also may indicate points for specialized sortingor exiting within the tubular recycling system. While it is notclear how these various complexes would assemble, the abilitiesof Rab11-FIP proteins to homo- and hetero-dimerize [21], alongwith the likely dimerization of Rab11a [38], all point to a vastcomplexity of possible regulatory trafficking protein assemblies.
Previous investigations have sought to classify Rab11-FIPproteins into classes based on the presence of specific motifs
including C2 domains in the Class I members (RCP, Rab11-FIP2 and pp75/Rip11/Rab11-FIP5) and EF hands in theClass II members (Rab11-FIP3 and Rab11-FIP4) [39]. Bythese criteria, Rab11-FIP1B would be a fourth member of theClass I Rab11-FIPs. By lacking both C2 domains and EFhands, Rab11-FIP1A and Rab11-FIP1D are members ofClass III Rab11-FIPs. Like other members of the Rab11-FIPproteins, the Rab11-FIP1A-D proteins with Rab11 bindingdomains co-localize with Rab11a and transferrin receptor inHeLa cells. In addition, fluorescent transferrin is traffickedinto membranous organelles expressing the EGFP-Rab11-FIP1A-D proteins that contain the Rab11 binding domain.While over-expression of the wild type Rab11-FIP1A-Dproteins containing the Rab11 binding domain do not affectrecycling of transferrin in HeLa cells, we have observedtubulation of the Rab11a-containing recycling system withoverexpression of Rab11-FIP1B and Rab11-FIP1C/RCP.Both of these splice variants contain C2 domains andprevious studies with other Class I Rab11-FIP proteins alsohave observed tubulation of the recycling system [25,39–41].While studies of dominant negative inhibitory truncatedconstructs of myosin Vb, Rab11-FIP2, rabbit Rab11-FIP1and RCP have emphasized collapse and tubulation as aninhibitory phenotype in the recycling system, the presentstudies demonstrate that tubulation without collapse is notassociated with alteration of recycling system function. Thus,tubulation itself may be more reflective of over-expression ofC2 domain containing proteins rather than true physiologicalroles attributable to endogenous proteins.
In the course of these studies to define novel Rab11-FIP1gene transcripts, we also have determined the existence of asubfamily of transcripts from the same gene on humanchromosome 8, which code for proteins lacking the Rab11binding domain. These proteins, because of their inability tobind Rab11, represent assemblies of several of the Rab11-FIP1exons into novel transcripts. The Rab11-FIP1E and Rab11-FIP1F transcripts code for the same protein open reading framebecause of the inclusion of an entire intron in the transcript,which codes for an immediate termination of the reading frame.While we originally believed that the transcripts represented anincomplete splicing of an hnRNA, we have now observed thesame sequence from multiple oligo-dT transcribed cDNAs frommultiple human cell types. The Rab11-FIP1E/F and Rab11-FIP1G protein transcripts contain the region originallydescribed as characteristic of rabbit Rab11-FIP1. This region,which is not well conserved among species, has no knownfunction or obvious structure. In contrast, Rab11-FIP1H hasmany of the structural elements of Rab11-FIPC/RCP andRab11-FIP1D, but lacks both the C2 domain and the Rab11binding domain. It is notable that both Rab11-FIP1G andRab11-FIP1H have a unique carboxyl terminal sequence. Whileit is not certain whether this sequence is required for targeting ofthese proteins, it is clear that the Rab11-FIP1E-H do not targetto the Rab11a-containing recycling system and over-expressionof Rab11-FIP1E-H do not alter the distribution of Rab11awithin HeLa cells. Although the distribution of the non-Rab11-binding EGFP-Rab11-FIP1H proteins was perinuclear, it did
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not overlap significantly with Rab11a or with markers of eitherthe Golgi apparatus or the endoplasmic reticulum. Thus, theroles of Rab11-FIP1E-H in the regulation of vesicle traffickingremain obscure.
In summary, we have described a number of novel transcriptsassociated with the human Rab11-FIP1 gene. These transcriptsencode four Rab11 binding proteins and three proteins that lackthe Rab11 binding domain. Comparison of endogenous Rab11-FIP1C/RCP with over-expressed EGFP-Rab11-FIP1B demon-strates that Rab11-FIP1C/RCP occupies a more restricteddistribution compared with EGFP-Rab11-FIP1B. The resultsreemphasize the diversity of Rab11 interacting proteins thatoccupy the plasma membrane recycling system. The studieshere, along with previous investigations, indicate that some ofRab11 family interacting proteins may occupy discrete sub-domains within the recycling system and mediate specializedtrafficking functions either within or out of the tubular recyclingsystem.
This work was supported by grants to JRG from NIHNIDDK (DK48370 and DK43405). We thank Dr. James N.Higginbotham and the VUMC Institutional Flow CytometryCore supported by the Vanderbilt-Ingram Cancer Center (P30CA68485) for assistance with analysis of transferrin trafficking.We thank Janine Ward for technical assistance with clonemanipulation. We thank Dr. Lynne A. Lapierre and Dr. CeciliaM. Larocca for advice and critical reading. We thank Dr. JosephT. Roland for assistance in preparation of this manuscript. Wethank Dr. Mary W. McCaffrey for the gift of the pLexA-Rab11plasmids.
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