Molecular Characterization of Rab11 Interactions with the Members

Molecular Characterization of Rab11 Interactions with the

Members of Family of Rab11-Interacting Proteins (FIPs)

Jagath R. Junutula1, Eric Schonteich2, Gayle M. Wilson2, Andrew A. Peden1, Richard H.

Scheller1 and Rytis Prekeris2*

1Genentech, Inc., 1DNA Way, South San Francisco, CA 94080, USA.

2School of Medicine, Department of Cellular and Developmental Biology,

University of Colarado Health Sciences Center,

4200 E. Ninth Ave., Room 4520, B111, Denver, CO 80262, USA.

*Corresponding author:

Tel: 303-315-1725

Fax: 303-315-4729

E.mail: [email protected]

JBC Papers in Press. Published on June 1, 2004 as Manuscript M404633200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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Running Title: Rab11-FIP interactions

SUMMARY

The Rab11 subfamily of GTPases plays an important role in vesicle trafficking from

endosomes to the plasma membrane. At least six Rab11 effectors (Family of Rab11

Interacting Proteins, FIPs) have been shown to interact with Rab11 and are hypothesized

to regulate various membrane trafficking pathways, such as transferrin recycling,

cytokinesis and EGF trafficking. In this study we characterized interactions of FIPs with

the Rab11 GTPase, using Isothermal Titration Calorimetric studies and mutational

analysis. Our data suggest that FIPs cannot differentiate between Rab11a-GTP and

Rab11b-GTP in vitro (50-100 nM affinity) and in vivo. We also show that while FIPs

interact with the GDP bound form of Rab11 in vitro, the affinity of binding (>1000 nM)

is not sufficient for FIP and Rab11-GDP interactions to occur in vivo. Mutational analysis

revealed that both the conserved hydrophobic patch and Tyr628 are important for the

GTP-dependent binding of Rab11 to FIP proteins. The entropy and enthalpy analyses

suggest that binding to Rab11a/b may induce conformational changes in FIP proteins.


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INTRODUCTION

Rab proteins constitute the largest family of small monomeric GTPases and play an

important role in membrane trafficking pathways. More than 60 Rabs have been

identified in the human genome (1,2) and many of them have been implicated in the

regulation of specific exocytic and endocytic transport steps. Several Rabs (Rabs 4, 5, 7,

9, 11,15, 22 and 25) have been shown to localize to endosomes, consistent with the

multiplicity of trafficking pathways mediated by this organelle (reviewed in 3,4). Rab7

and Rab9 associate with late endosomes and lysosomes, while the rest of the endosomal

Rabs have been shown to localize to either early or recycling endosomes.

Rab11a, Rab11b and Rab25 are three closely related Rabs that belong to the

Rab11 subfamily (1,2). Rab11 family proteins have been shown to play an essential role

in protein recycling from endosomes to the plasma membrane (5), polarized transport in

epithelial cells (6), as well as transport from endosomes to the trans-Golgi network (7),

insulin-dependent GLUT4 trafficking (8) and phagocytosis (9). While Rab11a and

Rab11b are ubiquitously expressed, Rab25 is present exclusively in the epithelial cells

(10,11), hence its proposed role in regulating membrane traffic pathways that are specific

to the epithelial cells. The differential roles of the Rab11a and Rab11b isoforms remain

unclear. While several Rab11 binding proteins have so far been isolated (12-15), the

differences in their binding to Rab11a and Rab11b isoforms have not been investigated.

Rabs cycle between inactive GDP- and active GTP-bound forms. In the GTP

bound form, they interact with effector proteins and each Rab-effector complex is

proposed to regulate a unique trafficking step/event such as vesicle docking, budding,

transport or fusion (3,16). Recently, several Rab11 effector proteins, Rip11, FIP2, Rab


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Coupling Protein (RCP), FIP3/eferin and FIP4 were identified using biochemical and

yeast two-hybrid methods(12-15). In addition to Rab11 binding, FIP3 and FIP4 also

interact with ARF GTPases, thereby coupling these small GTPase families, allowing for

potential cross talk between two signaling pathways (17). All FIPs have a conserved C-

terminal motif that is known as the Rab11/25 binding domain (RBD) (18). Based on the

presence of additional structural domains, FIPs are classified into three groups: class I

FIPs (Rip11, FIP2 and RCP) containing a C2 domain, class II FIPs (FIP3/eferin, FIP4)

containing EF hand motifs and class III FIP (FIP1), with no homology to known protein

domains (reviewed in 19).

Despite the identification of the RBD domain that mediates the interaction of FIPs

with Rab11, we are only beginning to understand the mechanisms of Rab11 and FIP

interactions. It remains unclear whether different FIPs exhibit preferences for interaction

with Rab11a or Rab11b isoforms. We still do not know whether these multiple effectors

are capable of binding to Rab11a or Rab11b with the affinities that are relevant to their

physiological concentrations in the cells. Interestingly, some recent work suggests that

FIP2 and RCP bind to Rab11a in a GTP-independent manner (14,20). In addition, while

several effector proteins have been identified for Rab11 little information is available on

the affinities and thermodynamic properties of Rab11-effector complex formation. Thus,

to understand the properties of Rab11 and FIP binding, we have performed Isothermal

Titration Calorimetric (ITC) analysis on the interaction of class I FIPs with GDP- and

GppNHp-bound forms of Rab11a and Rab11b. ITC analysis has been used previously to

study several protein-protein and protein-ligand interactions, as it directly measures

enthalpy and the binding constant involved (22,23). Our data indicate that all class I FIPs


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are capable of binding to the GppNHp-bound form of Rab11 with similar affinity. A

weak interaction is also seen with the GDP-bound form of Rab11, although it not

sufficient to mediate Rab11-GDP and FIP interactions in vivo. FIPs bind equally well to

both isoforms of Rab11 in vitro and in vivo, suggesting that Rab11a and Rab11b mediate

redundant and overlapping functions. The free energy (DG) for Rab11-FIP complex

formation is contributed by enthalpic factors and exhibits a strong enthalpy-entropy

compensation effect. Mutational analysis of the conserved amino acids in the RBD region

of Rip11 indicates that the hydrophobic patch is crucial for Rab11-FIP complex

formation. In addition to these conserved hydrophobic residues, Rip11-Tyr628 also plays

an important role in FIP association with Rab11. Finally, our data suggest that FIPs form

a high affinity homo-dimer, which interacts with two Rab11 molecules to form a hetero-

tetramer.

EXPERIMENTAL PROCEDURES

Plasmids and Reagents

GST gene fusion constructs were prepared by cloning GST-Rip11, GST-FIP2, and GST-

FIP3 into the baculo virus expression plasmid, pAcGHLT-B (BD Biosciences) and

Rab11a, 11b into pGEX-KG (Amersham Biosciences). GST and GFP fusions of Rip11-

F1 (Rip11 C-terminal fragment consisting amino acids 490-652) constructs were

previously described (18). pGEX-FIP2-F1 (aa 378-511) and pGEX-RCP-F1 (aa 479-659)

constructs were also prepared. GppNHp was purchased from Roche. Miscellaneous

chemicals were obtained from Sigma. Transferrin-Alexa647 (Tf-Alexa647) was


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purchased from Molecular Probes (Eugene, CA). Polyclonal rabbit anti-Rip11 antibody

was described previously (12). Polyclonal rabbit anti-RCP antibody was prepared by

immunization with recombinant RCP. Cell culture reagents were obtained from Life

technologies, unless otherwise specified.

Site directed mutagenesis, Expression and Purification of proteins

Site specific mutations were introduced into pGEX- or pEGFP-Rip11-F1 constructs using

a Stratagene PCR based mutagenesis kit. The GST-Rab11, GST-FIP2-F1, GST-RCP-F1,

and GST-Rip11-F1 constructs were transformed into BL-21 Codon Plus Escherichia coli

(Stratagene). GST fusion proteins were expressed and purified from E. coli as described

previously (24). The GppNHp-bound or GDP-bound forms of Rab11a and Rab11b

proteins were prepared by processing through a series of nucleotide (GppNHp or GDP)

exchange reactions in the presence of EDTA, as described previously for Rab5 GTPase

(25). Full length FIPs were cloned into baculo virus expression plasmid and the GST

fusion proteins were purified from insect cells using glutathione affinity column

chromatography. Protein concentrations were determined by the Bradford assay.

In vitro binding assays

Glutathione beads (50 ml) were coated with 5 mg of GST-fusion protein and incubated

with varying amounts of soluble protein in a final volume of 0.5 ml reaction buffer (50

mM Hepes, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 0.1% Triton X-100, 0.1% bovine

serum albumin, 1mM phenylmethylsulfonyl fluoride) either in the presence of GTPgS or

GDPbS (0.5 mM) where indicated. Samples were incubated at 4°C for 1 hour on a


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nutator with constant rotation. The samples were pelleted at 2000xg for 3 min, and

washed three times with 1ml of reaction buffer. Bound proteins were eluted with 1%

SDS, analyzed on SDS-PAGE and either stained with Coomassie Blue or immunoblotted.

Gel filtration analysis

The oligomeric status of Rab11, Rip11-F1, Rip11-F1 mutants and the Rab11-Rip11-F1

complex were determined by analyzing the proteins on Superdex-S200 column that was

connected to a FPLC equipped with UV280 monitor and mini DAWN detector (Wyatt

Technology). The native molecular weight of the protein was calculated from light

scattering analysis using the software provided (Wyatt Technology).

Isothermal Titration Calorimetry (ITC)

ITC experiments were performed using a VP-ITC calorimeter from Microcal, LLC

(Northampton, MA) as described in the instrument user’s manual. Rab11 (8 mM) was

loaded in the sample cell (in PBS containing 5 mM MgCl2 and 0.5 mM GppNHp or

GDP), volume 1.426 ml, and titrated with Rip11-F1, FIP2-F1, GST-RCP-F1 and Rip11-

F1 proteins in the same buffer with each 5-ml injection to a total of 40-50 injections. The

titrations were performed while samples were being stirred at 300 rpm at 25°C or at the

indicated temperature. An interval of 4 min between each injection was given for the

baseline to stabilize. The blank ITC titration was performed against buffer by injecting

the corresponding FIP that was used in Rab11 titration. The blank subtraction was done

for all the data used for analysis. The data were fitted via one set of sites model to

calculate binding constant (K) using Origin software, provided by Microcal, LLC. All the


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proteins used in the ITC studies were either in the thrombin-cleaved form (Rab11, Rip11-

F1 and FIP2-F1) or as a GST fusion form (GST-RCP-F1). The presence of GST did not

interfere with the binding studies.

Cell Culture, Transient transfection and Immunofluorescence

HeLa cells were cultured as described previously (18). The cells were transfected with

wild type and mutant GFP-Rip11-F1 constructs using Lipofectamine 2000 (Invitrogen,

Carlsbad, CA). After 18-24 hours post transfection, the cells were plated on collagen-

coated glass cover slips, grown overnight, fixed with 4 % paraformaldehyde, followed by

quenching with 0.1M glycine. The cells were permeabilized with PBS containing 0.4%

saponin, 2% FBS and 1% BSA for 30 min and followed by incubation with anti-Rip11 or

anti-Rab11 rabbit polyclonal antibodies (Zymed Laboratories). After extensive washing,

cells were incubated with Alexa-594 secondary antibodies (Molecular Probes) for 30

min, washed and mounted with Vectashield (Vector laboratories). Cells were imaged

with Zeiss Axiovert 200M deconvolution microscope.

RNA interference analysis

Rab11a and Rab11b isoforms were knocked-down using siRNA that were designed using

human Rab11a and Rab11b sequences (Rab11a 5’-aatgtcagacagacgcgaaaa-3’; Rab11b 5’-

aagcacctgacctatgagaac-3’). Rab11a, Rab11b, or mixture of Rab11a and Rab11b siRNAs

were co-transfected into HeLa cells using Lipofectamine2000 (Gibco BRL). Transfected

cells were incubated for 74 hours and analyzed for Rab11a and Rab11b expression by

western blotting or used for subcellular fractionation. Remaining cells were plated on


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collagen-coated glass cover slips, fixed, and analyzed by immunofluorescence

microscopy using Zeiss Axiovert 200M inverted deconvolution microscope. Primary

Rab11 antibodies were labeled with Alexa488 and Rip11 with Alexa594 using Zenon

labeling kit (Molecular Probes) and were used for staining HeLa cells.

Transferrin (Tf) uptake and recycling assays using FACS

HeLa cells (80-90% confluent T-175cm2 flask) were transiently transected with GFP

alone, GFP-Rip11-F1 or GFP-Rip11-F1-Y628A and –I629E mutants using

Lipofectamine 2000 (Invitrogen). The cells were incubated with the transfection mixture

(60mg DNA and 120ml of lipofectamine) for 3 hours and the media replaced. After 16

hours post transfection, the cells were trypsinized and resuspended first in complete

media and then subsequently in serum free media.

FACS-based Tf upate and recycling assays were done as described previously

(36). Briefly, for Tf uptake assays, the cells were incubated at 4oC for 30 min with 20

mg/ml of Tf-Alexa647, followed by incubating at 37°C for various time intervals in the

continuous presence of Tf-Alexa647. To measure plasma membrane associated TfR, cells

were washed and analyzed after incubation at 4oC. In all cases the reactions were washed

and terminated by pelleting and resuspending the cells in 3% paraformaldehyde. Cell-

associated Tf-Alexa647 was determined by FACS analysis. For recycling assays cells

were incubated at 4oC for 30 minutes followed by internalization for 20 min at 37oC in

continuous presence of Tf-Alexa647. Cells were then washed and incubated in complete

media supplemented with 50mg/ml unlabelled Tf for various times prior to fixation. The

experiment was terminated as described before and analyzed on a BD Calibur flow


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cytometer (BD Biosciences) equipped with 488nm and 647nm lasers, gating for

transfected cells (10,000 GFP positive cells) and the amount of Tf internalized

determined.

RESULTS

Rab11 effector proteins (FIPs) form homo-dimers.

It has been previously suggested that FIPs can form homo- and hetero-dimers, yet it

remains unclear whether dimerization is dependent on Rab11 binding (26). To test this

we have produced recombinant full-length GST-FIP fusion proteins in insect cells and

truncated FIP proteins in E. coli and tested their ability to form homo-dimers or hetero-

dimers with other FIP family member proteins in the presence or absence of Rab11a. As

shown in Figure 1, soluble full-length Rip11 bound to GST-Rip11. The binding was

specific to Rip11 (Figure 1A), as there was no binding observed with GST alone or with

other FIP family member proteins, FIP2 (Figure 1B) or FIP3 (Figure 1A). Similarly, FIP2

and FIP3 interacted with only FIP2 and FIP3, respectively, indicating that FIPs form only

homo-dimers in vitro (Figure 1A and 1B). Rab11a had no influence on the FIP homo-

dimerization, as FIPs formed homo-dimers both in its presence and absence (Figure 1A

and 1B). The deletion of the N-terminal region (1-490) of Rip11 had no effect on the

Rip11-Rip11 interaction, whereas deletion of the C-terminal 28 amino acids completely

abolished this interaction, indicating that the C-terminal region including the Rab11

Binding Domain (RBD, aa 630-652) is very important for homo-dimer formation (Figure

1A). These results were further supported by cross-linking experiments showing that

Rip11-F1 protein formed a dimer in solution (data not shown). To confirm that Rip11


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forms a homo-dimer, we performed gel filtration followed by light scattering analysis on

Rip11-F1 (Rip11-490-652) alone or complexed with Rab11 (Figure 2). Consistent with

cross-linking results Rip11-F1 eluted as a dimer (36 kDa), while Rab11 eluted as

monomer (24.3 kDa) and the Rab11-Rip11 complex as a hetero-tetramer of 85 kDa

(Rip11-F1 dimer + two Rab11 molecules).

Class I FIPs form strong GTP-dependent complexes with Rab11a and Rab11b

The binding affinity and thermodynamics of the interactions between Rab11 and FIP

complexes were characterized using ITC analysis. Figure 3A and 3B (upper panels) show

a typical calorimetric titration of Rip11-F1 with Rab11a at 25 °C in the presence of GDP

and GppNHp, respectively. The size of the injection peaks decreased gradually as the

injections progressed due to saturation of the binding sites. The lower half of each panel

in the Figure 3 shows the binding isotherm, in which the total heat per injection (kcal per

mole of Rip11-F1 injected) has been plotted against the molar ratios of Rip11-F1 and

Rab11a. Curve fitting the data using the identical site model (Origin software) resulted in

the following parameters: the binding constant (KB), stoichiometry (n), enthalpy (DH)

and entropy (DS). Both GDP- and GppNHp-dependent binding exhibited exothermic heat

responses. The binding stoichiometry for the Rip11-F1 and Rab11a interaction was 0.5,

indicating that one Rip11-F1 dimer bound to two Rab11a molecules. This agreed with the

gel filtration analysis (Figure 2). The affinity of Rip11-F1 for the GppNHp-bound form

of Rab11a (54 nM) was 17 times higher than the Rab11a-GDP-bound form (950 nM).

Similar results were obtained for the interaction of Rip11-F1 with Rab11b (Figure 3C and

3D). There was no exothermic heat response produced by the buffer control or with other


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Rab GTPases, indicating that the Rab11 and Rip11 interaction was specific and that ITC

based assays can be used to measure affinities between Rabs and their effector proteins

(Figure 3E, 3F and data not shown).

The ITC data suggest that Rip11 interacts with Rab11a and Rab11b in vitro with

similar affinity. To examine these interactions in vivo, we used siRNA designed against

Rab11a and Rab11b isoforms. As shown in Figure 4, knock-down of Rab11a or Rab11b

alone had no effect on Rip11 subcellular distribution, indicating that Rab11b can

compensate for Rab11a and vice versa. In contrast, knock-down of both Rab11 isoforms

(Figure 4, C and F) resulted in a redistribution of Rip11 from endosomes to the cytosol.

To confirm this, we fractionated cells into cytosol and membranes. As shown in Figure 5,

only double Rab11a and Rab11b knock-down resulted in translocation of Rip11 from

membranes to the cytosol.

To gain the additional insights into the interaction of Rab11 with FIP family

effectors, we have extended the ITC based study to other Rab11 effector proteins, FIP2

and RCP, belonging to the Class I group of FIPs. The interactions of FIP2 and GST-RCP

to Rab11a and Rab11b were studied under similar conditions to those for the Rip11-

Rab11 interaction as described above. It was evident from Table I that a strong GppNHp-

dependent interaction exists between FIP2 with Rab11a and 11b (~40 nM), while the

interaction with GDP-bound Rab11 is weak (> 1000 nM), similar to the conditions for

Rab11-Rip11 complex formation. Similar results were also obtained for the interaction

GST-RCP with Rab11, but with slightly decreased affinity (~2-3 fold) as compared to the

Rab11-Rip11 interaction (Table I).


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The interactions of Rab11 with FIPs are thermodynamically similar and

enthalpically driven

The enthalpy changes associated with the formation of the Rab11-FIP complex were

measured directly using ITC. The net DG and entropy (DS) were calculated using the

following equations: DG = -RTlnK and DG = DH - TDS, respectively. Negative enthalpy

(-12.8 kcal/mol for GDP and –21.4 kcal/mol for GppNHp) and entropy (-4.6 kcal/mol for

GDP and –11.6 kcal/mol for GppNHp) values were obtained for the GDP- as well as the

GppNHp-dependent Rab11-Rip11 complex formation, thus this interaction must be

driven by enthalpic rather than entropic factors, as the net favorable DG is contributed by

enthalpic factors (Table I). The enthalpy for the GppNHp-dependent Rab11-Rip11-F1

complex formation is about two times higher and the DG was about –1.5 to -2.1 kcal/mol

larger than that with GDP dependent Rab11-Rip11 at each temperature used in the study,

indicating that Rab11 binds to Rip11 in the presence of GppNHp with higher affinity

(Figure 6A). The enthalpy change upon binding showed a strong temperature

dependence, which was essentially linear (R= 0.99, Figure 6A). GDP and GppNHp-

dependent Rab11-Rip11 complex formation both displayed a strong enthalpy-entropy

compensation resulting in a relatively constant DG at all the temperatures tested (Figure

6B). The change in heat capacity (DCp) values were calculated by performing the linear

regression analysis on a DH versus T plot, yielding –0.84 kcal/(mol.K) and –0.97

kcal/(mol.K), respectively for both the GDP and GppNHp dependent Rab11-Rip11

complex formation. The large negative DCp values may be indicative of a hydrophobic

component to the binding and may mean that a conformational change accompanies the

association of Rip11 with Rab11.


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The conserved hydrophobic patch and Tyr628 are important for Rab11-Rip11

complex formation

The thermodynamic analysis of Rip11 and Rab11 interactions suggested that

hydrophobic amino acid residues may play an important role in Rab11-Rip11 complex

formation. The amino acid sequence alignment of FIP proteins in the RBD region

indicates that the conserved hydrophobic residues (marked with ‘ an asterisk ’ in Figure

7A) form a hydrophobic patch (Figure 7A) (18). To test the importance of these

hydrophobic residues we have introduced a conservative substitution at the central core

isoleucine residue (Ile629) with either valine or a non-conservative charged glutamic acid

residue. All the mutant proteins were analyzed using ITC to test their ability to bind to

Rab11. Consistent with the involvement of the “hydrophobic patch” in Rab11 binding,

the non-conservative replacement (I629E) abolished the Rip11 interaction completely

(more than 500 fold decreased affinity), while the conservative substitution I629V had

little effect on Rab11-Rip11 complex formation (Figure 7A and 7B, Table II).

In addition to the I629E mutation, we also mutated other highly conserved

residues, such as Tyr628, Asp630 and Glu638. Interestingly, the Y628A mutant weakly

interacted with GppNHp-bound form of Rab11 (1900 nM) (Figure 7B), whereas

substitution with Phe partially rescued this effect but still exhibited weak affinity (530

nM) compared to wild type Rip11-F1 protein (Figure 7C). The Ala substitution at highly

conserved negatively charged residues (Asp630 and Glu638) had no effect on GppNHp-

dependent Rab11-Rip11 complex formation (Table II). None of the Rip11 mutants used


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in this study had any effect on dimerization (as assessed by gel filtration analysis in

Supplemental Figure 1 and data not shown).

The inactive Rip11 mutants do not affect Transferrin uptake or recycling

The above described ITC binding studies showed that I629E mutation in the hydrophobic

patch and an Ala mutation at the conserved Tyr628, a neighboring residue to the

hydrophobic patch significantly reduced Rip11 interaction with the GppNHp-bound form

of Rab11 in vitro. To test whether these mutations have a similar effect on Rip11 binding

in vivo, we have transfected wild type and mutant GFP-Rip11-F1 constructs into HeLa

cells and analyzed their colocalization with endogenous Rab11. As shown in Figure 8,

the wild type Rip11-F1 (panels A-C) is localized to Rab11-positive compartments, which

were tubulated and aggregated due to Rip11-F1 overexpression, consistent with

previously published data (18). In contrast, the Rip11-F1 mutants are predominantly

cytosolic and have no effect on the cellular distribution of Rab11, indicating that these

mutants fail to interact with Rab11 in vivo (Figure 6, panels D-I), consistent with their

interaction in vitro. Interestingly, co-overexpression of Rab11 causes the cytosolic

Y628A (panels J-L) but not I629E mutant (data not shown) to be recruited onto a Rab11

compartment. Thus, the low affinity of Rip11-Y628A binding to Rab11 can be

compensated by the presence of excess Rab11.

We have previously reported that Rip11-F1 has a strong dominant negative effect

on Tf uptake and recycling, since the overexpression of truncated Rip11 sequestered

Rab11 and inhibited the formation of native Rab11-FIP complexes (18, 36). Here, we

have utilized a FACS-based assay to analyze the effects of overexpresion of GFP-Rip11-


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F1 mutants on Tf uptake and recycling (see methods section). Consistent with previous

report (18, 36), Rip11-F1 had an inhibitory effect on Tf-Alexa 647 uptake (Figure 9A).

This inhibition is most likely due to the inhibition of TfR recycling from endosomes to

the plasma membrane (Figure 9C), thus resulting in fewer TfR at the cell surface. Indeed,

overexpression of GFP-Rip11-F1 decreased the amount of TfR at the plasma membrane

(Figure 9B). In contrast, the Rip11-F1 mutants Y628A and I629E had little effect on Tf-

Alexa647 recycling (Figure 9A-C). These results indicate that the Rip11 mutants that

bind with low affinity to Rab11 in vitro also do not interact with Rab11 in vivo, and are

unable to compete with endogenous FIPs for Rab11.

DISCUSSION

Rab11 GTPases play an important role in regulating membrane trafficking pathways by

interacting with their effector proteins (FIPs). To date, six different FIP proteins have

been isolated and shown to interact with Rab11a (reviewed in 19). Furthermore, different

FIPs appear to play distinct roles in regulating membrane traffic (12-15,17,27). The

presence of several Rab11 effectors and two Rab11 isoforms complicates our ability to

understand the roles of Rab11 and FIPs. One possible mechanism is that various FIPs

differentially bind to Rab11a or Rab11b, thus regulating distinct membrane transport

steps or pathways. Alternatively, FIPs may bind to both Rab11 isoforms with similar

affinity, thus competing with each other for this interaction. Our ITC analysis indicates

that the affinities of both Rab11 isoforms to different FIPs are nearly identical. This was

further supported by RNAi analysis, where the absence of one isoform could compensate

for the other, as the knock down of either one of the Rab11 isoform had no effect on


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Rip11 localization, whereas knocking down both the isoforms redistributed the Rip11

into the cytosolic fraction. Thus, FIPs appear to play a role of scaffolding proteins that

may regulate Rab11 function by colocalizing them with various regulatory proteins.

Consistent with this idea, different FIPs are reported to interact with distinct sets of

proteins that are known to regulate various endocytic pathways. For instance, FIP2

interacts with myosin Vb, while FIP3 and FIP4 bind to ARF GTPases, thereby generating

putative Rab11-FIP2-Myosin Vb or Rab11-FIP3-ARF complexes to regulate different

transport steps and/or pathways (17,27).

It has been previously reported that FIPs may form homo- or even hetero-dimers

(26). This raises an interesting possibility of a cross-talk between different FIPs via

hetero-dimerization. Our data did show that FIPs form homo-dimers in vitro. The homo-

dimerization appears to be independent from Rab11 binding. Each of the dimer subunits

can independently interact with Rab11, forming a final hetero-tetramer that contains two

FIPs and two Rab11 molecules. Surprisingly, we saw very little evidence of hetero-

dimerization between different FIPs. However, we cannot fully discount the possibility

that FIPs hetero-dimerize in vivo. The functional meaning of FIP dimerization remains

unclear. The dimerization of FIP-Rab11 complexes on the opposing membranes may play

a role in transport vesicle docking at its target membrane. Indeed, it has been suggested

that dimerization of EEA1-Rab5 complexes mediates organelle docking during

homotypical early endosome fusion (28-30).

While FIPs interacted strongly with GTP-bound Rab11 (~50 nM), weaker (>1000

nM, Kd) GDP dependent interactions were also observed, which raised the possibility that

FIPs may also interact with GDP-bound Rab11 in vivo. We have determined the


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concentrations of Rab11, Rip11 and RCP in HeLa cells by western blot analysis using

available antibodies for these proteins. These analyses revealed that there is about 270

nM of total Rab11, 200 nM of Rip11 and 430 nM of RCP present in HeLa cells.

Therefore, in the cell, GDP dependent interactions will not be favored due to weaker

affinity and the realistic concentrations of available Rab11-GDP at any given time will be

much lower compared to the estimated total concentration as described above. Indeed, the

Rip11-Y629A mutant that binds to Rab11-GTP with an affinity comparable to that of

wild type Rip11 with Rab11-GDP showed a predominant cytosolic distribution and was

not capable of inhibiting TfR recycling. This approximately 20-fold difference in FIP

binding affinity appears to be sufficient to ensure the specific interaction between FIPs

and Rab11-GTP in vivo. Even though we are able to determine the total Rab11 and FIP

concentrations in the cell, they do not fully reflect the local effective concentration of

Rab11 and FIPs on endocytic membranes. Thus, it is possible that relatively low affinity

interaction between Rab11-GDP and FIPs could be compensated by high concentrations

of these proteins on the endosomes.

Thermodynamic properties for the formation of the Rab11-FIP complex described

in this paper showed invariably favorable enthalpies and unfavorable entropies,

indicating that the FIP and Rab11 interactions are energetically driven by exothermic

enthalpy. This is in contrast to most protein-protein interactions, which possess favorable

entropy because of the similarity of many protein interfaces to the interiors of proteins

(31). The favorable enthalpy may arise from the significant number of polar interactions

such as formation of salt bridges and hydrogen bonds. On the other hand, the negative

DCp, for both GDP- (-0.84 kcal/mol.K) and GppNHp- (-0.97 kcal/mol.K) dependent


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Rab11-Rip11 complex formation suggests the presence of significant hydrophobic

interactions. This is further supported by the presence of a conserved hydrophobic patch

among all FIPs. The substitution of the central core residue within this patch with a

charged glutamic residue severely affected the interaction of Rab11 with Rip11. The

large negative DCp may also be the result of reduction in conformational entropy that

occurs due to a structural change upon binding. Similarly, a large negative DCp was also

observed for the TCF4/b-Catenin interaction (32). b-Catenin ligands were shown to

undergo significant structural changes upon b-Catenin binding and the interfaces are

mixture of polar and non-polar contacts (32-35). Interestingly, both GDP- and GppNHp-

dependent Rab11-Rip11 interactions show similar negative DCp values and this is

probably true for all FIPs, as FIP2 and RCP showed a similar trend (unfavorable entropy

factor) indicating that FIP, but not Rab11, may undergo conformational changes upon

binding. Our thermodynamic as well as mutational analysis suggests that the Rab11-FIP

interface is probably contributed by both polar as well as non-polar interactions.

The RBD domain in FIPs is a highly conserved domain among different species

(reviewed in 19). It is predicted to exist in an alpha helical conformation with

hydrophobic and charged residues clustered on the opposite sides of the helix (18).

Consistent with the involvement of hydrophobic residues in mediating Rab11 binding,

mutations within the hydrophobic cluster inhibit Rip11 interaction with Rab11. In

addition, mutational analysis of the highly conserved Tyr628 indicates that this residue

may contribute to both hydrophobic and polar interactions. Surprisingly, mutations of the

highly conserved Asp630 and Glu638 had no effect on Rab11 and Rip11 interactions or

on homo-dimerization. Even though these residues do not directly participate in Rab11


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binding, they still could be important for some other functions, such as in determining the

specificity of Rab11 binding or in the Rab11-independent interaction between FIPs and

other binding proteins, such as myosin Vb and/or the ARF GTPases (17,27).

The data described in this paper is the first glimpse at understanding the

molecular properties of Rab11-FIP interactions, but, many questions remain to be

answered. Perhaps the most intriguing one is how cells regulate the timing and location

of Rab11 interactions with a specific FIP molecule. Since most of the cells express

several FIPs it is unlikely that they simply compete with each other for a limited number

of Rab11 molecules. The presence of multiple phosphorylation and Ca2+-binding motifs

suggests that additional cellular factors probably regulate Rab11 and FIP interactions in

vivo. The basic characterization of Rab11 and FIP interactions allows us to start

addressing these questions.

ACKNOWLEDGEMENTS

We thank Dr. Bob Kelley for the help with light scattering analysis and for his comments

on the manuscript, Dr. Bill Weis, Dr. Suzie Scales and Karen Ervin for the critical

reading of the manuscript and Dr. James Goldenring for anti-Rab11b antibodies. This

work was supported in part by grant J02JF37 from American Diabetes Association (to

RP) and grant DK064380 from NIH-NIDDK (to RP).


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ABBREVIATIONS

FIPs, Family of Rab11 Interacting Proteins; ITC, Isothermal Titration Calorimetry; RBD,

Rab11/25 binding domain; RCP, Rab Coupling Protein; Tf, Transferrin


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FIGURE LEGENDS

Figure 1. FIPs form homo-dimers in Rab11-independent manner. (A) Glutathione

beads coated with GST alone or various GST-FIPs were incubated with different FIPs,

either in the absence (A) or presence of recombinant Rab11a (B). Beads were washed and

the bound protein eluted with 1%SDS. The homo-dimerization of FIPs was visualized by

immunoblotting with anti-Rip11, anti-FIP2, or anti-FIP3 antibodies. Thrombin cleaved

full-length FIPs were used as the source of protein in the binding experiments.

Figure 2. Gel filtration analysis of Rab11-Rip11 complex and Rip11-F1 mutants.

The GppNHp-bound form of Rab11a, Rip11-F1, and Rab11a-Rip11 complex in the

presence of GppNHp were loaded onto Superdex-S200 gel filtration column. Absorbance

280 elution profiles were shown in solid lines and the calculated molecular weight using

light scattering data was shown in dotted symbols and the corresponding numbers are

indicated in the figure. The predicted monomeric molecular weight of Rab11a and Rip11-

RBD are 24.4 kDa and 18.3 kDa, respectively.

Figure 3. ITC binding analysis of the interaction of Rip11 with Rab11. Raw data

(upper panels) for ITC analysis were obtained after 42 successive 5-ml of injections of

110 mM of Rip11 into 8 mM of Rab in phosphate-buffered saline (PBS) containing 5 mM

MgCl2 and 0.5mM GppNHp or GDP. Nonlinear least-squares fit ( ____ ) of the heat

released as a function of the added ligand is also shown (lower panels). Rip11-F1 titration


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with the GDP-bound form (A) or GppNHp-bound form of Rab11a (B). Rip11-F1 titration

with the GDP-bound form (C) or GppNHp-bound form of Rab11b (D). Buffer titration

with the GDP-bound form (E) or GppNHp-bound form of Rab11a (F). The number of

moles of Rip11-F1 was calculated by considering it as a dimer, due to the gel filtration

results in Figure 2.

Figure 4. Rip11 interacts with Rab11a and Rab11b in vivo. HeLa cells were

transfected with Rab11a siRNA (A and B), Rab11b siRNA (C and D), or a mixture of

Rab11a and Rab11b siRNA (E and F). Cells were then fixed and stained with anti-

Rab11a (A), anti-Rab11b (C), anti-Rab11 (E), and anti-Rip11 (B, D and F) antibodies.

Asterisks mark the cells with Rab11a (A), Rab11b (B), Rab11a/b (C and F) knock-down.

Figure 5. Western blot analysis of Rab11 siRNA transfected cells. HeLa cells were

transfected in the absence (mock) or presence of Rab11a, Rab11b, or Rab11a and Rab11b

siRNA, cells were then incubated for 74 hours. (A) Triton X-100 extracts of these cells

were separated on SDS/PAGE and immunoblotted with anti-Rab11a, anti-Rab11b and

anti-Rab4 antibodies. (B) Rab11 siRNA transfected cells were lysed and fractionated into

cytosolic and membrane fractions. These fractions were then separated by SDS/PAGE

and immunoblotted with anti-Rip11 antibodies.

Figure 6. Effect of temperature on the thermodynamic parameters of the Rab11-

Rip11 complex. Panel A shows the temperature dependence of enthalpy (DH). The line

shows the best fit of the data for a DCp (slope) of –0.84 kcal/(mol.K) for Rab11-Rip11


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complex formation in the presence of GDP (open circles) and –0.97 kcal/(mol.K) in the

presence of GppNHp (solid circles). Panel B shows the enthalpy-entropy compensation

plot for GDP (open symbols) and GppNHp (solid symbols) dependent Rab11-Rip11

complex formation, respectively. The dependence of DG and DH on TDS is shown with

squares and circles, respectively. DH versus TDS yields the magnitudes of the slope and

intercept.

Figure 7. Mutational analysis in the conserved RBD region of FIP proteins. (A) The

amino acid sequence alignment of RBD region of FIP proteins. The residues marked with

asterisks form the hydrophobic patch. Mutations were introduced at the conserved

residues that are numbered here and characterized in this study. (B) shows ITC analysis

for Rip11-F1-Y628A (-n-) and Rip11-F1-I629V (-m-). (C) shows ITC analysis for

Rip11-F1-Y628F (-n-) and Rip11-F1-I629E (-m-). Upper panels in 5B and 5C

correspond to the I629V and I629E mutants, respectively. ITC experiments were carried

out as described in the legend to Figure 3.

Figure 8. Rip11-F1 mutants (I629E and Y628A) are not capable of binding to

Rab11 in vivo. (A-I) HeLa cells were transiently transfected with GFP-Rip11-F1 (panels

A-C), GFP-Rip11-F1-I629E (panels D-F) or GFP-Rip11-F1-Y628A (panels G-I). Rab11

was immunostained using an anti-Rab11 polyclonal antibody followed by Alexa 594 anti

rabbit secondary antibody. Panels C, F and I show merged images for A-B, D-E and G-H,

respectively. (J-L) HeLa cells were co-transfected with GFP-Rip11-F1-Y628A (green in


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J and L) and myc-Rab11a (red in K and L). Yellow in L represents the overlap between

myc-Rab11a and GFP-Rip11-F1-Y628A staining.

Figure 9. Inactive Rip11 mutants suppress the inhibitory Rip11-F1 effect on Tf

uptake and recycling. HeLa cells were either transfected with GFP alone, GFP-Rip11-

F1, I629E, and Y628A mutants and analyzed for Tf uptake (A), plasma membrane

associated TfR (B), and Tf recycling (C) using Tf-Alexa647, as described in the Methods

section. In panel A, plasma membrane bound Tf signal (as determined by cell associated

Tf-Alexa 647 signal at time 0) was subtracted from the total signal. In panel C, to

compare the rates of recycling data was expressed as a percentage of total endocytosed

(at time 0) Tf-Alexa 647.


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Table I: Thermodynamic parameters of the association of Rab11 with Rab11effector proteins

Rab/FIP-F1complex

Kd (nM)

Enthalpy (DH)(kcal.mol-1)

Entropy (TDS)(kcal.mol-1)

Free energy (DG)(kcal.mol-1)

Rip11 Titration

11A with GDP 948 -12.8 -4.6 -8.2

11A with GppNHp 54.4 -21.4 -11.6 -9.8

11B with GDP 797 -12.9 -4.6 -8.3

11B with GppNHp 45.9 -22.5 -12.5 -10

FIP2 Titration

11A with GDP 1317 -23.5 15.5 -8.0

11A with GppNHp 40 -27.4 -17.3 -10.1

11B with GDP 1106 -22.5 -14.4 -8.1

11B with GppNHp 44 -27.7 -17.6 -10.1

RCP Titration

11A with GDP 1178 -16.7 -8.6 -8.1

11A with GppNHp 107 -25.7 -16.2 -9.5

11B with GDP 1287 -17.1 -9.1 -8.0


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11B with GppNHp 173 -28.3 -19.1 -9.2

All the ITC experiments were carried out as described in the Methods section and a

representation of a single experiment for the Rab11-Rip11 interaction is shown in Figure

3. Each value is an average of three independent experiments and the standard error is

less than 5%. The number of binding sites ‘n’ was in the range of 0.45-0.55 for all the

interactions listed in the table.

Table II: Thermodynamic parameters for the association of Rab11 with Rip11-F1mutants.

Rip11-F1/Rab11Complex

Kd (nM)

Enthalpy (DH)(kcal.mol-1)

Entropy (TDS)(kcal.mol-1)

Free energy (DG) (kcal.mol-1)

Y628A with 11b 1900 -7.6 0.2 -7.8

Y628F with 11b 530 -19.2 -10.8 -8.4

I629V with 11a 160 -18.0 -8.7 -9.3

I629V with 11b 169 -17.0 -7.7 -9.3

I629E with 11a 30000 - - -

I629E with 11b 28500 - - -

D630A with 11b 38 -22.8 -12.7 -10.1

E638Awith 11b 50 -27.4 -17.4 -10.0

All the Rip11 mutations described in the table are in the context of Rip11-F1 construct

and Rab-Rip interactions were monitored only in the presence of GppNHp. The Rab11


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binding to I629E mutant did not reach saturation, therefore the Kd derived for I629E was

only a rough estimation.

Supplementary Information

Figure 1. Gel filtration analysis of Rip11-F1 mutants.

Rip11-F1 ( ____ ), Rip11-F1-I628V ( ____ ) and Rip11-F1-I629E ( ____ ). Absorbance

280 elution profiles were shown in solid lines and the calculated molecular weight using

light scattering data was shown in symbols.


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Scheller and Rytis PrekerisJagath R. Junutula, Eric Schonteich, Gayle M. Wilson, Andrew A. Peden, Richard H.

Rab11-interacting proteins (FIPs)Molecular characterization of Rab11 interactions with the members of family of

published online June 1, 2004J. Biol. Chem.

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Molecular Characterization of Rab11 Interactions with the Members