Role of Rab Proteins in Epithelial
Membrane Traffic
Sven C. D. van IJzendoorn,* Keith E. Mostov,{ and Dick Hoekstra*
*Department of Membrane Cell Biology, University of Groningen, Groningen
9713AV, The Netherlands
Department of Anatomy, University of California, San Francisco, California 94143
{
Small GTPase rab proteins play an important role in various aspects of
membrane traffic, including cargo selection, vesicle budding, vesicle motility,
tethering, docking, and fusion. Recent data suggest also that rabs, and their
divalent effector proteins, organize organelle subdomains and as such may define
functional organelle identity. Most rabs are ubiquitously expressed. However,
some rabs are preferentially expressed in epithelial cells where they appear
intimately associated with the epithelial-specific transcytotic pathway and/or tight
junctions. This review discusses the role of rabs in epithelial membrane transport.
KEY WORDS: Epithelial cell, Rab protein, Membrane traffic, Cell polarity, Small
GTPase. ß 2003 Elsevier Inc.
I. Introduction
Members of the small GTPase rab protein family play important roles in
membrane traYcking. Recent research has provided a vast amount of evidence that rab proteins regulate membrane organization and dynamics. Specific rab proteins are found associated with distinct organelles such as the
endoplasmatic reticulum (ER), Golgi apparatus, and endosomes. Most rab
proteins are ubiquitously expressed. However, cell types with a highly specialized, e.g., secretory, function often express additional rab proteins, express rab proteins in diVerent ratios, or have adapted the function of specific
rab proteins for diVerent needs. Selective expression and/or reorganization
of rab protein distribution and functioning may also govern organelle identity.
International Review of Cytology, Vol. 232
0074-7696/03 $35.00
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Copyright 2003, Elsevier Inc.
All rights reserved.
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VAN IJZENDOORN ET AL.
Epithelial cells are polarized cells that are characterized by the segregation
of their plasma membrane (PM) into an apical PM domain facing the lumen
and a basolateral PM domain facing the underlying tissue and neighboring
cells, each of which displays a distinct protein and lipid composition. Also
organelles such as the Golgi apparatus and the endosomal system are distributed in a polarized fashion, and the cytoskeleton displays a highly spatial
order in epithelial cells. Such a polarized phenotype allows epithelial cells to
perform their delicate function as a barrier between the body and the outside
world. Tightly regulated intracellular sorting and traYcking of membranes,
and functional tight junctions that prevent the intermixing of apical and
basolateral PM components, are required to secure this apical–basolateral
polarity (Mostov et al., 2003). A variety of rab proteins, some of which are
ubiquitously expressed and some of which appear specifically expressed in
epithelia, are known to play a crucial role in epithelial traYcking and, thus,
epithelial functioning. These rab proteins are discussed in this review.
II. Membrane Traffic in Epithelial Cells
In order to better understand the role of rab proteins in epithelial membrane
traYc and polarity, we will first outline the vesicular membrane traYc itineraries for basolateral and apical proteins and lipids in epithelial cells as they
are currently understood (Fig. 1).
From the endoplasmatic reticulum (ER), newly synthesized proteins are
delivered to the Golgi apparatus in transport vesicles. Following their sequential passage through diVerent Golgi stacks, basolateral and apical PM
proteins are sorted at the trans-Golgi network (TGN) and packaged into
specific transport carriers for eYcient delivery to the respective surface
domains (Fig. 1; 1). Transport from the TGN to the PM of some basolateral
proteins may involve prior passage through endosomes. The sorting
of proteins and lipids is achieved by their segregation into distinct domains
within the organelle membrane. Sorting of basolateral proteins is mediated
by well-described sorting signals encoded in their cytoplasmic domains that
typically include tyrosine, dileucine, and monoleucine motives and clusters of
acidic amino acids. Basolateral sorting signals are recognized by cytosolic
proteins, including the m1b adaptin subunit (Fölsch et al., 1999; Gan et al.,
2002; Simmen et al., 2002, Mostov et al., 2000). m1b adaptin is part of the AP-1
adaptor complex that also binds to clathrin, in this way causing basolateral
proteins to become concentrated in clathrin-coated vesicles (Hirst and
Robinson, 1998). Sorting of apical proteins is less understood but may be
governed by carbohydrate (N-glycan, O-glycan) modifications in the ectodomain (ScheiVele et al., 1995; ScheiVele and Fullekrug, 2000) and is generally
ROLE OF Rab PROTEINS
61
FIG. 1 Schematic outline of the diVerent transport pathways in epithelial cells involved in
plasma membrane asymmetry.
thought to involve their association with (glyco)sphingolipid and cholesterolenriched microdomains called rafts, either directly [e.g., glucosylphosphatidylinositol (GPI)-anchored proteins] or indirectly via binding to other raft
proteins such as lectins (Simons and Ikonen, 1997; Maier et al., 2001, Aı̈t
Slimane and Hoekstra, 2002). It should be noted, however, that some nonraft-associated proteins are sorted to the apical surface via less understood
cytoplasmic apical-sorting signals (Gokay et al., 2001; Takeda et al., 2003;
Jacob and Naim, 2001). DiVerent apical-sorting mechanisms possibly
include the segregation of apical cargo into distinct apical PM-targeted
vesicular carriers (Jacob and Naim, 2001).
There are also examples of raft-associated proteins traveling to the basolateral surface (Sarnataro et al., 2002; Aı̈t Slimane et al., 2003), indicating
that raft association per se is thus not suYcient for apical targeting. Moreover, nonpolarized fibroblasts also sort apical and basolateral proteins, which
are either delivered to the ‘‘uniform’’ PM (Yoshimori et al., 1996; Keller
et al., 2001; Tuma et al., 2002) or selectively retained intracellularly. Interestingly, protein glycosylation, which mediates apical delivery in epithelial
cells, can provide a signal for surface transport in nonpolarized fibroblasts
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VAN IJZENDOORN ET AL.
(ScheiVele and Fullekrug, 2000). Upon epithelial polarization, basolateral
and apical proteins are selectively targeted to the lateral and apical PM
domain, respectively, as demonstrated by advanced confocal and timelapse internal reflection fluorescence microscopy in living cells (Kreitzer
et al., 2003). The molecular mechanisms that govern targeting following
sorting are largely obscure, but, at least for apical proteins, appear to involve
the concerted action of microtubules and the asymmetric distribution of
docking and membrane fusion machineries, including syntaxin 3 (Kreitzer
et al., 2003). The multiprotein exocyst complex appears to mediate the
targeting of basolateral proteins to the tight junction area (Lipschutz and
Mostov, 2002) and also interacts with microtubules (Vega and Hsu, 2001).
Where at the cell surface apical or basolateral protein-containing vesicles are
eventually targeted in polarized epithelia is subject to cell type (e.g., hepatocytes target many apical proteins to the basolateral surface prior to apical
delivery) and diVerentiation state of the cells (Zurzolo et al., 1992; van
Adelsberg et al., 1994). In addition, extracellular cues such as interaction
between epithelial cells or interaction with extracellular matrix (ECM) components and, subsequently, rearrangements of the microtubule and actin
network play an important role in defining sites at the cell surface for vesicle
targeting (van Adelsberg et al., 1994; Yeaman and Nelson, 1997).
The large variety of transport vesicles with diVerent lipid and protein
composition known to exit from the Golgi/TGN implies a highly organized
and dynamic membrane architecture/organization of this organelle. Factors
that aVect organelle membrane curvature, e.g., the formation of tubular or
globular extensions, can facilitate or frustrate the interaction of those membranes with cytosolic proteins (e.g., clathrin) required to form specific transport vesicles (Cluett et al., 1993). Furthermore, cytoplasmic phospholipase
A2 (PLA2) activity contributes to the formation of membrane tubules from
the Golgi and endosomes, and PLA2-induced endosomal tubules have been
reported to be involved in the recycling of the transferrin receptor to the PM
(Drecktrah and Brown, 1999; de Figuieredo et al., 2001). Thus, although
polarized sorting of proteins and lipids is mediated by their recruitment into
membrane domains, the nature and presumed plasticity of such domains and
their interaction with other intracellular components that govern their fate
remain largely obscure.
Upon arrival at the cell surface, many proteins and lipids are internalized
via a process called endocytosis (Fig. 1; 2). Endocytosis comprises a variety
of distinct ways by which molecules can enter the cell, including clathrin- and
raft-mediated routes and pinocytosis (Mukherjee et al., 1997). Apical endocytic activity is generally lower than that of the basolateral surface. Polarized
hepatocytes, which sort many apical proteins to the basolateral surface prior
to apical delivery, may sort diVerent apical proteins at the basolateral PM
into distinct endocytic pathways. Similarly, nonpolarized cells can sort
ROLE OF Rab PROTEINS
63
proteins, which are delivered to either the apical or the basolateral surface
when expressed in epithelial cells, into distinct endocytic pathways (Tuma
et al., 2002; Aı̈t Slimane et al., 2003). The entire surface area of a typical cell
turns over every hour. In epithelial cells, endocytosis is a prerequisite for the
exchange of apical and basolateral PM components and, in this way, communication between the two surface domains. Vesicular transport between
the basolateral and the apical surface, i.e., endocytosis of a macromolecule at
one side and its exocytosis at the other side, is called transcytosis and is
crucial for the proper functioning of polarized epithelial cells. Importantly,
the need to maintain PM polarity in the face of continuous PM turnover
requires the ability of the endosomal system to sort and retarget internalized
proteins and lipids.
The endocytic system in most cells is organized following a common
concept (Mukherjee et al., 1997; Sachse et al., 2002) that allows for basic
housekeeping functions. In polarized epithelial cells the endocytic system
appears more complex due to the need to meet epithelial-specific functions.
Two distinct endocytic pathways, i.e., apical and basolateral derived, operate
in polarized epithelial cells. The basolateral endocytic pathway, followed
by the polymeric Ig receptor (pIgR) and its ligand dimeric IgA (dIgA),
is well characterized (Leung et al., 2000; Wang et al., 2000; Brown et al.,
2000; Apodaca et al., 2001), while less is known about apical endocytosis
and the fate of apically internalized molecules (Altschuler et al., 1999;
van IJzendoorn and Mostov, 2000, Rahner et al., 2000; Tuma et al., 1999).
Following internalization, basolateral and apical PM components are first
delivered to basolateral and apical early sorting endosomes, respectively
(Fig. 1; 2), of which the former have been studied most extensively. In
these acidic, irregularly shaped, tubular–vacuolar structures, fluid and membrane components (e.g., epidermal growth factor receptor, growth hormone
receptor) that are destined for the late endosomal/lysosomal degradative
pathway are sorted from molecules (e.g., transferrin receptor, asiologlycoprotein receptor) that need to be recycled to the PM. Endosomal acidification, generated by vacuolar ATPases, is essential for the dissociation and
sorting of ligands that bind to their receptor in a pH-dependent manner.
While sorting along the degradative pathway appears to involve the vacuolar
part of early sorting endosomes, recycling to the PM is mediated by the
tubular parts. Recycling can occur directly from the early sorting endosomes
or, alternatively, following a subsequent transfer of proteins and lipids to the
mildly acidic recycling endosomes (Fig. 1; 3). Recycling endosomes display a
multibranching tubular morphology (up to 3 mm long; Tooze and
Hollinshead, 1991; Stoorvogel et al., 1996) and typically appear concentrated
around the centrosome or microtubule-organizing center. The basolaterally
endocytosed transferrin receptor is likely to be recycled to the basolateral
surface (Fig. 1; 4) via an AP1-clathrin-mediated mechanism that recognizes
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VAN IJZENDOORN ET AL.
the basolateral sorting signal in its cytoplasmic domain (Stoorvogel et al.,
1996; Odorizzi et al., 1996; Futter et al., 1998; Gibson et al., 1998; Gan et al.,
2002). The function of the recycling endosomes is not known, but likely
reflects an elaboration of the endocytic system that allows for the storage
and/or sorting and targeting of proteins and lipids to specific sites at the cell
surface.
So far, the endosomal system in either polarized epithelial or fibroblastic
cells shows no major diVerences. In polarized epithelial cells, however, recycling endosomes extend well into the apical cytoplasm, which may be related
to the repositioning of the centrosome facing the apical PM domain upon
polarity development. Based on biochemical and (fluorescence and electron)
microscopical data, it is believed that apical and basolateral endocytosed
proteins share the same population of recycling endosomes, which therefore
are referred to as common (recycling) endosomes (Apodaca et al., 1994;
Barosso and Sztul, 1994; Knight et al., 1995; Odorizzi et al., 1996; Brown
et al., 2000; Wang et al., 2000; Leung et al., 2000; van IJzendoorn and
Hoekstra, 1999; SheV et al., 2002). An equivalent compartment exists
in polarized hepatocytes, called the subapical compartment (SAC)
(van IJzendoorn and Hoekstra, 1999; van IJzendoorn et al., 2000; van
IJzendoorn and Mostov, 2000; Ihrke et al., 1999; Rahner et al., 2000). In
the common endosomes, basolateral and apical proteins and lipids are sorted
and recycled to the proper surface domain. TraYcking from common endosomes in the basolateral-to-apical direction has been studied in some detail,
whereas virtually nothing is known in the apical-to-basolateral direction. The
molecular mechanism that controls the sorting and targeting of proteins
from the recycling endosomes to the apical surface remains still largely
obscure, but is likely to involve the MAL2 proteolipid (de Marco et al.,
2002) and detergent-insoluble, (glyco)sphingolipid/cholesterol-enriched
rafts (Puertollano and Alonso, 1999; Hansen et al., 1999), thus resembling
apical-sorting machineries in the biosynthetic pathway (see earlier discussion). The enrichment of raft markers, e.g., caveolin, sphingolipids, and
cholesterol, in common recycling endosomes (Gagescu et al., 2000; Wüstner
et al., 2002; see also Holtta-Vuori et al., 2002) may support raft-based apical
sorting and targeting. Indeed, proteins that are typically sorted to the basolateral or apical PM domain in epithelial cells are also sorted in recycling
endosomes of nonpolarized cells, and this sorting reflects their association
with cholesterol-containing rafts (Major et al., 1998; Chatterjee et al., 2001;
Fivas et al., 2002).
In addition, (glyco)sphingolipid segregation has been demonstrated within
the common endosome/SAC membrane in polarized hepatocytes (van
IJzendoorn and Hoekstra, 1998, 1999, 2000), which gives rise to diVerent
transport vesicles with distinct morphology that are enriched in specific
sphingolipid species, i.e., glucosylceramide (GlcCer) or sphingomyelin
ROLE OF Rab PROTEINS
65
(SM)/galactosylceramide (GalCer) (Maier and Hoekstra, 2003). In polarized
hepatocytes, GlcCer-enriched vesicles are targeted from the common endosome/SAC to the apical surface, whereas SM/GalCer-enriched vesicles are
targeted from the SAC to the basolateral domain. Interestingly, during cell
polarity development, vesicles containing SM/GalCer are rerouted from the
SAC to the newly formed apical domain along a pathway followed by
transcytosing dIgA–pIgR, but distinct from apically recycling GlcCer. Inhibition of this rerouting perturbs polarity development, suggesting a central
role for SAC in polarity development (van IJzendoorn and Hoekstra, 1999,
2000). This was further supported by the observation that circulating cytokines of the interleukin-6 family that promote fetal liver development stimulate cell polarity development via reorganizing polarized membrane traYc at
the SAC (van der Wouden et al., 2002). The plasticity of polarized epithelial
membrane traYc is also reviewed in Mostov et al. (2003). In addition to
spingolipid raft-based sorting, polarized sorting from the common endosomes requires a brefeldin A-sensitive process that may involve g-adaptin
(Wang et al., 2001), actin filaments (SheV et al., 2002), and microtubules
(Gibson et al., 1998).
It is not clear whether the transcytotic pathway constitutes an entirely
novel route or is a combination of the basolateral and apical endocytic
recycling pathways. Transcytosing pIgR–dIgA, destined for delivery at the
apical surface, moves from the common endosomes first to nearly neutral
apical recyling endosomes (ARE, Fig. 1; 5), which is relatively depleted of the
basolateral recycling transferrin receptor (Brown et al., 2000; Wang et al.,
2000), prior to apical delivery (Apodaca et al., 1994; Barosso and Sztul, 1994;
Gibson et al., 1998). Controversy exists whether the ARE represents a
distinct compartment or may be a specialized (sub)domain of the common
endosomes (Rojas and Apodaca et al., 2002; SheV et al., 1999, 2002).
The diVerence in pH value between common endosomes and the ARE
strongly suggests that they represent distinct compartments. In case the
ARE does represent a (sub)domain of the common endosome, it may reflect
an epithelial-specific adaptation of the recycling endosomes carrying out in
a highly regulated fashion the basolateral–apical exchange of PM components. Indeed, the observation that the binding of dIgA to pIgR at the
basolateral surface dictates the de novo formation of morphologically
distinct cup-shaped dIgA–pIgR-containing ARE, derived from the common
endosome (Gibson et al., 1998), would be in support of this view. Also
the ectopic expression of the MAL protein has been shown to induce the
formation of vesicular structures at the TGN, involved in apical sorting
(Puertollano et al., 1997).
Taken together, in addition to the biosynthetic pathways that display
plasticity with regard to routing transport vesicles to defined areas at the
cell surface, the endosomal system appears as a highly complex network of
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VAN IJZENDOORN ET AL.
connected compartments and/or domains that can be dynamically shaped
to meet cell-specific requirements. Evidence suggests that a group of small
GTPase rab proteins interact in a highly concerted manner to control
the functional identity of (recycling) endosomal elements, as well as their
intercommunication and exchange of membrane components.
A. Characteristics of Rab Proteins
Rab proteins form the largest branch of the Ras-like small GTPase family.
The human genome is predicted to contain about 60 Rab genes. Rab
proteins seem to be involved in nearly every aspect of membrane traYc:
vesicle formation, motility, tethering, docking, and fusion events. Rab proteins act by virtue of their guanine nucleotide-specific interaction with eVector proteins, which include a variety of proteins that appear unrelated to each
other, ranging from large protein complexes (e.g., TRAPP, exocyst) and
cytoskeleton elements (e.g., kinesin, myosin V) to lipid kinases (e.g., phosphatidylinositol 3-kinase). The nucleotide state, i.e., the balance between
GTP binding and hydrolysis, and thus the activity of rab proteins, is regulated by GTPase-activating factors (GAPs) and guanine nucleotide exchange
factors (GEFs). However, some proteins interact with rab proteins in a
nucleotide-independent manner, e.g., calmodulin with rab3 or rab11a with
rab11-FIP2, and may aVect rab protein function in a diVerent way. Most rab
proteins studied so far appear to control a specific step in membrane traYc,
which is reflected by their evenly specific subcellular localization. While many
rab proteins appear ubiquitously expressed, the expression of others appears
restricted to specific (epithelial) cell types and/or is (developmentally) regulated. Moreover, some rab proteins appear to display diVerent functions
when expressed in diVerent cell types.
B. Rab Proteins in Epithelial Cells
Several rab proteins have been reported to be exclusively expressed in or to
perform specific functions in epithelial cells. These include rab17, rab18,
rab11a, rab25, rab4, rab3b and rab3d, rab8, and rab13. While the latter
two are likely to play a role in polarized sorting in the biosynthetic pathway,
the others all localize to the transcytotic pathway, in particular to the
common and the apical recycling endosomes, with variable degrees of overlap. Several eVector proteins have been identified, some of which may bind to
more than one of these rab proteins or may interact with each other. As noted
earlier, this again underscores the complexity of the endocytic system
in epithelial cells and the importance of rab proteins in organizing and
ROLE OF Rab PROTEINS
67
regulating membrane traYc events. It also suggests that rab proteins function
in a highly concerted manner, possibly orchestrated by (divalent) rab eVector
proteins. This section discusses each of these rab proteins in detail (see also
Table I).
1. Rab17
Rab17 was identified as an epithelial-specific small GTPase (Lütcke et al.,
1993). Thus, Northern blot analysis on various organs revealed that the
rab17 mRNA is present in liver, intestine, and kidney, but not in organs
that lack epithelial cells or in fibroblasts (Lütcke et al., 1993). Rab17 expression is induced upon the diVerentiation of epithelial cells from their mesenchymal precursors. Rab17 may therefore provide regulatory mechanisms that
are necessary for epithelial-specific (i.e., apical versus basolateral) traYcking
functions. Immunofluorescence and immunoelectron microscopy have
shown that rab17 localizes to pericentrosomal apical endosomal tubules,
most likely the common endosome and/or the apical recycling endosome
(see Section I), and the basolateral surface. Rab17 colocalizes with dIgA–
pIgR at both locations (Hansen et al., 1999; Zacchi et al., 1998; Hunziker and
Peters, 1998) and was found associated with immuno-isolated, dIgA–pIgRcontaining transcytotic 60- to 100-nm vesicles (Jin et al., 1996), suggesting a
role for this rab protein in transcytosis.
The involvement of rab17 in transcytosis has been demonstrated by two
independent studies that have taken the approach of overexpressing mutated
rab17 proteins (Zacchi et al., 1998; Hunziker and Peters, 1998). In both
studies, rab17-positive subapical compartments were accessible for basolaterally endocytosed dIgA–pIgR, as well as the transferrin receptor, suggesting
that rab17 preferentially associates with the common endosome where dIgA–
pIgR and transferrin receptor become segregated (see Section I), representing
a crucial sorting step in the transcytotic pathway. Indeed, overexpression of a
mutant rab17 that is defective in either GTP hydrolysis or GTP binding
increases the apical delivery of the transferrin receptor and a FcLR 5-27
chimeric receptor from, presumably, the common endosomes in polarized
Eph4 cells. These mutants also increase apical recycling but not apical or
basolateral endocytosis (Zacchi et al., 1998). In polarized kidney epithelial
(MDCK) cells, overexpression of wild-type rab17 inhibits the basolateralto-apical transcytosis of dIgA–pIgR, whereas polarized transport in the
biosynthetic pathway remains unaVected (Hunziker and Peters, 1998). The
apparent discrepancy may reflect diVerences in cell type and/or transcytosed
receptor studied. Nevertheless, these data indicate a role for rab17 in the
regulation of polarized membrane transport through the common endosome
in epithelial cells.
TABLE I
Overview of Rab Proteins Involved in Epithelial Membrane Transport
Rab
protein
Location in
epithelial cells
Function in
epithelial cells
Effectors expressed in
epithelial cells
Rab17
Common endosomes
ARE
Basolateral PM
Basolateral recycling
apical transport
n.d.a
Lütcke et al. (1994)
Zacchi et al. (1998)
Hunziker and Peters (1998)
Rab18
Apical PM
Basolateral PM
n.d.
n.d.
Lütcke et al. (1994)
McMurtie et al. (1997)
Rab11a
ARE
Apical transport
Myosin Vb
Rip
Rab11a-FIP1
Rab11a-FIP2
Rab11a-FIP3
Rab11a-FIP4
Lapierre et al. (1999)
Prekeris et al. (2000)
Hales et al. (2001)
Casanova et al. (1998)
Wang et al. (2000)
Rab25
ARE
Apical transport
See rab11a
Casanova et al. (1998)
Wang et al. (2000)
Rab4
Common endosome
Apical transport
n.d.
Mohrmann et al. (2002)
Rab3
Transcytotic vesicles
Apical transport
pIgR?
Rabphilin-3
Van IJzendoorn et al. (2002)
Weber et al. (1994)
Larkin et al. (2001)
Rab13
Tight junctions
Tight junction establishment
Tight junction trafficking
d-PDE
Zahraoui et al. (1994)
Marzesco et al. (1998)
Rab8
TGN
Basolateral transport
n.d.
Huber et al. (1993)
References
ARE
Secretory vesicles
a
Not determined.
ROLE OF Rab PROTEINS
69
Interestingly, rab17 associates with the perinuclear recycling endosomes
when expressed ectopically in nonpolarized cells (Zacchi et al., 1998), suggesting that the recycling endosomes in nonpolarized cells correspond to
the common endosomes in polarized epithelial cells (van IJzendoorn and
Hoekstra, 1999; van IJzendoorn et al., 2000). Possibly, the induction of rab17
expression upon mesenchymal-to-epithelial transition provides the cells with
regulatory mechanisms to control apical versus basolateral sorting and/or
targeting. Thus far, there have been no rab17 eVector proteins identified
to date. The identification of these and/or rab17 activators is expected to
provide further insight into rab17 function in epithelia.
2. Rab18
Rab18, like rab17, is highly expressed in epithelial cells (Lütcke et al., 1994;
McMurtrie et al., 1997). Rab18 is also expressed in human umbilical vein
endothelial cells (which are also polarized cells with a fixed apical and
basolateral surface), peripheral blood mononuclear cells (Schafer et al.,
2000), human skeletal muscle cells (Bao et al., 1998), and the brain (Yu
et al., 1993). In polarized epithelial cells, rab18 localizes to both apical and
basolateral domains (Lütcke et al., 1994), suggesting a role in transcytosis,
similar to rab17. However, in contrast to rab17, rab18 is not associated with
immuno-isolated dIgA–pIgR-containing transcytotic vesicles (Jin et al.,
1996). To date, no functional studies with rab18 have been reported.
3. Rab11a
Rab11 has been reported to mediate polarized membrane recycling and
cytoskeleton reorganization toward the posterior pole in oocytes, and in
this way contributes to the generation of asymmetric plasma membrane
domains essential for proper oogenesis (Dollar et al., 2002). A specific role
for rab11a in epithelial cells is indicated by the observation that this small
GTPase specifically localizes to apical vesicle populations in discrete epithelial cell populations (Goldenring et al., 1996; Calhoun and Goldenring,
1996). In gastric parietal cells, rab11a is present in subapical tubulovesicles
that are involved in transport to the apical domain. Indeed, rab11a was
found to redistribute from subapical tubulovesicles in resting cells to the
apical domain during cell stimulation (Calhoun et al., 1998). Similarly,
rab11a concentrates with syntaxin 3 at the apical plasma membrane upon
stimulation of a regulated exocytic pathway (Castle et al., 2002). Expression
of a dominant-negative rab11a mutant, rab11aN124I, in gastric parietal cells
inhibits the stimulatory recruitment of the Hþ-Kþ-ATPase from the subapical tubulovesicles to the apical surface (Duman et al., 1999). This further
supports the involvement of rab11a in apical directed transport. In other
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VAN IJZENDOORN ET AL.
polarized epithelia, the rab11a-specific compartment was identified as the
apical recycling endosome (see Section I) and is accessible for basolaterally
endocytosed dIgA–pIgR (Casanova et al., 1999; Wang et al., 2000) and
apically internalized membrane-associated markers (Rahner et al., 2000),
but not for the basolaterally internalized transferrin receptor (Brown et al.,
2000; Wang et al., 2000). The latter is in contrast to the localization and
function of rab11a in nonpolarized fibroblasts, where rab11a is involved in
the recycling of transferrin receptors from recycling endosomes. Overexpression of a dominant-negative rab11a mutant deficient in GTP binding inhibits
trancytsosis and apical recycling of dIgA–pIgR but has no eVect on the
basolateral recycling of the transferrin receptor (Wang et al., 2000).
These data suggest a functional role for rab11a in regulating transport
between the apical recycling endosome and the apical surface and underscore
the segregation of rab11a from the transferrin receptor recycling pathway in
polarized epithelial cells. Interestingly, overexpression of a constitutive active
rab11a mutant inhibits basolateral-to-apical transcytosis but does not aVect
apical recycling, the latter in contrast to dominant-negative rab11a (Wang
et al., 2000). Possibly, apical transcytosis requires GTP–GDP cycling on
rab11a, whereas apical recycling does not, similarly as proposed previously
in gastric parietal cells (Calhoun et al., 1998), suggesting that apical recycling
and transcytosing cargo are retained in separate cargo vesicles (Barosso and
Sztul, 1994; van IJzendoorn and Hoekstra, 1999, 2000).
Although the association of rab11a with the apical recycling system in
polarized epithelial cells is well established, still little is known about its
function. However, several rab11a eVector proteins have been identified:
rab11-binding protein (rab11BP) or rabphilin 11 (Zeng et al., 1999;
Mammoto et al., 1999), myosin Vb (Lapierre et al., 2001), and a family of
four rab11a-binding proteins (see also Table I). The latter consists of rab11family-interacting protein 1 (rab11-FIP1), rab11-FIP2, rab11-FIP3, and
rab11-interacting protein (Rip11) (Hales et al., 2001). Rab11-FIP1–3 and
Rip11 all interact with GTP-rab11a, as well as with rab11b and rab25 (see
later). Moreover, the rab11 coupling protein, another rab11 eVector, but not
rab11-FIP2, rab11-FIP3, or Rip11, also interacts with rab4 (Wallace et al.,
2002).
In addition, some of the rab11a eVectors can either self-interact or interact
with each other (Wallace et al., 2002). Like myosin Vb, rab11-FIP1, rab11FIP2, and Rip11 colocalize with rab11a in the ARE in kidney epithelial cells
and in the subapical tubulovesicular compartments in parietal cells (Prekeris
et al., 2000; Hales et al., 2001). The distribution of rab11-FIP2 is somewhat
diVerent, which may reflect its potential association with diVerent pools of
rab11a family members, i.e., rab11b or rab25 (see later). Rab11-FIP1 and
rab11-FIP2 redistribute with rab11a to the apical plasma membrane of
parietal cells upon cell stimulation (Hales et al., 2001). Rab11-FIP proteins
ROLE OF Rab PROTEINS
71
may regulate the localization of rab11 by recruiting it to distinct membraneous organelles (Meyers and Prekeris, 2002). In addition, rab11-FIP2 has
been proposed to act as an adaptor protein that promotes complex formation
with rab11 and a-adaptin in fibroblasts (Cullis et al., 2002). In polarized
epithelial cells, rab11-FIP2 associates with both rab11a and the rab11a
eVector protein myosin Vb and regulates dIgA–pIgR traYcking (Hales
et al., 2002). Thus, overexpression of a dominant-negative rab11-FIP2 causes
the accumulation of rab11a and inhibits apical recycling and transcytosis of
dIgA–pIgR. It has been proposed that a multimeric protein complex, consisting of rab11a, rab11-FIP2, and myosin Vb, controls apical traYcking in
epithelial cells (Hales et al., 2002).
The recruitment of Rip11 to the ARE is mediated by rab11a, which may
stabilize Rip11 association with the membrane (Meyers and Prekeris, 2002),
and through a Mg2þ-dependent interaction of its C2 domain with neutral
phospholipids (Prekeris et al., 2000). Overexpression of Rip11 aVects endosomal membrane morphology in fibroblasts, and a role for Rip11 in transport from the ARE to the apical plasma membrane domain has been
demonstrated (Prekeris et al., 2000; Meyers and Prekeris, 2002).
Rab11-FIP4 is also a rab11a eVector protein, but, unlike the other rab11FIPs, does not seem to be involved in mediating transport. Rather,
rab11-FIP4 aVects the organization and morphology of recycling endosomes
(Wallace et al., 2002). Although the role of this rab eVector protein in epithelial cells awaits further investigation, it is suggested that diVerent rab11a
eVector proteins may control diVerent aspects of endosome functioning.
Rabphilin 11 interacts with mamalian sec13, the yeast counterpart of
which is involved in vesicle formation. The interaction between rabphilin
11 and sec13 is modulated by rab11a, which, in this way, may aVect membrane traYc (Mammoto et al., 2000).
Finally, it is of interest that overexpression of rab11a in nonpolarized cells,
results in a deposition of cholesterol and sphingolipids in recycling endosomes (Holtta-Vuori et al., 2002), suggesting a role for rab11a in cholesterol
transport and underscoring a role for recycling endosomes in regulating lipid
traYcking (see also van IJzendoorn and Hoekstra, 1999; van IJzendoorn
et al., 2000). Because lipid sorting, as well as rab11a regulation of membrane
traYc, takes place at common endosomes/ARE in polarized epithelial cells, it
will be of interest to determine how polarized cholesterol/sphingolipid
traYcking and rab11a may be functionally connected in these cells.
4. Rab25
Rab25, like rab17 (see earlier discussion), is specifically expressed in epithelial cells. Rab25 shows 63% identity and is thus closely related to rab11
(Goldenring et al., 1993). Rab25 and rab11a share considerable overlap in
72
VAN IJZENDOORN ET AL.
subcellular localization. Both rab proteins are present in the subapical tubulovesicles in parietal cells (Calhoun and Goldenring, 1997) and in the ARE in
polarized epithelial cells (Casanova et al., 1999; Wang et al., 2000). Overexpresson of wild-type rab25 or a GTPase-deficient rab25 mutant dramatically slows the rate of dIgA–pIgR transcytosis as well as apical recycling. In
contrast, the basolateral recycling of transferrin receptor is unaVected
(Casanova et al., 1999; Wang et al., 2000). These data indicate that rab25
plays a role in the regulation of apical PM-directed transport through the
ARE in epithelial cells. Overexpression of rab25 alters the distribution of
rab11a (Casanova et al., 1999). Possibly, rab25 in this way aVects the localization and functioning of the rab11a protein in epithelial cells when compared to nonpolarized cells (see preceding section). It will be of interest to see
how the ectopic expression of rab25 in fibroblasts aVects endosome and
rab11a function. Because the eVector domains of rab11a and rab25 are
90% conserved, both rab proteins interact with similar or identical eVector
proteins (Hales et al., 2001; Table I).
Disruption of the microtubule network results in the dispersion of rab25/
rab11a-positive compartments, whereas microtubule stabilization with taxol
causes the rab25/rab11a compartments to redistribute to the apical corners of
the cells (Casanova et al., 1999). These data suggest that rab25 and rab11a
may interact with cytoskeleton motors to regulate the movement of vesicles
or endosomes along microtubules. Lapierre et al. (2001) identified the unconventional myosin Va as an eVector protein that binds to both rab25 and
rab11a. Overexpression of a dominant-negative myosin Va tail chimera
prevents the exit of basolaterally endocytosed dIgA–pIgR from the apical
recycling system, causing its accumulation in the pericentrosomal region, but
does not aVect basolateral recycling of transferrin receptors (Lapierre et al.,
2001). Given that within the same polarized epithelial cell at least five
interacting proteins (rab11-FIP1–3, Rip11, and myosin Vb) can interact
with both rab25 and rab11a, it will be of interest to determine how and
where these rab eVector protein complexes are located within the recycling
endosomal system (spatial segregation in subdomains?) and how such
protein complexes are established as a function of time.
5. Rab4
In polarized epithelial cells, rab4 localizes to endosomal compartments,
accessible for transcytosing dIgA–pIgR and transferrin receptors, distal
from the early sorting endosomes and therefore most likely resembling the
common endosome (Mohrmann et al., 2002). Overexpression of rab4 or a
GTPase-deficient rab4 mutant increases the amount of basolaterally internalized transferrin receptors and their targeting to the apical domain, as
observed previously with brefeldin A (BFA; Wang et al., 2001). These data
ROLE OF Rab PROTEINS
73
suggest that rab4 functions in polarized traYcking through or from the
common endosomes. Interestingly, overexpression of rab4 in conjunction
with BFA treatment does not result in a synergistic eVect, suggesting that
they act in the same pathway (Mohrmann et al., 2002). In addition, cross-talk
may thus exist between rab4 and BFA-aVected processes, including
ADP-ribosylating factor (ARF)-interacting proteins. This is supported
by the observation that the rab4 eVector rabaptin also binds to g-adaptin,
which is involved in ARF-dependent vesicle budding from endosomes
(Stoorvogel et al., 1998), including the common endosome (Futter et al.,
1998; Gibson et al., 1998).
6. Rab3
The rab3 family consists of four members: rab3a, rab3b, rab3c, and rab3d.
Rab3 family members are typically involved in the process of regulated
secretion and, accordingly, are expressed predominantly in cells with specialized secretory functions, such as neurons and endocrine cells (Lledo et al.,
1994; Geppert and Sudhof, 1998). Interestingly, rab3b and rab3d have been
reported to also perform epithelial-specific functions (Weber et al., 1994;
Larkin et al., 2000; van IJzendoorn et al., 2002; Smythe, 2002). Rab3d
appears to be associated with the transcytotic pathway, followed by the
polymeric immunoglobulin receptor. Thus, rab3d was found in a hepatocyte
membrane fraction enriched in transcytotic vesicles, and immunoisolation of
rab3d-containing vesicles were found to be enriched in transcytosed pIgR. In
addition, rab3d-positive vesicles localize near the apical PM and in the apical
cytoplasm of polarized hepatocytes, and experimental perturbation of transcytosis causes the accumulation of rab3d in the subapical cytoplasm (Larkin
et al., 2000). Another rab3 family member, rab3b, is preferentially expressed
in cultured epithelial cells and native epithelial tissue, including the liver,
intestine, and nephron (Weber et al., 1994). Kirk and collegues showed that
rab3b localizes in the apical region near the tight junction area in epithelial
cells with high secretory capacity. This specific localization pattern was
shown to be dependent on cell–cell contact. Thus, when cells lose contact
with neighboring cells upon extracellular calcium removal, rab3b redistributes to the cell periphery. The reestablishment of cell–cell contact by readministering extracellular calcium causes the rab3b to be recruited again to the
tight junction area. These characteristics suggest a role for rab3b in apical
and/or tight junction traYc in epithelial tissues. Data from studies in which
rab3b was overexpressed in PC12 cells suggested that this GTPase may
influence cell signaling pathways that, in turn, modulate cytoskeleton
arrangement and junctional protein targeting (Sunshine et al., 2000).
Detailed information about a role of rab3b in apical traYcking and
transcytosis was described in polarized epithelial kidney (MDCK) cells
74
VAN IJZENDOORN ET AL.
(van IJzendoorn et al., 2002). In these cells, rab3b was found associated with
vesicles that were concentrated in the apical cytoplasm and near the centrosome. These vesicles contain transcytosing pIgR, but not markers of early
endosomes, late endosomes/lysosomes, or Golgi. GTP-bound rab3b interacts directly with a 14 amino acid stretch in the cytoplasmic domain of pIgR
that also contains the basolateral sorting signal, suggesting that rab3b may
modulate the polarized transport of the receptor. Intriguingly, rab3b and
pIgR do not interact and localize to distinct locations when dimeric IgA
(dIgA) is allowed to bind pIgR (van IJzendoorn et al., 2002). Following its
sorting and delivery to the basolateral surface, pIgR can bind dIgA circulating in the basolateral medium. The receptor–ligand complex is subsequently
internalized and transcytosed to the apical surface where the dIgA and the
extracellular portion of the receptor are cleaved oV and released in
the extracellular space. Also nonbound pIgR is transcytosed, albeit with
lower eYciency, and more of the nonbound pIgR is recycled to the basolateral
domain.
In canine kidney epithelial (MDCK) cells, binding of dIgA to pIgR at the
basolateral surface stimulates transcytosis (i.e., delivery from the common
endosome/ARE to the apical surface (Luton et al., 1998, 1999; Luton and
Mostov, 1999) of the dIgA–pIgR complex. Stimulation of dIgA–pIgR transcytosis requires a dIgA-elicited signaling cascade that involves dimerization
of the pIgR, PLCg activity, protein kinase C, the nonreceptor tyrosine kinase
p62yes, and an increase in intracellular calcium (Song et al., 1994; Cardone
et al., 1994, 1996; Singer and Mostov, 1998; Luton et al., 1998, 1999; Luton
and Mostov, 1999). In addition, the dIgA–pIgR complex must be sensitized
in order to respond to this signaling cascade, which requires Arg657 in the
cytoplasmatic domain of pIgR. The observed dissociation of rab3b and
dIgA-bound pIgR, and presumably the preceding GTP hydrolysis on
rab3b, was found to require the signaling cascade elicited by dIgA via pIgR
and, moreover, rab3b–pIgR dissociation does not occur when Arg657 is
mutated to Ala657. Importantly, overexpression of a GTP-locked rab3b
mutant inhibits the dIgA-stimulated transcytosis of pIgR. Rab3b-GTP,
when bound to pIgR, may stimulate pIgR recycling to the basolateral surface, whereas when dissociated from pIgR upon GTP hydrolysis, it allows
the apical delivery of dIgA–pIgR. These data suggest that pIgR controls its
apical delivery via interaction with rab3b.
Rab3b, like the other rab3 family members, interacts with calmodulin.
This interaction is dependent on calcium but independent on the nucleotide
conformation of the rab3 protein (Park et al., 1997; Coppola et al., 1999;
Sidhu and Bhullar, 2001). Calmodulin may cause the dissociation of rab3, as
well as Ra1A (see Section IIIF), from the membrane (Park et al., 1997). A
mutation in rab3 that prohibits binding to calmodulin does not inhibit the
binding of rab3 to its eVectors rabphilin and RIM, nor does it aVect the
ROLE OF Rab PROTEINS
75
subcellular location of the rab3 protein (Coppola et al., 1999). However, this
mutant does inhibit the ability of GTP-rab3 to inhibit exocytosis of catecholamine- and insulin-secreting cells. Whether the calmodulin–rab3b interaction
plays a role in polarized transport in epithelial cells remains to be investigated. However, the pIgR is a major calmodulin-binding protein in an
endosome fraction of rat liver that is enriched in recycling receptors
(Enrich et al., 1996). Moreover, calmodulin was reported to bind to the
basolateral sorting receptor of the pIgR in a calcium-dependent manner
(Chapin et al., 1996), similar as rab3b (van IJzendoorn et al., 2002). It is
not known whether binding of GTP-rab3b to the pIgR aVects the binding of
calmodulin, and vice versa. Calmodulin antagonists inhibit transcytosis of
dIgA–pIgR in kidney epithelial cells (Apodaca et al., 1994; Enrich et al., 1996)
and polarized hepatocytes (van IJzendoorn and Hoekstra, 1998), resulting in
a concommitant increase in basolateral recycling (Enrich et al., 1996). In
addition, calmodulin antagonists cause the appearance of exceptionally large
endosomal structures (Apodaca et al., 1994) and inhibit endosome fusion
(Colombo et al., 1997), which points to a role of calmodulin in regulating the
function of the endocytic compartment in epithelial cells (Apodaca et al.,
1994; Enrich et al., 1996).
Calmodulin-dependent kinases have been reported to phosphorylate rabphilin-3, a rab3 eVector protein (Kato et al., 1994). The rab3–rabphilin-3
system may control a-actinin-regulated reorganization of actin filaments
(Kato et al., 1996). Furthermore, the coating of exocytic vesicles with actin
filaments has been shown to correlate with the release of rab3D in pancreatic
acinar cells, which is required for the movement of these vesicles to the site of
fusion with the apical plasma membrane (Valentijn et al., 2000). It will be of
interest to determine whether such mechanisms may play a role in apical
transport in epithelial cells. This is not unprecedented, as traYcking in the
early steps of the endocytic pathway in epithelial cells, i.e., from apical or
basolateral early endosomes to the recycling endosomes, depends on the
actin-based mechanoenzyme myr4, a member of the unconventional myosin
V superfamily that uses calmodulin as its light chain, and polymerized actin
(Huber et al., 2000). Moreover, polarized sorting from the common endosome
was reported to depend on intact actin filaments (SheV et al., 2002).
7. Rab13
Rab13, which is closely related to the yeast sec4 protein, is localized in close
proximity to the tight junctions of epithelial cells of various origin (Zahraoui
et al., 1994). This specific localization requires intact tight junctions. Thus,
upon disruption of tight junctions, or in cells devoid of tight junctions, rab13
is distributed throughout the cytoplasm of the cells. Conversely, rab13 is
recruited from the intracellular pool to the junctional complex upon cell–cell
76
VAN IJZENDOORN ET AL.
contact formation and was reported to play a role in the early maturation of
the tight junction during mouse preimplantantion development (Sheth et al.,
2000). Tight junctions are required for maintaining cell surface asymmetry
(apical vs basolateral). They act as a selective barrier that restricts the paracellular leakage of solutes. Tight junctions also perform a ‘‘fence’’ function,
preventing the intermixing of basolateral and apical proteins and lipids.
Finally, tight junctions have been proposed to act as a targeting patch for
the delivery of specialized cargo vesicles (Louvard, 1980; Zahraoui et al.,
2000). Indeed, the exocyst complex, which is a sec4 eVector (Guo et al., 1999)
and targets basolateral secretory vesicles to the site of exocytosis, localizes to
the tight junction area in various epithelia (Lipschutz et al., 2000; Lipschutz
and Mostov, 2002).
Almost 30 proteins have been described that are associated with tight
junctions and can be grouped in four major categories (Anderson, 2001).
The first group consists of peripherally associated scaVolding proteins such
as ZO-1, ZO-2, and ZO-3 that organize and connect junctional transmembrane proteins (e.g., occludin and claudin) with cytoplasmic proteins and the
underlying cytoskeleton. The second group consists of numerous signaling
proteins. The third group are those proteins involved in membrane traYc
[e.g., exocyst, vesicle-associated protein (VAP)-33], and the fourth group
consists of junction adhesion molecule (JAM), occludin, and claudin that
create the paracellular barrier. The establishment and proper functioning of
tight junctions most likely require the careful orchestration and timely recruitment and clustering of all these proteins. Rab13 may regulate the
assembly of functional tight junctions in epithelial cells (Marzesco et al.,
2002). Thus, overexpression of a constitutively active (GTP-locked) rab13
mutant, rab13Q67L, aVects the transepithelial resistance and increases the
paracellular flow of small tracers, indicating impaired gate and fence functions. Indeed, partial mislocalization of apical and basolateral protein has
been observed in cells overexpressing the rab13 mutant. In addition, this
mutant induces a disorganization of the tight junction strand network and,
importantly, delays the recruitment of claudin-1 from the intracellular pool
to the area of cell–cell contact. Possibly, rab13 plays an important role in
coordinating the recruitment of claudin-1 and, to a lesser extend, ZO-1 to
specific sites on the lateral membrane. This would support the hypothesis
that rab proteins act by controlling the assembly of protein complexes, in this
case to build functional tight junctions. The involvement of rab13 in the
polarized delivery of basolateral and/or apical proteins has not been
reported.
In order to elucidate in further detail the function of rab13, a yeast twohybrid screen was performed to search for putative rab13 eVectors, i.e.,
proteins that interact specifically with the GTP-bound form of rab13. A 17kDa protein has been identified, the rod cGMP phosphodiesterase d subunit
ROLE OF Rab PROTEINS
77
(d-PDE). Immunolocalization experiments show that this protein is associated with vesicles that localize closely to the plasma membrane of epithelial
cells. d-PDE is able to extract rab13 from cellular membranes and may be
involved in the dissociation and recycling of rab13 from its target membranes
(Marzesco et al., 1998). A role for d-PDE in the establishment and/or
maintenance of tight junctions has not been reported.
8. Rab8
In polarized epithelial cells, rab8 localizes to the Golgi region, vesicular
structures, and the basolateral plasma membrane (Huber et al., 1993). This
rab protein has been found to be highly enriched in basolateral vesicles that
carry the vesicular stomatitis virus glycoprotein (VSV-G), but is absent from
vesicles that carry the hemagglutinin protein (HA) of influenza virus to the
apical surface. A peptide derived from the hypervariable COOH-terminal
region of rab8 inhibits the basolateral delivery of Golgi-derived VSV-G but
has no eVect on the apical delivery of HA in an in vitro transport assay. Rab8
thus plays a role in polarized, i.e., basolateral, transport in epithelial cells. In
autosomal-dominant polycystic kidney disease (ADPKD) epithelial cells,
rab8 is redistributed from the perinuclear Golgi region to disperse vesicles
(Charron et al., 2000a). A similar observation has been made for sec6 and
sec8, which are components of the exocyst complex, which mediates basolateral transport, and is a known rab (sec4) eVector. The perturbed localization of rab8 may account for the impairment of membrane transport between
the Golgi and the basolateral plasma membrane in ADPKD epithelial cells,
resulting in the accumulation of the VSV-G protein in the Golgi complex. In
addition, ADPKD epithelial cells display a compromised cytoarchitecture
(Charron et al., 2000b), presumably due to alterations in the cytoskeleton
organization.
Also, nonepithelial cells such as fibroblasts are capable of sorting ‘‘apical’’
and ‘‘basolateral’’ proteins, e.g., VSV-G and HA, respectively, in the TGN
into distinct carrier vesicles (Yoshimori et al., 1996; see also Section I). Overexpression of wild-type rab8 or a GTP-locked mutant of rab8, rab8Q67L,
infibroblasts results in a dramatic change in cell morphology. Thus, processes
are formed, which is the result of a reorganization of actin filaments and
microtubules (Peränen et al., 1996). Intriguingly, newly synthesized VSV-G is
preferentially delivered to the rab8-induced processes, suggesting that rab8
provides a link between the formation of actin-dependent cell protrusions
(i.e., membrane polarization) and polarized membrane traYcking (Peränen
et al., 1996).
In a search for rab8-interacting proteins by the yeast two-hybrid system, a
tumor necrosis factor (TNF)-a-induced coiled-coil protein, FIP-2 (not related to rab11-FIP2), has been identified, which binds to GTP-bound rab8
78
VAN IJZENDOORN ET AL.
(Hattula and Peränen, 2000). FIP-2 localizes to the cytosol, the Golgi region,
and the basolateral plasma membrane in epithelial cells, similar to rab8. Its
Golgi localization has been proposed to require intact Golgi function but not
structure (Stroissnigg et al., 2002). Overexpression of FIP-2 promotes the
formation of cell protrusions in fibroblasts, similar to that observed with the
GTP-locked rab8 mutant. The FIP-2 mediated change in cell shape is inhibited by a dominant-negative rab8 mutant, rab8T22N, which is in a GDPbound conformation, suggesting that FIP-2 may act upstream of rab8
(Hattula and Peränen, 2000). Interestingly, the subcellular localization of
FIP-2 may diVer between epithelial cells that are highly secretory and those
that are primarily absorptive (Li and Gallin, 2002). Thus, in polarized
hepatocytes, FIP-2 is primarily associated with the apical, bile canalicular
surface. In contrast, in depolarized hepatocytes, FIP-2 redistributes to
cytoplasmic structures, presumably Golgi elements.
The subcellular localization of rab8 in polarized hepatocytes is not known.
Possibly, FIP-2, in cooperation with rab8, is involved in regulating the spatial
organization of the cytoskeleton that, by forming a scaVold for the assembly
of protein complexes that are involved in apical–basolateral plasma membrane segregation and the targeting of vesicles to defined regions of the cell
surface, underlies cell polarity. FIP-2 also binds to and may recruit huntingtin, a protein proposed to play a role in membrane traYcking, to rab8positive vesicles (Hattula and Peränen, 2000). GTP-Rab8 also binds to a
stress-activated protein kinase, rab8ip/germinal center (GC) kinase, involved
in TNF-a-mediated processes (Ren et al., 1996), suggesting that rab8 plays a
role in regulating membrane traYcking linked to TNF-a-mediated processes
such as diVerentiation. Intriguingly, filopod fomation by TNF-a requires the
interaction between RalA, a Ras-related small GTPase involved in controlling actin cytoskeleton remodeling and vesicle transport, and a member of
the exocyst complex, sec5 (Sugihara et al., 2002). RalA has been shown to
regulate the targeting of basolateral proteins in polarized epithelial cells
(Moskalenko et al., 2002).
Rab proteins are typically activated by rab guanine exchange factors
(GEFs). Hattula and collegues (2002) have identified a coiled-coil protein,
rabin8, that stimulates nucleotide exchange on rab8 and thus is likely a rab8
GEF. Rabin8 localizes to the cortical actin but redistributes to rab8-specific
vesicles when cells expressed a dominant-negative rab8 mutant, rab8T22N.
Association of rabin8 with rab8-specific vesicles promotes their polarized
transport. Overexpression of rabin8 in fibroblasts results in remodeling of the
actin cytoskeleton and the formation of polarized cell surface domains
(Hattula et al., 2002). Possibly, rab8 activation by rabin8 links vesicles
carrying basolateral proteins to the actin cytoskeleton for polarized targeting
to the plasma membrane.
ROLE OF Rab PROTEINS
79
III. Concluding Remarks
Many rab proteins are now known that perform functions that are likely to be
specific for epithelial cells. One of the currently most intriguing and striking
observations is the involvement of a variety of rab protein in the endocytic/
transcytotic transport pathway, where more rab proteins are acting than
compartments have been identified (Table I). The endocytic system in polarized epithelia has received a great deal of interest as it is likely to play a central
role in the establishment and maintenance of cell polarity. Indeed, adaptation
and plasticity of the endosomal system (SheV et al., 2002a,b), in particular the
recycling endosomes, appear of crucial importance to store and/or sort and
target plasma membrane proteins and lipids, either as cargo or as integral
components of epithelial junctional structures. In light of recent insight in
nonpolarized cells (Sönnichsen et al., 2000; Zerial and McBride, 2001; de
Renzis et al., 2002; Miaczynska and Zerial, 2002), it is anticipated that rab4,
rab17, rab11a, rab25, and rab3 form overlapping domains within the epithelial endosomal system through interactions with divalent rab eVector proteins. In this way, the concerted action of these rab proteins may control
endosome compartmentalization and, as such, coordinate the various steps
(i.e., vesicle formation, movement, docking/fusion) of protein and lipid transfer to various destinations in epithelial cells. The identification of divalent rab
eVectors in epithelial cells and the detailed investigation of their functioning
by proteomics and advanced microscopy are expected to provide exciting new
insight in membrane dynamics and epithelial cell polarity.
The potential of rab proteins to act as membrane organizers not only requires
energy and guanine nucleotide-dependent protein–protein interactions, but also
local phosphoinositide lipid metabolism and protein–lipid interactions (Zerial
and McBride, 2001). Phosphoinositide lipid metabolism has been functionally
linked to sphingolipid and cholesterol-enriched rafts, and rab11 has been shown
to modulate cholesterol transport and metabolism. Cholesterol and sphingolipid metabolism and traYcking are also closely related (Hoekstra and van
IJzendoorn, 2000). Moreover, phosphoinositide lipid domains, sphingolipid/
cholesterol rafts, and rab proteins all are intimately connected to actin filament
dynamics, which is involved in membrane dynamics and epithelial polarity at
many levels. It will be a challenge to unravel the complex web that functionally
connects rab domains, sphingolipid/cholesterol domains, and cytoskeleton
organization in the establishment and maintenance of epithelial cell polarity.
Acknowledgments
Sven C. D. van IJzendoorn is supported by a fellowship from the Royal Dutch Academy of
Sciences (KNAW). Keith Mostov is supported by grants from NIH.
80
VAN IJZENDOORN ET AL.
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