Molecular Biology of the Cell
Vol. 19, 2059–2068, May 2008
Rab8 Regulates Basolateral Secretory, But Not Recycling,
Traffic at the Recycling Endosome
Lauren Henry and David R. Sheff
Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, IA 52242
Submitted September 17, 2007; Revised February 5, 2008; Accepted February 11, 2008
Monitoring Editor: Jennifer Lippincott-Schwartz
Rab8 is a monomeric GTPase that regulates the delivery of newly synthesized proteins to the basolateral surface in
polarized epithelial cells. Recent publications have demonstrated that basolateral proteins interacting with the ?1-B
clathrin adapter subunit pass through the recycling endosome (RE) en route from the TGN to the plasma membrane.
Because Rab8 interacts with these basolateral proteins, these findings raise the question of whether Rab8 acts before, at,
or after the RE. We find that Rab8 overexpression during the formation of polarity in MDCK cells, disrupts polarization
of the cell, explaining how Rab8 mutants can disrupt basolateral endocytic and secretory traffic. However, once cells are
polarized, Rab8 mutants cause mis-sorting of newly synthesized basolateral proteins such as VSV-G to the apical surface,
but do not cause mis-sorting of membrane proteins already at the cell surface or in the endocytic recycling pathway.
Enzymatic ablation of the RE also prevents traffic from the TGN from reaching the RE and similarly results in mis-sorting
of newly synthesized VSV-G. We conclude that Rab8 regulates biosynthetic traffic through REs to the plasma membrane,
but not trafficking of endocytic cargo through the RE. The data are consistent with a model in which Rab8 functions in
regulating the delivery of TGN-derived cargo to REs.
The Rab proteins are small monomeric GTPases of the Ras
family. More than 60 known Rabs collectively regulate the
flow of nearly all membrane traffic within the cell (Pfeffer,
1994; Gurkan et al., 2005; Jordens et al., 2005). Of these,
mammalian Rab8 has been associated with the flow of
newly synthesized proteins to the basolateral surface in
polarized epithelial cells (Huber et al., 1993b; Moritz et al.,
2001). In yeast, the Rab8 homolog, sec4, is also required for
the targeting of membrane traffic to the plasma membrane
(Guo et al., 1999). It has been suggested that Rab8 may be
active in delivery of secretory cargo from the TGN to the
plasma membrane. (Chen et al., 1993; Huber et al., 1993b).
Studies of vesicular stomatitis virus protein G (VSV-G) de-
livery from the trans-Golgi network (TGN) have suggested
that this process is regulated by Rab8, as dominant-negative
(DN) mutants of Rab8 interfere with the delivery of basolat-
eral cargo (Huber et al., 1993b). Rab8 is not localized to the
TGN; rather it is localized at or near the recycling endosome
(RE) in both polarized and nonpolarized cells (Hattula et al.,
2006; Roland et al., 2007). The function of Rab8 at the RE is
likely to regulate recycling of membrane traffic, although its
role in polarized sorting is not clear. Rab8 is not required for
the delivery of all basolateral proteins, but appears to be
obligatory in the case of those that bear a tyrosine-depen-
dent basolateral-sorting determinant capable of interacting
with the clathrin adapter ?1-B (Ang et al., 2003). The baso-
lateral TGN-to-plasma membrane trafficking pathway is not
as direct as once thought. Recent reports demonstrate clearly
that ?1-B–dependent basolateral traffic from the TGN passes
through the RE en route to the plasma membrane (Ang et al.,
2004). This finding has prompted us to reevaluate the role of
Rab8 in basolateral trafficking, in an effort to determine
whether Rab8 controls passage from the TGN to the RE or
from the RE to the plasma membrane.
In polarized kidney epithelia, such as Madin-Darby ca-
nine kidney (MDCK) cells, newly synthesized basolateral
plasma membrane proteins are delivered “directly” to the
basolateral plasma membrane without first passing through
the apical membrane (Drubin and Nelson, 1996; Keller and
Simons, 1997; Mostov et al., 2000; Nelson, 2003; Hua et al.,
2006). With the notable exceptions of NgCAM, and poly-
immunoglobulin receptor, apical proteins are likewise de-
livered to the apical plasma membrane without first being
delivered to the basolateral surface (Breitfeld et al., 1989,
Anderson et al., 2005; Hua et al., 2006; Paladino et al., 2006).
Delivery to apical and basolateral domains is via separate
transport vesicles (Wandinger-Ness et al., 1990; Sztul et al.,
1991). This is in contrast to the situation in hepatocytes,
where the majority of both the apical and basolateral pro-
teins are first delivered basolaterally, after which the apical
membrane proteins are endocytosed and sorted to the apical
surface (Simons and Wandinger-Ness, 1990; Ihrke et al.,
1998). However, even direct delivery of nascent proteins to
the basolateral surface implies only that these proteins are
not first delivered to the apical surface; it does not preclude
passage of these proteins through REs en route from the
TGN to the basolateral surface. Thus, regulators of basolat-
eral sorting, for example, Rab8 and the exocyst complex,
could potentially act at the level of the TGN, the RE, the
This article was published online ahead of print in MBC in Press
on February 20, 2008.
Address correspondence to: David R. Sheff (david-sheff@uiowa.
Abbreviations used: CHO, Chinese Hamster ovary; MDCK, Madin-
Darby canine kidney; MDCKT, MDCK cell with transferrin recep-
tor; RE, recycling endosome; RFP, red fluorescent protein; TER,
trans epithelial resistance; TGN, trans-Golgi network; Tfn, trans-
© 2008 by The American Society for Cell Biology 2059
plasma membrane, or the pathways that link these or-
ganelles (Peranen et al., 1996; Grindstaff et al., 1998).
Sorting within the RE is a multistep process. Arriving
ligands are first separated into distinct apical and basolateral
subdomains within the RE, with ligands destined for each
pathway thereby being concentrated (Thompson et al., 2007).
Apical and basolateral transport vesicles then bud from
these ligand-enriched domains, a process that results in high
fidelity of cargo sorting, at least along the basolateral trans-
port pathway (Sheff et al., 1999). Basolateral delivery of
secretory proteins bearing tyrosine-based basolateral sorting
determinants (such as transferrin receptor [TfnR], E-cad-
herin, and pIgR, as well as asialoglycoprotein receptor) in-
volves sequential delivery from the TGN to the RE to the
plasma membrane (Stoorvogel et al., 1989; Futter et al., 1995;
Leitinger et al., 1995; Ang et al., 2004; Murray et al., 2005;
Cresawn et al., 2007). Rab8 is known to regulate traffic along
the TGN–RE–plasma membrane pathway, but whether this
involves traffic into, through or out from the RE is not clear.
Rab8 is also involved in AMPA recruitment to dendritic
spines from the RE of neurons and thus may serve in both
sorting and storage functions of the RE (Huber et al., 1993a;
Gerges et al., 2004).
Given the diverse roles of Rab8 in a variety of contexts, it
could potentially act 1) in the transport of vesicles from the
TGN to the RE, perhaps controlling fusion into the RE; 2) in
organizing the sorting of basolateral traffic into subdomains
of the RE as vesicular traffic enters this structure; or 3) in
budding from the RE and delivery of cargo to the plasma
membrane, including the penetration of the actin-filament
web near the plasma membrane(Peranen et al., 1996; Hattula
et al., 2002). In an effort to resolve these issues, we examined
the requirement for Rab8 in the polarized sorting of endo-
cytic ligands and secreted proteins in MDCK cells. We find
Rab8 to be associated with the basolateral secretion of pro-
teins and the development of cell polarity. Surprisingly we
find that Rab8 is not associated with endocytic recycling or
basolateral delivery once cell polarity is established. Our
results indicate that Rab8 is most likely involved in the
TGN-to-RE transport step rather than in sorting at the RE or
MATERIALS AND METHODS
All chemicals used were of analytical grade or better and purchased from
Sigma-Aldrich (St. Louis, MO) or Fisher Chemical (Fairlawn, NJ). Alexa-488
Tfn and Alexa-488 goat anti-mouse antibody were purchased from Molecular
Probes (Invitrogen, Carlsbad, CA). Mouse monoclonal anti-gp114 was clone
Y652, a gift from the Mellman laboratory.
Cell Lines and Constructs
MDCK cells stably expressing the human transferrin receptor (MDCKT) in
pCB6 were previously described (Sheff et al., 1999). DsRFP-Rab8 and dsRFP-
Rab8 Q67L in adenoviral constructs and pShuttle-CMV as well as YFP-VSV-G
tsO45 adenovirus construct were a kind gift from the Mellman laboratory.
Adenovirus was produced in A-293 cells. Lentiviral constructs were made by
first subcloning into the TA cloning vector pENTR/D Topo (Invitrogen) using
a PCR product with primers CACCATGGCCTCCTCCGAGG and TCACA-
GAAGAACACATCGGAA. The red fluorescent protein (RFP)-Rab8 con-
structs were then transferred to pLenti4/V5 DEST using a clonase reaction
according to manufacturer’s directions (Invitrogen). Lentivirus was produced
in F-293 cells with a Virapower support kit (Invitrogen) to supply packaging
plasmids. Monoclonal antibodies against P58 and P114 were a kind gift from
the Mellman laboratory.
Tfn Labeling and Kinetics
Tfn was labeled with125I using Iodo-Gen reagent (Pierce Chemical, Rockford,
IL) as described (Podbilewicz and Mellman, 1990). MDCKT cells were grown
to confluence on a 10-cm cell culture Petri dish. The cells were trypsinized and
plated 1:1.1 into 24-mm Transwell filter inserts with 0.4-?m pore size (Corn-
ing Life Sciences, Corning, NY). For adenoviral and some lentiviral expres-
sions, virus was applied 24 h after plating cells onto the filters. For day 3
lentiviral expression, the virus was applied 72 h after plating. All virus was
applied in a minimal volume of unsupplemented DMEM media (Invitrogen/
Invitrogen) for 1 h. Then growth media (DMEM with 8% fetal calf serum,
pen/strep, and added glutamine) was substituted. All cells were induced
with 5 mM butyrate overnight and then placed in butyrate-free medium for
4 h before analysis on day 4. Binding and recycling of Tfn as well as data
handling, equations, and fitting of mathematical models was as described in
detail previously (Sheff et al., 1999, 2002). In particular,125I-labeled Tfn was
selectively bound to the basolateral surface of the cells on ice for 45 min. After
cells were washed on ice, the attached Tfn was chased into the cells with
media containing 0.1 mg/ml unlabeled Tfn for up to 1 h. Internalization rates
were derived from the clearance of acid-labile, labeled Tfn from the cell
surface over 4 min. Recycling and transcytosis data were determined from
counts released into the media at various times. These values were then used
for further curve fitting of recycling data. k?1is also derived from clearance
values and indicates Tfn that is internalized and returns to the surface without
passing through an acidic compartment, remaining bound to the receptor.
Initial values for k4were derived from recycling rates at times ?6 min (little
contribution from the RE). Remaining values were calculated by iterative
curve fitting, first by eye and then by minimizing the sum squared error (SSE)
for each data set (Daniel, 1987). The SSE was derived using the following:
SSE ? ? ((average value of data at a given time point) ? (value predicted by
model at the same time point))2.
The SSE is useful for fitting a given model to a given data set by altering the
values of the model variables (in this case the rate constants) because the
better the fit, the smaller the SSE becomes. In this case a unique minimum SSE
could be derived using each data set and each model. To determine whether
a statistically significantly better fit was obtained by adding another param-
eter to the model (adding a value for k6), we used the SSE derived for the best
fit without k6and the SSE derived for the best fit of a model including a value
for k6to derive the value of F, in Fischer’s F test using the following formula:
F ? (((SSE1? SSE2)/(df1? df2))/(SSE2/df2)) (see Motulsky and Ransnas,
1987), where df represents the degrees of freedom (number of independent
data points ? number of parameters fit by the model). Because F values vary
with the number of degrees of freedom, a more useful comparison is to derive
the probability p, that the additional parameter added to the model actually
results in a better fit. For any given value of F and number of degrees of
freedom df, a value p is obtained from a standard table (Daniel, 1987).
Recycling endosomes were specifically ablated using HRP-Tfn (Pierce Chem-
ical) essentially as described in Ang et al. (2004). Briefly, MDCKT monolayers
on Transwell filters (Corning Life Sciences) were labeled with horseradish
peroxide (HRP)-Tfn, 0.010 mg/ml, in DMEM for 45 min at 37°C. The label was
chased with label-free medium for 25 min. Cells were placed on ice and
washed with PBS2?(phosphate-buffered saline) three times. The filters were
placed in PBS with 0.1 mg/ml 3,3?-diaminobenzidine (Sigma) with or without
0.025% H2O2. The reaction was stopped with PBS/bovine serum albumin
(BSA; 1%, wt/vol).
YFP-VSV-G adenovirus was applied to MDCKT cells grown on Transwell
filters 1 d after plating. Sixteen hours after infection, cells were shifted to 40°C
and kept at that temperature until polarization and other manipulations were
complete. To release the VSV-G, cells were moved to 31°C for 1 h, followed by
1 h at 37°C, followed by fixation.
Microscopy and Labeling
For localization of RFP-Rab8 in nonpolarized cells, Alexa-488 Tfn was bound
to the cell surface on ice for 30 min and then chased into the cell at 37°C in the
absence of further label for 25 min. Cells were fixed in 4% paraformaldehyde.
For gp114 labeling, cells were first fixed and then permeabilized with 0.1%
saponin in 2% BSA/PBS. Y652 antibody supernatant was applied at 1:100
with an Alexa 488 secondary label at 1:200. Images were acquired with a Zeiss
Axiovert 200M microscope Jena Germany) equipped with a Hamamatsu
(Hamamatsu, Japan) Orca ER cooled CCD camera and a 63? water immer-
sion objective (Zeiss) using chroma filters optimized for fluorescein isothio-
cyanate (FITC) and rhodamine. For polarized MDCKT cells, labeling was
from the basolateral surface only but otherwise as above. Images were ac-
quired on a Zeiss LSM-50 confocal microscope with a Zeiss Axiovert 100M
stand and a 63? oil immersion lens. Acquisition was in multitrack mode
using excitation wavelengths of 488 and 543 with FITC and rhodamine filters.
A stack of 30 images was taken over the cell height (typically 15 ?m) with the
raster zoomed to 2?. For three-dimensional (3D) reconstruction, the image
stack was cropped to a single cell and then processed with Volocity (Impro-
vision, Coventry, United Kingdom) software. An X-Z image was produced by
cropping the image stack in the X-Y plane. X-Z images were produced using
Zeiss LSM-510 software to acquire an X-Z image with 30 Z-sections.
L. Henry and D. R. Sheff
Molecular Biology of the Cell2060
To initiate our investigation of whether Rab8 acts on traffic
passing from the Golgi to the RE versus on traffic passing
from the RE to the plasma membrane, we first examined
basolateral recycling of Tfn through the RE. MDCK cells
stably expressing the human transferrin receptor (MDCKT)
were grown in Transwell filter inserts and 24 h after seeding,
were infected with an adenoviral construct expressing wild-
type RFP-Rab8. This resulted in high expression levels in
more than 85% of cells (as assessed visually, see Supplemen-
tary Figure 1). After an additional 3 d in culture during
which the cells were allowed to complete their polarization,
Tfn recycling was analyzed using125I-labeled Tfn (see Ma-
terials and Methods). Apical and basolateral media were col-
lected at the indicated time points, and the total125I in each
was measured (Figure 1A). In control cells, the recycled Tfn
(released from the basolateral surface) represented 89.6 ?
3.3% of the total, whereas transcytosed Tfn (released from
the apical surface) represented 6.1 ? 1.6%, an outcome that
is consistent with previous studies (Sheff et al., 1999, 2002). In
contrast, when wild-type RFP-Rab8 was overexpressed, only
56.7 ? 2.7% of Tfn was recycled, whereas 39.0 ? 2.0% was
transcytosed to the apical surface. These results suggested
that overexpression of Rab8 as the cells were polarizing (and
while at the time of the assay) led to mis-sorting of basolat-
eral traffic in the endocytic system.
The kinetic data obtained from this initial experiment
were used to theoretically analyze which of the pathways
involved in endocytic recycling (shown in Figure 1B) is most
likely perturbed. A mathematical model of traffic recycling
through the endocytic system was fit to our Rab8 overex-
pression data, an approach we have used previously to
localize endocytic trafficking defects that arise due to treat-
ment with aluminum fluoride or latrunculin B (Sheff et al.,
1999, 2002). Using first-order rate constants for various en-
docytic trafficking pathways present in polarized cells
(k1–k5, shown in Figure 1B), we were able to fit curves to the
recycling data for both controls and cells overexpressing
Rab8 (Figure 1C). The values derived from a best fit for all
rate constants are shown in Table 1. Internalization rates (k1)
were determined directly as surface clearance over the first
4 min. The rate constant (k1) for internalization of125I-Tfn
was 0.853/min in control cells, whereas it was 0.488/min in
RFP-Rab8–expressing cells (data not shown). This difference
reflects a decrease in the rate of clearance from the cell
surface after normalization for the number of receptors.
There was also a difference in k?1, which represents the rate
of return to the plasma membrane for Tfn that has not
encountered an acidic compartment (k?1, 0.219 in control
cells, 0.0 in infected cells). This discrepancy may be directly
attributed to the lower internalization rate, which makes
such traffic less likely. The values of k1and k?1were then
used to derive the remaining rate constants, by fitting the
curves for the mathematical model to the recycling data
(Table 1). Fitting of the model to experimental data were
initially done by eye, after which the fit was fine-tuned by
minimizing the SSEs for the fit of the model to each data set
(see Materials and Methods). This best fit was made by itera-
tively altering values for all the rate constants involved, thus
simultaneously exploring if changes in traffic between the
plasma membrane and early and REs could explain the data.
Multiple independent starting points were used for each
curve fitting to ensure that a unique solution set with min-
imal SSE could be derived. Although there is no absolute
SSE value for a good fit, a smaller SSE represents a better fit
of the model to a given data set. The SSE for the fit of the
model to control cells was 69.8. Surprisingly, the SSE for
RFP-Rab8–overexpressing cells was 185.7. The large error in
ing of Tfn traffic by disrupting cell polarity. (A) Effect of Rab8
overexpression.125I-Tfn bound to the basolateral surface of MDCKT
cells on ice was internalized at 37°C, and label released into the
apical and basolateral media was measured at the times indicated.
Blue circles, recycling into the basolateral medium in control cells;
blue squares, transcytosis to the apical medium in control cells; red
circles, recycling into the basolateral medium in cells overexpress-
ing Rab8; and red squares, transcytosis into the apical medium in
cells overexpressing Rab8. (B) Cartoon of possible Tfn recycling
pathways, whose rate constants were used in the mathematical
model. The rapid recycling pathway includes k1and k4. The long
recycling pathway includes k1 and k3. k6indicates hypothetical
mis-sorting pathway that would occur only in cells that are not
properly polarized so that the apical and basolateral surfaces are
functionally equivalent. BEE, basolateral early endosomes. (C) Fit of
mathematical model to data in A using rate constants in Table 1,
assuming k6? 0. Arrow indicates area of poor fit. Data points are as
in A; blue line models recycling in control cells; brown line models
transcytosis in control cells, red line models recycling in cells over-
expressing Rab8; and green line models transcytosis in cells over-
expressing Rab8. (D) Fit of mathematical model to data in A when
loss of cell polarity is assumed. Data points and lines are as in C.
Error bars, SD for experimental data. n ? 9 for all data points.
Rab8 overexpression before polarization causes mis-sort-
Table 1. Values used for fit of kinetic model to Tfn recycling data
Sum-squared error: 69.85, 185.7, and 21.2.
Rab8 in Recycling Endosomes
Vol. 19, May 2008 2061
fit with respect to the data from the RFP-Rab8–overexpress-
ing cells corresponded to a poor fit of the model to measure-
ments of apical transcytosis at time points below 15 min
(arrow in Figure 1C). Mathematical modeling allowed us to
quantitatively compare a large number of possible Rab8
actions on the recycling pathways, for example, whether
Rab8 resulted in a change in RE-to-plasma membrane traffic,
to a change in early endosome to RE traffic, to both, or to
neither. This allowed us to exclude changes in pathways
k1–k5as explanations for the effect of Rab8 on recycling
Normally, polarized cells contain separate apical and ba-
solateral early endosomes that serve each surface domain
(Bomsel et al., 1989). However, cells that are improperly
polarized allow the delivery of basolaterally internalized Tfn
from the basolateral early endosomes to the apical surface, a
pathway denoted as k6in Figure 1B (Sheff et al., 2002).
Addition of this pathway to the model for Rab8-infected
cells resulted in a significantly better fit (arrow in Figure 1D
shows area of improved fit), with an SSE of 21.3 (see Table 1;
Daniel, 1987). To determine the statistical significance of this
improvement, we used Fischer’s F test (see Materials and
Methods) In this case F ? 61.7. Given the degrees of freedom
available from our data set and including k6in our model,
the probability is p ? 0.001, that the added pathway (k6) is
statistically justified by the improvement in fit of the model
to the experimental data. (Daniel, 1987; Motulsky and Ran-
snas, 1987). Other differences in the overall model included
a diminishment of basolateral traffic from the RE (k3, 0.061 in
control cells vs. 0.033 in Rab8-overexpressing cells), and
increased apical traffic from the RE (0.005 in control cells vs.
0.0236 in Rab8-overexpressing cells).
The kinetic results are consistent with Rab8 overexpres-
sion disrupting the apical-basolateral polarization of ex-
pressing cells, but could also represent a Rab8-infected
monolayer that may not be intact. A leaky monolayer would
give traffic recycled to the basolateral membrane direct ac-
cess to the apical medium. Such a paracellular pathway
would be topologically equivalent to k6. To investigate the
possibility that our results were due to monolayer leakiness,
we applied125I-Tfn to the apical chamber of the Transwell
system and measured leakage into the basolateral chamber
at 4°C. Negligible leakage in monolayers of control cells, as
well as in cells overexpressing RFP-Rab8 (0.09% for control
cells, 0.1% for RFP-Rab8 expressing cells, and 10% for
EDTA-treated cells), suggested that paracellular leakage was
not abnormally high. To further test the integrity of the
monolayer, we tested the transepithelial resistance (TER) at
37°C across the monolayer grown in a 12-mm transwell.
Control cells had a specific TER of 104 U`?cm2(excluding the
TER of the transwell membrane itself), and the RFP-Rab8–
transfected cells had an almost identical TER of 95.3 U`?cm2,
again suggesting that paracellular leakage was not a factor
in our results. Taken together, the results so far suggested
that RFP-Rab8 overexpression during the time when cells
would normally polarize leads to a profound disruption of
apical-basolateral polarity in RFP-Rab8–expressing cells.
A defect in cell polarization could result from a disruption
in the delivery of newly synthesized proteins, the recycling
of polarized proteins, or both. Expression of RFP-Rab8 1 d
after plating may disrupt the ability of cells to achieve api-
cal-basolateral polarity as well as the ability of endosomes to
maintain polarity if it is established. To differentiate between
these possibilities, we sought to examine the effect of per-
turbing Rab8 both before polarization had occurred and
afterward, in fully polarized cells. To this end, we used a
lentivirus vector, which allowed for expression of Rab8 mu-
tants in nondividing, fully polarized MDCKT monolayers.
Although the lentiviral constructs were expressed in ?90%
of all cells (as judged by appearance of RFP in random fields
examined by microscopy), protein expression per cell was
less than that achieved with the adenoviral constructs used
in the original experiments. In fact, expression levels of the
wild-type Rab8 lentiviral construct were not sufficient to
consistently alter Tfn traffic. We therefore utilized GTPase
deficient (Q67L) DA Rab8 (DA-Rab8) and GDP-binding
(T22N) DN RFP-Rab8 (DN-Rab8) constructs. These con-
structs are based on homology with the Ras GTPase. As
previously described, the Q67L mutant binds GTP, whereas
the T22N mutant does not (Peranen et al., 1996). Both were
expressed as lentiviral constructs. Rab proteins operate by
cycling between GTP bound at the membrane and GDP
bound in the cytosol. Therefore, although phenotypes of DA
and DN mutants may vary, both would be expected to
interfere with traffic along the regulated pathway, as evi-
denced by disruption of endoplasmic reticulum (ER)-to-
Golgi traffic by the DA mutant of Sar1p GTPase (Oka and
Nakano, 1994; Kimura et al., 1995). When applied to MDCKT
cells 24 h after plating (before the cells are polarized), the DA
and the DN constructs each led to significant Tfn mis-
sorting, although the mis-sorting at these expression levels
(Figure 2, A and B) was not as great as that observed when
wild-type Rab8 was expressed using adenovirus (Supple-
mentary Figure 1). All Tfn recycling assays were performed
polarity is established. All panels compare recycling (circles) and transcytosis (squares) of
overexpression mutants (red) after 4 d of polarization. (A) Lentiviral DA-Rab8 application 24 h after cell plating results in significant
mis-sorting to the apical surface. (B) Lentiviral DN-Rab8 application 24 h after cell plating results in significant mis-sorting to the apical
surface. (C) Lentiviral DA-Rab8 application 3 d after cell plating does not result in mis-sorting. (D) Overlay of results from control cells in
A with those obtained for cells infected with DA-Rab8 in A and C, to directly compare the effects of infection after 24 h (red) versus 3 d
Dominant-active (DA) and -negative (DN) Rab8 mutants cause mis-sorting of Tfn traffic when applied before, but not after, cell
125I-Tfn in control cells (blue) and Rab8
L. Henry and D. R. Sheff
Molecular Biology of the Cell2062
4 d after plating, for consistency between assays and to
ensure that all cells were polarized.
The DA-Rab8 construct was next applied to MDCKT
monolayers 3 d after plating; this time point was chosen
because the cells would be in the process of becoming po-
larized, but polarization would be completed before the
construct was fully expressed (Thompson et al., 2007). Con-
sistent with the infection efficiency of this virus, ?90% of the
cells infected 3 d after plating expressed DA-Rab8 (as as-
sessed by visual evaluation of RFP in cells in random mi-
croscope fields; Supplementary Figure 1). On the next day (4
d after plating), the expression level of DA-Rab8 was the
same as that achieved in the previous experiment; however,
Tfn traffic was not affected (Figure 2C). A direct comparison
of cells infected with DA-Rab8 on days 1 and 3 (Figure 2D)
illustrates the dramatic reduction of the Rab8 mutant effect
after the cells have polarized. These results suggested that,
although Rab8 is important for the establishment of cell
polarity, it is not necessary for endocytic sorting and recy-
cling of plasma membrane components. The possibility re-
mained that even in polarized cells, Rab8 may affect secre-
tory traffic passing from the Golgi to the RE (or to the plasma
membrane), but not traffic arriving from the endocytic recy-
cling pathway. These results, confirmed the kinetic analysis
above, suggesting that the observed effects of wild-type
Rab8 overexpression (when it occurs before polarization,
i.e., 24 h after plating) on endocytic Tfn traffic were an
indirect effect of a defect in cell polarization.
Because our results suggested that endocytic recycling
traffic may not be directly affected by Rab8 activity, we
wanted to confirm that our Rab8 constructs were correctly
localized to the RE. Thus, wild-type RFP-Rab8 was ex-
pressed in MDCK cells using adenovirus (infected 24 h after
plating). Tfn was bound to its receptors on ice and internal-
ized for 25 min to specifically label the RE (Sheff et al., 1999;
Thompson et al., 2007). Cells expressing relatively low levels
of RFP-Rab8 were selected for visualization, and the RFP
signal was found to colocalize with that of the internalized
Alexa-488 Tfn (see X-Z reconstruction, Figure 3A). Similar
results were obtained for DA-Rab8 expressed with lentivirus
(data not shown).
We next visualized the distribution of the human Tfn
receptor (TfnR) in the infected MDCKT cells. TfnR is nor-
mally distributed such that 93–95% of the receptor at the
plasma membrane is at the basolateral surface, whereas
5–7% is present at the apical surface (Fuller and Simons,
1986; Sheff et al., 1999). We used confocal microscopy to
compare the localization of TfnR in cells infected with DA-
RFP-Rab8 to that in uninfected cells. A lower multiplicity of
infection (MOI) was used so that both infected and control
cells would be present within a single field. Cells infected
24 h after plating were allowed to polarize before they were
surface-labeled with Alexa-488 Tfn. In the control cells, Tfn
labeling was confined to the basolateral surfaces (Figure 3, B
and C, cells without red). In cells expressing DA-Rab8, how-
ever, apical labeling was observed, consistent with the pres-
ence of a defect in polarization (Figure 3B, arrows). In con-
trast, when the MDCKT cells were infected after polarization
(3 d after plating), the distribution of TfnR was not disturbed
by DA-Rab8 expression, nor was TfnR detectable at the
apical surface (Figure 3C, arrows). These results suggested
that TfnR polarity is sensitive to Rab8 disruption only before
cell polarity is established.
Why would sensitivity to DA-Rab8 end with the estab-
lishment of cell polarity? It is possible that as the cell be-
comes polarized, redundant mechanisms ensure normal
basolateral delivery, even in the presence of DA-Rab8. Al-
ternatively, DA-Rab8 may affect the delivery of basolateral
proteins from the secretory system, but not the recycling of
basolateral proteins already at the plasma membrane, so that
proteins mis-sorted during secretory delivery are resorted
correctly during endocytic recycling. To differentiate be-
tween these possibilities, we used a construct in which YFP
was fused with the temperature-sensitive VSV-G mutant,
ts045. This mutant is trapped in the ER at the nonpermissive
temperature of 40°C, but upon switching to the permissive
temperature (31°C), a wave of the protein is released for
processing through the Golgi. VSV-G was expressed in
MDCKT cells using an adenoviral construct. Cells were infected
24 h after plating and were shifted to the nonpermissive
temperature (40°C) 48 h after plating. Cells were allowed to
polarize at the nonpermissive temperature until 4 d after
plating, and then were shifted to 31°C for 1 h. Because it was
possible that membrane trafficking may not proceed nor-
mally at this lower temperature, the cells were then shifted
to 37°C for an additional 1 h. As is apparent from Figure 4
that the shift to 37°C did not result in sequestration of the
VSV-G construct within the cell. We then tested the effect of
DA-Rab8 expression by infecting the cells with DA-Rab8 (at
40°C) 3 d after plating. A low MOI was again used so that
both control and DA-Rab8–expressing cells could be viewed
in the same field. Further, we chose a time shortly after
arrival of VSV-G at the cell surface so that there would not
be time for correction of mis-sorted proteins through endo-
cytic recycling and sorting. In this way, we could selectively
observe VSV-G that had entered the secretory pathway after
DA-Rab8 was expressed in cells that were fully polarized.
fore, but not after, cell polarity is established. (A) X-Z reconstruction
of an MDCKT monolayer infected with wild-type RFP-Rab8 (red)
24 h after plating and labeled with Alexa-488 Tfn internalized for 25
min (green, marks the RE). (B) X-Z reconstruction of MDCKT mono-
layer infected with DA-RFP-Rab8 (red) 24 h after plating and sur-
face labeled on ice with Alexa-488 Tfn (green) on day 4. Arrows
indicate mis-sorted TfnR. (C) MDCKT cells infected with DA-Rab8
(red) 3 d after plating and surface labeled on ice with Alexa-488 Tfn
(green) on day 4 (green). Arrows indicate cells that express DA-
Rab8 but did not mis-sort TfnR. Bar, 10 ?m.
DA-Rab8 causes mis-sorting of TfnR when applied be-
Rab8 in Recycling Endosomes
Vol. 19, May 20082063
We found that, in control cells ?80% of VSV-G was de-
livered to the plasma membrane (Figure 4, A and B, cells
with no red DA-Rab8), predominantly to basolateral areas
and to a few puncta near the cell apex. To determine
whether these puncta were external or internal, we also
performed live-cell surface labeling, using an antibody that
recognizes the VSV-G ectodomain. In control cells, some
apical VSV-G staining was observed, (Figure 4A, blue in
three cells in the top left of the field) but this was rare,
involving ?8% of VSV-G–infected cells (assessed by count-
ing affected cells in microscope fields). In contrast, 100% of
the cells expressing DA-Rab8 (Figure 4, A and B, arrows)
stained for VSV-G on their apical surfaces (Figure 4, A and
B, blue). In X-Z cross sections, VSV-G was visualized at the
apical surface of these cells (white arrows in Figure 4), as
well as in internal puncta (Figure 4B, green/yellow puncta).
These findings are consistent with mis-sorting of newly
synthesized basolateral VSV-G to the apical surface in the
presence of Rab8.
The introduction of Rab8 3 d after plating caused mis-
sorting of newly synthesized VSV-G, but this may also have
reflected a general loss of apical-basolateral polarity in the
treated cells. Although this seemed unlikely because endo-
somal recycling was unaffected under these conditions, we
confirmed that the cells were polarized by staining for the
endogenous proteins GP114 and P58. Because both of these
proteins are made constitutively and have long half-lives,
the bulk of the protein should have been delivered and
polarized before DA-Rab8 was introduced. Further, we ex-
pected that defects in polarization would be corrected grad-
ually, through the normal endocytic sorting process. GP114
is a marker for the apical plasma membrane and has been
reported to be delivered normally in the presence of Rab8
mutants. We confirmed this observation here (Figure 4C).
P58 is the beta subunit of the Na?/K?ATPase and is nor-
mally expressed basolaterally. Like the TfnR (Figure 3), P58
was normally distributed in cells expressing DA-Rab8.
Taken together with the results above, these findings sug-
gested that DA-Rab8 expression selectively disrupts the de-
livery of newly synthesized basolateral proteins without
affecting established cell polarity or the fidelity of endocytic
Basolaterally targeted VSV-G traffic is reported to pass
through the RE. Our results suggested that DA-Rab8 dis-
rupts secretory traffic without affecting the recycling path-
way. This could result from either a disruption of TGN-
to-RE traffic or a bypass of the RE by direct TGN-to-plasma
membrane traffic. To determine if traffic disruption at the RE
could be responsible for these results, we took an organelle-
ablation approach, using Tfn-HRP to ablate the RE (Hopkins,
1983; Stoorvogel et al., 1988; Pond and Watts, 1997). This
approach was previously used in glass-grown (incompletely
polarized) MDCK cells to ablate the RE, which resulted in
trapping of VSV-G secretory traffic at the Golgi (Ang et al.,
2004). The method has also been used to ablate REs in fully
polarized MDCK cells, although in the latter case apical
mis-sorting of VSV-G was not directly examined (Ang et al.,
2004; Cresawn et al., 2007). In the current study, MDCKT
monolayers were incubated with Tfn-HRP conjugate for 45
min, followed by a chase with serum-free medium for 25
min to allow the RE to be targeted this is a longer chase
period than used by Ang et al. (2004). The cells were then
treated with diaminobenzidine (DAB) and H2O2for 1 h on
ice, to allow for the formation of an insoluble precipitate in
the HRP-containing compartment. This results in the effec-
tive ablation of the RE, and importantly, prevention of all
trafficking to and from the affected structure. To ensure that
only the RE was ablated, we internalized Alexa 546-Tfn for
8 min (Figure 5A) and checked for the usual labeling of early
endosomes and REs. Although REs as well as early endo-
somes were clearly visible in control cells (Figure 5, A and B,
large arrows, no H2O2), only peripheral small endosomes
could be discerned in cells that had undergone the full
ablation treatment (Figure 5A, small arrows). We define
early endosomes and REs functionally so that early endo-
somes are those that contain Tfn internalized for 2.5 min,
whereas REs contain Tfn internalized for 25 min. To further
confirm that the perinuclear REs had been ablated, we used
a double-labeling protocol, internalizing Alexa 488-Tfn for
25 min and Alexa 546-Tfn for 2.5 min in the same cells. This
allowed visualization of functional early endosomes and
REs. Perinuclear REs were clearly visible in control cells
(Figure 5C, green), but were absent in the cells that had
undergone the ablation treatment (Figure 5D; note absence
of green). Early endosomes were visualized by Tfn internal-
ized for 2.5 min in both control and ablated cells (Figure 5,
B and C, red), indicating that early endosomes were still
present and functional. Because Alexa 488-Tfn was no
longer visible in the ablated cells, it must have recycled out
of the cells. Such rapid recycling would be consistent with
direct recycling from the early endosomes to the plasma
membrane (pathway k4in Figure 1B) rather than through
the RE (pathways k2and k3in Figure 1B).
To determine if cell polarity was acutely disrupted by the
ablation technique, we again visualized the endogenous
proteins GP-114 and P58. GP114 was apically localized in
both the control and RE-ablated cells (Figure 5, E and F).
Similarly, P58 was basolaterally localized in both control
and RE-ablated cells (Figure 5, G and H). These results
suggested that ablation of the RE did not acutely disrupt
preformed cell polarity, consistent with our observations for
sorting of VSV-G. (A) 3D reconstruction from a z-stack of confocal
images spanning the full height of the cells. The image is rotated as
shown by the axes at the lower left. GFP-VSV-G (green)–expressing
MDCKT cells were infected with lentiviral DA-Rab8 at low MOI
(red). Cells were kept at the nonpermissive temperature until 4 d
after plating and then shifted to the permissive temperature as
described in the text. The apical surface of live cells was labeled with
an antibody against the VSV-G ectodomain (blue). All cells express-
ing DA-Rab8 (arrows) mis-sorted VSV-G to the apical surface. A few
cells not expressing DA-Rab8 also mis-sorted VSV-G to the apical
surface (top left) (B) Three X-Z reconstructions of confocal line-scan
z-stacks from other fields of MDCKT cells labeled as in A, with
arrows indicating mis-sorted VSV-G. (C) X-Z reconstruction of
MDCKT cells expressing DA-Rab8 (red) as in A and labeled for
apical marker GP114 (green). (D) X-Z reconstruction of MDCKT
cells expressing DA-Rab8 (red) as in A and labeled for basolateral
marker P58 (green). Bars, 10 ?m.
DA-Rab8 Rab8 applied 3 d after plating causes mis-
L. Henry and D. R. Sheff
Molecular Biology of the Cell 2064
To determine if polarized delivery of newly synthesized
proteins was affected by RE ablation, we again used VSV-G.
Tfn-HRP was internalized, at the nonpermissive tempera-
ture, in MDCKT cells expressing VSV-G. After ablation, the
cells were shifted to 31°C for 1 h and then to 37°C for 1 h. In
control cells, VSV-G was delivered to the basolateral surface
(Figure 5I, green arrow), but in cells that had undergone
ablation treatment, VSV-G was observed in many intracel-
lular structures. In the ablated cells, some of the VSV-G
appeared to be at or near the apical surface (Figure 5J, red
arrow). To determine if the VSV-G had been delivered to the
apical surface, live cells were labeled with an antibody di-
rected against the VSV-G ectodomain, as had been done for
DA-Rab8–expressing cells. Little or no surface labeling for
VSV-G was observed in control cells (Figure 5K), but at least
some apical VSV-G signal was visible in virtually all of the
ablated cells (Figure 5L, red arrow). These results suggested
that, when the RE is not available to receive newly synthe-
sized traffic from the TGN, at least some of that traffic is
rerouted to the apical plasma membrane. Together these
results suggest that both HRP-mediated ablation of the RE
and the expression of DA-Rab8 both lead to the diversion of
secretory traffic to the apical plasma membrane at the RE.
Because its discovery, Rab8 has been associated with the
regulation of basolateral traffic in polarized cells. However,
rather than being localized to the plasma membrane, Rab8 is
present in the peri-Golgi area, which includes the Golgi,
TGN, and RE. It is now clear that basolateral cargo from the
TGN—including the TfnR, VSV-G, asialoglycoprotein recep-
tor, pIgR, and e-cadherin—passes through the RE. Our ob-
servations and those of others are consistent in that Rab8
appears to be associated with the RE rather than the Golgi.
In contrast to basolateral traffic, apical traffic is Rab8-inde-
pendent and does not appear to pass through the RE, but
rather through other apical endosomal organelles. Together,
these findings suggest that basolateral delivery is a two-step
process that involves sorting at the TGN, along with what
may be a secondary, or quality-control, level of sorting at the
RE (Figure 6). Our findings are consistent with Rab8 con-
trolling the delivery of basolateral secretory traffic from the
TGN to the RE, rather than functioning later in the process
of delivery to the surface.
Rab8 Does Not Regulate Basolateral Endocytic
Given that Rab8 is associated with the RE, it could be in-
volved in trafficking to the RE, sorting within the RE, or
trafficking out of the RE. The most direct way to examine
both sorting within the RE and basolateral traffic out of the
RE was to monitor endocytic recycling of Tfn through the RE
(Figure 6). We found that the overexpression of wild-type
Rab8 and the introduction of Rab8 mutants before cell po-
larity was established had substantial effects on endocytic
traffic, whereas the introduction of DA-Rab8 after the cells
were polarized did not disrupt endocytic traffic. This latter
finding was surprising in light of the fact that Rab8 had
previously been reported to be important for basolateral
delivery in general.
We sought to resolve why Rab8 mutants had an effect on
basolateral traffic when introduced before polarization, but
not after polarization. To this end we used a kinetic analysis
of the trafficking that was induced by Rab8 overexpression
before polarization. This analysis allowed us to test a large
number of hypothetical variations in trafficking against the
experimental data, using a quantitative mathematical
model. Quantification allowed us to examine changes in
trafficking not only into or out of the RE, but also along any
of the other known recycling pathways, alone or in combi-
nation, and then test which solution set of hypothetical
changes fit most closely to the experimental data. We started
sized VSV-G. All panels show MDCKT cells (4 d after plating) that
have internalized Tfn-HRP into the RE. (A, C, E, G, I, and K) Control
cells treated with DAB but not H2O2; (B, D, F, H, J, and L) cells
treated with DAB and H2O2. (A and B) Basolaterally applied Alexa-
546 Tfn internalized into MDCKT cells for 8 min labels early (small
arrow) and recycling (large arrow) endosomes. REs are not visible in
RE-ablated cells. (C and D) Basolaterally applied Alexa-546 Tfn
internalized into control MDCKT cells for 25 min labels RE (large
arrows). Small peripheral early endosomes are visible in RE-ablated
cells (small arrows). (E and F) In MDCKT cells labeled for Gp-114,
both control and RE-ablated cells are labeled apically. (G and H) In
MDCKT cells labeled for p58, both control and RE-ablated cells are
labeled basolaterally. (I and J) MDCKT cells expressing VSV-G
tsO45 released at the permissive temperature for 2 h after RE
ablation. Green arrow in I indicates normal basolateral distribution
of VSV-G. Red arrow in J indicates putative apical VSV-G in RE-
ablated cells. (K and L) MDCKT cells as in I and J containing cellular
GFP-VSV-G (green) and with surface VSV-G labeled by anti-VSV-G
ectodomain antibody (red). Red arrow indicates mis-sorted apical
VSV-G in ablated cells. Bars, 10 ?m.
Ablation of the RE causes mis-sorting of newly synthe-
Solid arrows represent secretory pathways. Open arrows represent
recycling pathways. EE, early endosome; RE, recycling endosome.
Cartoon of trafficking pathways that pass through the RE.
Rab8 in Recycling Endosomes
Vol. 19, May 20082065
with data sets of wild-type Rab8 overexpression (Rab8 ex-
pressed 24 h after plating), which showed clear disruption in
the polarized delivery of endocytosed Tfn. Contrary to our
initial expectations, we did not find a significant disruption
of trafficking at the RE, or even a combination of disruptions
along normal trafficking pathways, that could explain the
overall loss of polarized sorting in the endocytic pathway.
Rather, a model in which Rab8 overexpression disrupted the
apical-basolateral polarization of the affected cells was most
consistent with the data. This depolarization resulted in
aberrant delivery, with the cargo of basolateral early endo-
somes being mis-routed to the apical surface. Immunofluo-
rescence examination of DA-Rab8–expressing cells in an
otherwise normal MDCKT monolayer confirmed that these
cells were not normally polarized when the DA-Rab8 was
introduced before cell polarization was complete. Together
these results lead us to conclude that Rab8 did not directly
regulate endocytic recycling in individual endocytic com-
partments, but rather, it did disrupt the establishment of cell
polarity. In this way, cargo from the basolateral early endo-
somes is no longer restricted to either basolateral recycling
or delivery to the RE. Instead, this basolateral cargo can be
delivered directly to the apical surface of the poorly polar-
ized cell. This is consistent with Rab8 functioning in the
secretory pathway but not in the endocytic, pathway. Be-
cause the secretory and endocytic pathways to overlap after
passing through the RE (Figure 6), these results would sug-
gest that Rab8 acts on secretory traffic in transit from the
TGN to the RE.
Rab8 Regulates Biosynthetic Basolateral Traffic Both
Before and after Cell Polarize
One alternative to this conclusion, is that Rab8 function
shifts as the cells polarize. Perhaps Rab8 controls a direct
TGN to plasma membrane pathway in cells before they
achieve polarity, but this becomes less important afterward.
A similar suggestion has been made for the clathrin adapter
?1-B, which is required for biosynthetic and recycling baso-
lateral delivery of TfnR before cells are polarized, but not for
biosynthetic delivery after polarization (Gravotta et al.,
2007). We therefore tested whether DA-Rab8 had an effect
on biosynthetic delivery, when introduced 3 d after plating
the cells. Under these conditions, changes in endocytic func-
tion were not detected. Using a pulse of temperature-sensi-
tive GFP-VSV-G, we were able to detect VSV-G mis-sorting
when DA-Rab8 was introduced after polarization. The pulse
was after expression of DA-Rab8 and was sufficiently short
that endocytic resorting of VSV-G to the correct plasma
membrane domain could not have occurred. This finding
supports a model in which Rab8 continues to be required for
the basolateral delivery of newly synthesized proteins even
in polarized cells. The fact that endogenous basolateral as
well as apical markers were normally distributed may be
explained by their relatively long half lives, so that normal
sorting and delivery occurred before DA-Rab8 was ex-
pressed. Additionally, as these proteins were in the cell for
many hours, there would be endocytic resorting of any
mis-directed protein, not affected by DA-Rab8.
Rab8 Acts on the TGN-to-RE Pathway Not on a Separate
TGN-to-Plasma Membrane Pathway
Much of our interpretation relies upon the assumption that
most, or all, of the basolateral biosynthetic traffic passes
through the RE. This assumption is supported by both old
and recent observations. It is well established that the traf-
ficking of newly synthesized and recycling basolateral mem-
brane proteins relies upon the same set of basolateral target-
ing determinants (Matter et al., 1993). Recently, a study using
a slowed-down transport system and HRP ablation of the
RE in glass-grown MDCK cells has taken this understanding
further, showing the RE to be an intermediate in the baso-
lateral secretory pathway. Ablation of the RE using HRP
resulted in the intracellular trapping of VSV-G in the major-
ity of cells, although some cells did express VSV-G on their
surfaces (Ang et al., 2004). Because the cells were not fully
polarized, it is difficult to interpret whether this represented
mis-sorting or a bypass of the ablated RE. Further analysis of
basolateral and apical trafficking in fully polarized, filter-
grown MDCK cells also found passage through the RE to be
an essential step in the delivery of VSV-G to the basolateral
surface in fully polarized MDCK cells (Cresawn et al., 2007).
In blocking delivery of basolateral secretory traffic to the
RE, we observed mis-sorting to the apical surface. This
finding differs somewhat from those published by both Ang
and Cresawn (Ang et al., 2004; Cresawn et al., 2007). These
discrepancies may be attributable, in part, to the fact that we
used fully polarized MDCK monolayers and used Ab detec-
tion of apically mis-sorted VSV-G rather than YFP detection
alone. Additionally, we used a longer (25 min) chase period
to ensure that REs were ablated. Both recycling through
early endosomes and the Golgi morphology (data not
shown) were unaffected by this procedure. Under these
conditions, normal cell polarity was maintained, but newly
synthesized VSV-G was mis-sorted to the apical surface.
Others have demonstrated that the apical pathway does not
pass through the RE. The findings presented here establish
that when the RE is not available to receive traffic, mis-
sorting from the TGN to the apical surface occurs. This traffic
may also pass through an endocytic compartment serving
the apical surface only. Such would also appear to be the
case when Rab8 mutants are used to disrupt TGN-to-RE
On the basis of our assessments of morphology, endocytic
recycling traffic, and secretory traffic, we would suggest a
model of the system in which Rab8 regulates traffic from the
TGN to the RE along the TGN–RE–plasma membrane route.
This interpretation implies that basolateral trafficking out of
the RE and fusion at the plasma membrane are not Rab8
dependent. Thus, one potential role for Rab8 might be to
mediate fusion events between transport vesicles generated
at the TGN and the RE. Although we favor this interpreta-
tion, another possible interpretation is that TGN traffic
through the RE is directed to a unique subdomain of the RE
that exits in a Rab8-dependent manner. If this is the case, then
Tfn-HRP ablation would fill the organelle and thus disrupt
TGN–RE–plasma membrane traffic, whereas exit of Tfn and
Rab8 dependent secretory traffic from the RE to the plasma
membrane would be along separate pathways. Differentia-
tion between these possibilities will have to await further
analysis. It is thus not inconceivable that Rab8 may accom-
pany a variety of cargoes out of the TGN, even to differing
targets (Deretic et al., 1995; Moritz et al., 2001). For example,
in addition to basolateral traffic, Rab8 has been implicated in
TGN-to-primary cilium traffic (Nachury et al., 2007). Rab8 is
dispersed in cells from patients with the ciliopathies auto-
somal dominant polycystic kidney disease and Bardet-
Biedel syndrome (Charron et al., 2000; Nachury et al., 2007).
The primary cilium appears to be a domain separate from
either apical or basolateral plasma membrane domains,
which shares some characteristics with the basolateral do-
main (Rogers et al., 2004). It is possible that Rab8-mediated
L. Henry and D. R. Sheff
Molecular Biology of the Cell 2066
secretory traffic to the primary cilium also passes through an
endocytic compartment or may even be sorted from baso-
lateral traffic not at the TGN, but later in the RE. These
possibilities will require further investigation.
We are especially indebted to Agnes Ang, and Ira Mellman (both of Genen-
tech) for providing the dsRFP-Rab8 constructs as well as the YFP-VSV-G
tsO45 mutant construct. We also thank Heike Folsch and Bettina Winkler for
consultation and advice. This work was supported in part by a grant to D.S.
from the American Heart Association (0335078N).
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