The Rockefeller University Press $30.00
J. Cell Biol. Vol. 188 No. 1 21–28
J.A. Follit and L. Li contributed equally to this paper.
Correspondence to Gregory J. Pazour: email@example.com
L. Li’s present address is Merck Research Laboratories, Boston, MA 02115.
Y. Vucica’s present address is CSL Bioplasma, Broadmeadows, Victoria
Abbreviation used in this paper: CTS, ciliary targeting sequence.
The primary cilium is a ubiquitous eukaryotic organelle that
plays vital roles in the development of mammals and in the etiol-
ogy of diseases such as polycystic kidney disease and blindness.
It is thought that primary cilia function as cellular antennae
to monitor the extracellular environment and report this infor-
mation back to the cell. This small organelle is composed
of hundreds of proteins assembled onto a microtubule-based
cytoskeleton that projects from the surface of the cell and is
surrounded by an extension of the plasma membrane. Although
contiguous with the plasma membrane, the ciliary membrane is
unique, as cells have the ability to localize receptors and other
membrane proteins specifically to this domain. This polarized
distribution of proteins is required for the cilium to carry out its
sensory function, but little is known about how the cell achieves
To learn more about the mechanism of ciliary targeting of
membrane proteins, we characterized the ciliary targeting se-
quence (CTS) in fibrocystin. Fibrocystin is the gene product of
the human autosomal recessive polycystic kidney disease gene,
PKHD1 (Onuchic et al., 2002; Ward et al., 2002). Patients with
defects in this gene develop severe cystic kidney disease along
with defects in the lung, pancreas, and liver. Fibrocystin is a
large (>4,000 residues), single-pass transmembrane protein that
is predicted to be entirely extracellular except for a short 190
residue C-terminal tail. Fibrocystin has been localized to cilia
and centrosomes in mammalian cells (Ward et al., 2003, 2006;
Menezes et al., 2004; Wang et al., 2004; Zhang et al., 2004), and
a Chlamydomonas reinhardtii homologue was found in cilia
(Pazour et al., 2005).
Results and discussion
The cytoplasmic tail of fibrocystin contains
a ciliary targeting signal
To understand how fibrocystin is targeted to cilia, we character-
ized its CTS. To date, CTSs have been identified in a small num-
ber of proteins, but comparison of these does not reveal common
motifs. However, all are found in intracellular domains (Pazour
and Bloodgood, 2008). Thus, we reasoned that even though
fibrocystin is large, it is mostly extracellular with only a short
cilium. To further our understanding of this process, we
dissected the ciliary targeting sequence (CTS) of fibro-
cystin, the human autosomal recessive polycystic kid-
ney disease gene product. We show that the fibrocystin
CTS is an 18-residue motif localized in the cytoplasmic
tail. This motif is sufficient to target green fluorescent
protein (GFP) to cilia of ciliated cells and targets GFP
ensory functions of primary cilia rely on ciliary-
localized membrane proteins, but little is known
about how these receptors are targeted to the
to lipid rafts if the cells are not ciliated. Rab8, but
not several other Rabs implicated in ciliary assembly,
binds to the CTS in a coimmunoprecipitation assay.
Dominant-negative Rab8 interacts more strongly than
wild-type or constitutively active Rab8, and coexpres-
sion of this dominant-negative mutant Rab8 blocks traf-
ficking to the cilium. This suggests that the CTS functions
by binding regulatory proteins like Rab8 to control traf-
ficking through the endomembrane system and on to
The cytoplasmic tail of fibrocystin contains a ciliary
John A. Follit, Lixia Li, Yvonne Vucica, and Gregory J. Pazour
Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605
© 2010 Follit et al. This article is distributed under the terms of an Attribution–
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T H E J O U R N A L O F C E L L B I O L O G Y
JCB • VOLUME 188 • NUMBER 1 • 2010 22
found in the endoplasmic reticulum. In nonciliated cells, JAF16
remains in the endoplasmic reticulum, whereas JAF99 is found
throughout the cell in small punctate spots (Fig. 1 A, a; and
Fig. S1). These results indicate that a CTS is located within the
C-terminal 193 residues of fibrocystin.
To determine which part of the cytoplasmic tail is respon-
sible for ciliary targeting, we constructed a series of GFP fusions
containing smaller portions of the tail and quantitated their ability
to localize to cilia (Fig. 1, A and B). This analysis indicated that
18 residues near the N-terminal end of the cytoplasmic tail were
sufficient to target GFP to the cilium of ciliated cells or to punc-
tate spots in nonciliated cells. The large size of fibrocystin pre-
vented us from determining whether these residues are required
for trafficking of native fibrocystin to cilia. Thus, it is currently
unknown if this is the only CTS within the protein. If the GFP
cytoplasmic tail, and this is the likely position of its CTS.
To test this idea, we made two constructs fusing the C-terminal
end of fibrocystin to reporter proteins (Fig. S1 A). In the first
(JAF16), we fused the C-terminal 503 residues of fibrocystin
to a fragment of CD8. This construct contains the extracellular
domain of CD8 fused to fibrocystin just before its membrane-
spanning domain and is predicted to have the same membrane
topology as native fibrocystin. CD8 is a well-characterized
membrane protein often used in chimerics to identify targeting
domains (Xia et al., 2001). In the second construct (JAF99), we
fused the last 193 residues of fibrocystin to the C-terminal end
of GFP. This construct lacks most of the predicted membrane-
spanning residues but contains the entire cytoplasmic tail.
After transfection into cells, both constructs can localize to cilia
(Fig. 1 A, a; and Fig. S1 B). In addition to cilia, JAF16 also is
Figure 1. Characterization of the CTS of fibro
cystin. (A, a–f) Selected examples showing the
distribution of subfragments of the cytoplasmic
tail. Two different cells are shown for each con-
struct. (a–f’) The first images (a–f) show a cili-
ated cell with the cilium marked with arrows,
whereas the second images (a’–f’) show a
nonciliated cell. Insets show the cilia (red) and
GFP-CTS (green) channels alone. The amino
acid fragments fused to GFP are listed at the
bottom of each image and are shown graphi-
cally in B. (B) Graphical representation of the
constructs and quantification of the ability of
the constructs to function. The numbers on
the left represent the amino acids included in the
construct, and the box denotes the limits of the
CTS. The graph shows the mean amount of
GFP fluorescence per micrometer in cilia from
25 transfected cells. (C) Alignment of the CTS
(box) and surrounding sequence of vertebrate
fibrocystins (Mm, mouse; Rn, rat; Hs, human; Pt,
chimp; Gg, chick). (D and E) Alanine-scanning
mutagenesis of the CTS. (D) Sequence and
quantification of the ability of the mutated
CTSs to direct GFP to cilia. Quantification is
described as in B. WT, wild type; CCC, LV,
WF, KKS, KTRK, and IKP indicate which amino
acids were mutated in each construct. Error
bars indicate SEM. (E) Images illustrating the
cellular distribution of constructs described in D.
Arrows mark cilia. Bars, 5 µm.
Ciliary targeting sequence of fibrocystin • Follit et al.
CTS-GFP–expressing cells were labeled with fluorescent cholera
toxin B (Fig. 2 B). Cholera toxin B binds GM1 gangliosides
and is a marker for membrane domains enriched in these lipids.
In nonciliated cells, there is strong colocalization between the
CTS-GFP spots and the cholera toxin-binding sites (Fig. 2 B, a).
The colocalization is also observed if the cholera toxin is cross-
linked with an antibody and then fixed (Fig. 2 B, b). Cholera
toxin B labels the cilium (Fig. 2 B, c), confirming a previous
report that this organelle is enriched in GM1 gangliosides (Janich
and Corbeil, 2007). SNAP25 has been reported to localize to
cilia (Low et al., 1998), but the lipid raft targeting sequence of
SNAP25 was not sufficient to target GFP to cilia (Fig. 2 B, d),
indicating that a lipid raft targeting sequence alone is not suf-
ficient for ciliary targeting.
Cysteine residues near blocks of basic amino acids are often
palmitoylated (Bijlmakers and Marsh, 2003). The fibrocystin
CTS contains three cysteine residues followed by a block of basic
residues (Fig. 2 A), and mutational analysis indicated that these
cysteines are critical to CTS function (Fig. 1 D). To test whether
the CTS cysteines are palmitoylated, we grew cells expressing
either the wild-type or CCC-mutated GFP-CTS in radioactive pal-
mitate, immunoprecipitated the GFP-CTS, and looked for the
incorporation of isotope. The wild-type protein but not the CCC-
mutated version incorporated radioactive palmitate (Fig. 2 C).
This indicates that the CTS includes a palmitoylation motif, and
because the mutation of the cysteines blocks function, this sug-
gests that palmitoylation is important for targeting this protein
to the cilia. Acylations like palmitoylation and myristoylation
are common modifications of ciliary membrane proteins. The
opsin photoreceptor contains two cysteine residues that are pal-
mitoylated and needed for proper targeting to the cilium (Tam
et al., 2000). A Trypanosoma ciliary calcium-binding protein
contains a palmitoylated cysteine and a myristoylated glycine
fusion construct did not contain the 18-residue CTS, the GFP
was distributed throughout the cell or concentrated in the nucleus.
We did not carefully demarcate the nuclear targeting sequence, but
the fusion containing residues 63–193 was able to efficiently con-
centrate in the nucleus, whereas the 40–68 fusion could localize
to the nucleus but was also found in the cytoplasm. Other work
also mapped a nuclear targeting sequence to the region between
residues 80 and 104 (Hiesberger et al., 2006).
Examination of the CTS in fibrocystins from other spe-
cies indicates that it is highly conserved in mammals and mod-
erately conserved in chicken (Fig. 1 C). The sequence is not
conserved in the fibrocystin-related protein, fibrocystin-L from
mammals, or other species. BLAST searches with the CTS did
not identify any novel proteins containing similar sequences.
The fibrocystin CTS does not contain a VxP motif that has been
proposed to be a generic CTS (Deretic et al., 2005; Geng et al.,
2006) nor does it contain an Ax[S/A]xQ motif identified in sev-
eral G protein–coupled receptors (Berbari et al., 2008). To fur-
ther our understanding of the CTS, we used alanine-scanning
mutagenesis to mutate small blocks of residues (Fig. 1, D and E).
Quantification of the effects of these mutations (Fig. 1 D) shows
that most residues are important for function. The CCC and KTRK
residues are most critical because mutating these to alanines al-
most completely blocks CTS function. At the other extreme, the
LV mutation does little to the CTS function, whereas the other
mutations reduce the ability to traffic to cilia but do not completely
Although we did not detect any significant homology be-
tween the fibrocystin CTS and other nonfibrocystin sequences,
we noted similarity between the CTS amino acid composi-
tion and a lipid raft targeting sequence in SNAP25 (Fig. 2 A;
Salaün et al., 2005). This suggested that the punctuate spots to
which the CTS localized might be lipid rafts. To test this, live
Figure 2. The CTS is associated with lipid.
(A) Comparison of the CTS from fibrocystin to
the lipid raft targeting sequence of SNAP25
(Gonzalo et al., 1999). (B) Colocalization
of GFP-CTS with lipid rafts. (a–a) Live cells
stained with Alexa Fluor 594–conjugated
cholera toxin. In nonciliated cells, cholera
toxin shows extensive colocalization with the
GFP-CTS in the cell body (the arrows point
at one example). (b–b) Cholera toxin was
cross-linked by antibody before fixation, which
caused the toxin and GFP to cluster (arrows).
(c–c) In ciliated cells, GFP and cholera toxin
colocalize in the cilium (arrows). (d–d) The
lipid raft targeting sequence of Snap25
does not target GFP to the cilium (arrows).
Bar, 5 µm. (C) Tritiated palmitate is incorpo-
rated into the wild-type but not the cysteine-
mutated CTS (arrow). (D) The CTS cysteines
mediate interaction with membranes. Cells
expressing either wild-type or cysteine-mutated
CTS-GFP were lysed and fractionated by an
JCB • VOLUME 188 • NUMBER 1 • 2010 24
where it was loaded (Fig. 2 D). This indicates that the palmi-
toylated cysteines link the GFP-CTS to the membrane. Thus,
even though the protein no longer contains a transmembrane
domain, it remains associated with the membrane.
To understand the cellular compartment to which the
GFP-CTS localized, we labeled cells expressing the GFP-CTS
with a variety of compartmental markers (Fig. S2). No significant
colocalization was seen with Golgi, lysosome, and most endo-
some markers, but colocalization was seen with markers for the
recycling endosome. This suggests that the palmitoylated GFP-
CTS has affinity for the membranes of this compartment, but
whether the native protein is trafficked through the recycling endo-
some remains to be determined.
Trafficking of the CTS is regulated by Rab8
Work in frog retina indicates that Rab8 plays a key role in traf-
ficking of opsin to the outer segment (Moritz et al., 2001), and
recent work in mammalian cell culture has indicated that Rab8
and other Rab family proteins are critical for ciliary assembly
(Nachury et al., 2007; Yoshimura et al., 2007). To test whether
Rab8 plays a role in the trafficking of the fibrocystin CTS,
we generated cell lines expressing Flag-tagged wild-type Rab8,
dominant-negative Rab8T22N, and constitutively active Rab8Q67L
(Fig. 3). These mutations are often used to perturb the GTP/
GDP cycle of small G proteins. The T22N mutation is thought to
keep the protein in the GDP-bound state, which binds guanine
exchange factors to inhibit their activity on native substrates.
The Q67L mutation reduces GTP hydrolysis, keeping the pro-
tein in the GTP-bound state (Feig, 1999). As previously reported
(Nachury et al., 2007), wild-type Flag-Rab8 and Flag-Rab8Q67L
localized to cilia, whereas Flag-Rab8T22N did not localize to
the cilia that formed on these cells (Fig. 3 A). Cells expressing
Flag-tagged wild-type Rab8 and Rab8Q67L ciliated fairly well,
but cells expressing Rab8T22N did not ciliate nearly as well
(wild type, 68 ± 12%; T22N, 2.7 ± 0.6%; Q67L, 46 ± 3.5%).
The fibrocystin GFP-CTS did not traffic to the cilia that formed
on the Flag-Rab8T22N cells (Fig. 3, B and C) but was trafficked
to cilia on cells expressing Flag-Rab8 and Flag-Rab8Q67L.
Interestingly, the amount of the GFP-CTS trafficked to cilia was
higher in cells that expressed the wild-type Flag-Rab8 than in
cells that were not transfected or in cells expressing either of the
mutant forms. This suggests that Rab8 is a limiting factor in the
amount of GFP-CTS that can be trafficked to cilia. The observa-
tion that the mutant forms either do not show the enhancement
(Q67N) or reduce the amount of trafficking (T22N) suggests that
Rab8 needs the normal GTP/GDP cycle to function properly.
The CTS interacts with Rab8
Because Rab8 appeared to regulate the trafficking of the fibro-
cystin CTS, we sought to understand how this might be functioning
and asked whether Rab8 or other Rabs could be physically con-
nected to the CTS (Fig. 4). To do this, we coexpressed the cyto-
plasmic tail of fibrocystin with a series of Rab proteins (Rab6,
Rab8, Rab11, Rab17, and Rab23) that have been implicated in
trafficking of ciliary membrane proteins (Deretic and Papermaster,
1993; Moritz et al., 2001; Yoshimura et al., 2007). We also in-
cluded IFT20, which is not a Rab protein, but is an intraflagellar
(Godsel and Engman, 1999) that are needed for targeting to
It is likely that the palmitoylated cysteines of fibrocystin
serve to link the GFP-CTS to lipid membranes, and this is re-
sponsible for the puncta that are observed in nonciliated cells.
The fact that the GFP-CTS is evenly distributed in the cells
when the cysteines are mutated (Fig. 1 E) supports this idea, but
we tested this more directly by floatation analysis. Membranes
from cells expressing either wild-type or the CCC-mutated
GFP-CTS were loaded on the bottom of an OptiPrep gradient
and centrifuged. Membranes floated up and carried along the lipid
raft marker flotillin-2. The wild-type GFP-CTS was also carried
up, whereas the CCC-mutated protein remained at the bottom
Figure 3. Effect of Rab8 on the trafficking of the fibrocystin CTS. (A) Flag-
tagged Rab8 and Rab8Q67L are localized to cilia of IMCD3 cells,
whereas Rab8T22N is not. Insets show the red (Flag) channels of the cilia.
Bar, 10 µm. (B) The fibrocystin CTS targets GFP to cilia in control cells and
cells expressing wild-type Flag-Rab8 and Flag-Rab8Q67L but not in cells
expressing Flag-Rab8T22N. Bar, 2 µm. (C) Quantification of ciliary GFP-
CTS fluorescence. Quantification was performed as described in Fig. 1.
Error bars indicate SEM. Significance is shown as compared with control:
*, P < 0.02; **, P < 0.001.
25 Ciliary targeting sequence of fibrocystin • Follit et al.
transport subunit that we have implicated in trafficking proteins
to the ciliary membrane (Follit et al., 2006, 2008). None of these
proteins were completely colocalized with the GFP-CTS in non-
ciliated cells. However, Rab8 showed some colocalization, whereas
Rab11 and Rab17 showed the most colocalization (Fig. S3).
The colocalization with Rab11 and Rab17 is consistent with the
results of the compartmental analysis (Fig. S2), as these two
G proteins localize to the recycling endosome (Zerial and
McBride, 2001). Rab8 was the only one of these Rabs to localize
to cilia (Fig. 3 B and not depicted).
The ability of these proteins to interact with the GFP-CTS
was tested by a coimmunoprecipitation assay. In this assay, the
GFP-CTS was coexpressed with each of the Flag-tagged Rabs
in mouse kidney cells. The Rabs were precipitated via the Flag
tag, and the precipitates were probed for the GFP-CTS. No interac-
tion was seen between the GFP-CTS and Rab6, Rab11, Rab17,
Rab23, or IFT20, but a significant amount of the GFP-CTS was
precipitated by Rab8 (Fig. 4 A). This analysis was performed
with the entire cytoplasmic tail, so we asked whether the Rab8-
binding site overlapped the essential 18-residue CTS motif
within the tail. To do this, we tested selected deletion constructs
described in Fig. 1 B. The Rab8-binding site is located within
the 18-residue minimal CTS, as fragments that contain this se-
quence are coprecipitated (Fig. 4 B, lanes 1, 3, and 5), whereas
the C-terminal 130 residue (Fig. 4 B, lane 2), which does not
target to cilia (Fig. 1, A and B), is not precipitated. The CTS is
not simply a Rab8-binding site, as deletions of single residues
from either end of the minimal CTS blocked the ability of the
peptide to direct GFP to the cilium (Fig. 1, A and B) but did not
block the binding to Rab8 (Fig. 4 B, lanes 4 and 6).
We next examined the effect of the alanine-scanning mu-
tations on the ability of the CTS to be coprecipitated by Rab8.
Interestingly, there is a good correlation between the function of
the CTS to traffic to the cilium (Fig. 1 D) and its ability to bind
to Rab8 (Fig. 4 C). For example, the LV mutation does little to
block function of the CTS, and this mutated protein binds Rab8.
However, the CCC and KTRK mutations are most disruptive to
the CTS and most significantly decrease binding to Rab8. Other
mutations had intermediate effects on the targeting ability and
have intermediate effects on the ability to bind Rab8. The KKS
mutation is an exception, as this is fairly detrimental to CTS
function but still binds to Rab8. This suggests that additional
proteins may bind the CTS and require these residues for activity.
To begin to understand whether the interactions between
the CTS and Rab8 may be regulated by the GTP/GDP state of
Rab8, we compared the ability of the constitutively active and
dominant-negative mutations of Rab8 to bind to the CTS (Fig. 4 D).
Interestingly, the dominant-negative form bound more CTS
Figure 4. Rab8 interacts with the fibrocystin CTS. (A) Flag-tagged Rab
proteins were coexpressed with the GFP-CTS (1–193) and precipitated.
The precipitates were analyzed by Western blotting with Flag and GFP anti-
bodies. Positive interactions have a band in the GFP Western blot (WB).
(B) The deletion constructs (Fig. 1 B) were tested for the ability to inter-
act with Rab8. (C) The alanine scan mutations (Fig. 1 D) were examined
for their ability to interact with Rab8. WT, wild type; CCC, LV, WF, KKS,
KTRK, and IKP indicate which amino acids were mutated in each construct.
(D) The ability of Rab8T22N and Rab8Q67L mutants to bind the GFP-
CTS were compared with wild-type Rab8 in an analogous assay. Arrows
mark the predicted size of the full-length proteins. IP, immunoprecipitation;
*, nonspecific band precipitated by the Flag antibody.
JCB • VOLUME 188 • NUMBER 1 • 2010 26
with 5-GGGAATTCCTGAGCTGTCTCGTTTGCTG-3 and 5-GGGAATT-
CTTACTGGATGGTTTCTGGTGG-3 and cloned into the EcoRI site of
pEGFP-C2 (BD) to create pJAF99. This plasmid contains the last 193 amino
acids of fibrocystin fused in frame to the C-terminal end of GFP. Deletion
constructs were generated by similarly amplifying smaller fragments and
cloning them into this vector. Point mutations were generated by inverse
PCR. All plasmids were confirmed by DNA sequencing. Rab8, Rab8T22N,
and Rab8Q67L were cloned into p3×FlagMyc-CMV26 (Sigma-Aldrich) for
expression in mammalian cells.
All cell culture work used mouse kidney IMCD3 (Rauchman et al., 1993)
cells cultured in 47.5% DME and 47.5% F12 supplemented with 5% FBS
and penicillin/streptomycin (Cellgro) at 37°C in 5% CO2. For transfection,
cells were electroporated using a Gene Pulser Xcell (200 V; 50-ms pulse;
4-mm cuvette; Bio-Rad Laboratories). Stable cell lines were selected with
400 µg/ml G418 (Sigma-Aldrich). Clonal lines were isolated by dilution
cloning after drug selection.
Percent ciliation was determined by counting cilia on cells stained
with an IFT27 antibody after being grown for 48 h in low serum (0.25%).
Results reported are the percentage of cilia ± standard deviation from three
independent experiments in which >500 cells were counted.
Endocytosis assays were performed on IMCD3 cells electroporated
with JAF99 48 h after electroporation. Cells were washed with KRH
(125 mM NaCl, 5 mM KCl, 1.3 mM CaCl2, 1.2 mM MgSO4, 25 mM Hepes,
pH 7.4, 2 mM sodium pyruvate, and 0.5% bovine serum albumin), incu-
bated with Alexa Fluor 568–labeled EGF or transferrin for 5 min at 37°C,
washed with KRH, and incubated at 37°C in KRH (Leonard et al., 2008).
Coverslips were periodically removed and fixed during the chase period.
For visualization of lipid domains, live cells grown in glass-bottom
dishes (MatTec) were incubated for 10 min at 37°C with Alexa Fluor 594–
conjugated cholera toxin B (Invitrogen). Excess toxin was washed out, and
the cells were imaged at 37°C in medium lacking Phenol red. Alternatively,
cells were incubated for 10 min at 4°C with the cholera toxin followed by
15 additional min at 4°C with cholera toxin antibody before fixing and
staining, following the manufacturer’s specifications (Vybrant Lipid Raft
Labeling kit; Invitrogen).
Lipids were floated using the protocol of Macdonald and Pike
(2005). In brief, cells were lysed by passing them through a needle, the
postnuclear supernatant was mixed with OptiPrep (Sigma-Aldrich) to a
final concentration of 25%, placed at the bottom of a centrifuge tube, and
a gradient of 0–20% OptiPrep was layered on top. The gradient was centri-
fuged at 52,000 g for 90 min, and fractions were collected and analyzed
by Western blotting.
To determine whether the CTS was palmitoylated, IMCD3 cells were
electroporated with the wild-type (1–22) and equivalent cysteine-mutated
GFP-CTS (1–22CCC) constructs. After 24 h, the medium was changed to
one containing dialyzed serum (Invitrogen) supplemented with 0.25 mCi
tritiated palmitate (PerkinElmer). After 16 h, cells were lysed, the GFP-CTS
was precipitated with the JL8 GFP antibody (BD), and the eluates were sep-
arated by SDS-PAGE. The gel was fixed in 2:9:9 acetic acid/methanol/
water for 1 h followed by 1 M sodium salicylate for 30 min, dried, and ex-
posed to film.
Immunoprecipitations were performed using anti-Flag resin. To do
this, cells expressing the tagged constructs were rinsed with cold PBS
lysed in cell lytic M + 0.1% NP-40 (Sigma-Aldrich) and 0.1% CHAPSO
(Bio-Rad Laboratories) with Complete Protease Inhibitor (Roche) at 4°C
and clarified by centrifugation (18,000 g for 10 min). Clarified lysates
were incubated with agarose beads coupled with Flag M2 antibody
(Sigma-Aldrich) for 1 h. Flag beads were washed three times with wash
buffer (50 mM Tris and 150 mM NaCl, pH 7.4) plus 1% NP-40. Bound
Flag proteins were eluted with 200 µg/ml 3× Flag peptide (Sigma-
Aldrich). Purified proteins were separated by SDS-PAGE and electropho-
retically transferred to Immobilon-P (Millipore). After transfer, the
membranes were incubated with antibodies to GFP (JL8; BD) and Flag
(F1804; Sigma-Aldrich) followed by an HRP-conjugated anti–mouse IgG
antibody (Thermo Fisher Scientific). The HRP conjugates were detected on
film (BioMax XAR; Kodak) after LumiGLO (KPL) treatment.
Cells were transfected by electroporation and seeded on coverslips. After
24 h, serum was reduced to 0.25% to promote ciliation, and the cells
were grown for an additional 24–96 h before being fixed with parafor-
maldehyde and stained with primary antibodies as described previously
(Follit et al., 2006). Primary antibodies were detected by Alexa Fluor
than either the wild-type protein or the constitutively active form.
This suggests that exchange of a GDP with a GTP would release
Rab8 from the CTS. This behavior is different from classical Rab
effectors, which bind more strongly when bound to GTP (Zerial
and McBride, 2001) but is similar to what was previously
observed in the interaction between Rab5 and the angiotensin
receptor (Seachrist et al., 2002). In the case of the angiotensin
receptor, it was proposed that the receptor anchors Rab5-GDP on
the surface of the carrier vesicle so that once GTP is exchanged
for GDP, efficient vesicle fusion could occur. Similarly, the fibro-
cystin CTS may bind Rab8-GDP to increase its local concentra-
tion to allow for efficient execution of the next step in transport
when GTP exchange occurs. Based on work in frog photorecep-
tors, it is likely that the regulated step is the fusion of carrier
vesicles at the base of the cilium, as expression of dominant-
negative Rab8 mutants caused small vesicles to accumulate around
the base of photoreceptors (Moritz et al., 2001). This process
may be regulated by the BBSome, as the guanine exchange
factor Rabin8 is associated with the BBSome, and this protein
would be expected to exchange GDP with a GTP on Rab8
(Nachury et al., 2007).
Comparison of ciliary targeting to apical
and basolateral targeting
Compared with trafficking to the ciliary membrane, much
more is known about trafficking to apical and basal-lateral
membranes (Rodriguez-Boulan et al., 2005). Basolateral tar-
geting sequences consist of short motifs that interact with the
adaptor protein complexes that bind to clathrin coats as part of
the sorting mechanism. Apical targeting motifs are much more
diverse and do not share significant sequence homology with
each other but have been proposed to function by directing
proteins to lipid rafts that are preferentially sorted to the apical
membrane (van Meer and Simons, 1988). Ciliary targeting is
similar to apical targeting in that no sequence similarity is seen
between the known CTSs (Pazour and Bloodgood, 2008), and
at least for fibrocystin and the Trypanosoma calcium-binding
protein (Tyler et al., 2009), association with a lipid raft appears
to be required for proper targeting. Thus, it appears that ciliary
targeting is related to apical targeting but has additional com-
ponents to direct the proteins into the ciliary membrane. It
is interesting to note that on vertebrate epithelial cells, the
ciliary membrane is a subdomain of the apical membrane, so
perhaps it is logical that trafficking to these two domains share
Materials and methods
The cytoplasmic tail of mouse fibrocystin (GenBank/EMBL/DDBJ acces-
sion no. NM_153179.2) was amplified from mouse kidney cDNA
using 5-GGGAATTCGCTTTGACTGTGACATTTTCAGTCCTAG-3 and
5-GGGAATTCTTACTGGATGGTTTCTGGTGG-3 and fused to the CD8
open reading frame to create JAF16. The CD8 open reading frame was
amplified from pCMS-CD8-NR1C (provided by H. Xia, Stanford University,
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antibodies used included anti-acetylated tubulin (6111; Sigma-Aldrich),
anti-Flag (Sigma-Aldrich), anti-CD8 (Invitrogen), EEA1 (provided by
S. Corvera, University of Massachusetts Medical School, Worcester, MA),
giantin (provided by M. Fritzler, University of Calgary, Calgary, Alberta,
Canada), golgin97 (CDF4; Invitrogen), flotillin-2 (BD), and mouse IFT20 and
IFT27 (Follit et al., 2006).
Wide-field images were acquired by a camera (Orca ER; Hama-
matsu Photonics) on a microscope (Axiovert 200M; Carl Zeiss, Inc.)
equipped with a 100× Plan Apochromat 1.4 NA objective (Carl Zeiss,
Inc.). If comparisons are to be made between images, the photos were
taken with identical conditions and manipulated equally. For the quantifi-
cation of GFP in the cilia, the length, area, and mean fluorescence inten-
sity of the cilia were measured using the measurement tools of Openlab
(PerkinElmer). Numbers reported are fluorescence intensity per micrometer
Online supplemental material
Fig. S1 documents the initial pair of constructs used to identify the fibrocystin
CTS. Fig. S2 examines the cellular compartment to which the GFP-CTS
localizes in nonciliated cells. Fig. S3 documents the cellular distribution of
the Flag-tagged proteins examined in Fig. 4. Online supplemental material is
available at http://www.jcb.org/cgi/content/full/jcb.200910096/DC1.
We thank Drs. N. Kennedy for assistance with the palmitoylation assay,
D. Navaroli for assistance with the endocytosis assays, J. Jonassen for statistical
analysis, and H. Xia, M. Fritzler, and S. Corvera for reagents.
This work was supported by grants from the National Institutes of Health
(GM060992), the Worcester Foundation for Biomedical Research, and the
Polycystic Kidney Foundation.
Submitted: 16 October 2009
Accepted: 30 November 2009
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