The Chlamydomonas reinhardtii BBSome is an IFT cargo required for export of specific signaling proteins from flagella.
ABSTRACT In humans, seven evolutionarily conserved genes that cause the cilia-related disorder Bardet-Biedl syndrome (BBS) encode proteins that form a complex termed the BBSome. The function of the BBSome in the cilium is not well understood. We purified a BBSome-like complex from Chlamydomonas reinhardtii flagella and found that it contains at least BBS1, -4, -5, -7, and -8 and undergoes intraflagellar transport (IFT) in association with a subset of IFT particles. C. reinhardtii insertional mutants defective in BBS1, -4, and -7 assemble motile, full-length flagella but lack the ability to phototax. In the bbs4 mutant, the assembly and transport of IFT particles are unaffected, but the flagella abnormally accumulate several signaling proteins that may disrupt phototaxis. We conclude that the BBSome is carried by IFT but is an adapter rather than an integral component of the IFT machinery. C. reinhardtii BBS4 may be required for the export of signaling proteins from the flagellum via IFT.
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ABSTRACT: The transition zone (TZ) is a specialized region of the cilium characterized by Y-shaped connectors between the microtubules of the ciliary axoneme and the ciliary membrane . Located near the base of the cilium, the TZ is in the prime location to act as a gate for proteins into and out of the ciliary compartment, a role supported by experimental evidence [2-6]. The importance of the TZ has been underscored by studies showing that mutations affecting proteins located in the TZ result in cilia-related diseases, or ciliopathies, presenting symptoms including renal cysts, retinal degeneration, and situs inversus [7-9]. Some TZ proteins have been identified and shown to interact with each other through coprecipitation studies in vertebrate cells [4, 10, 11] and genetics studies in C. elegans . As a distinct approach to identify TZ proteins, we have taken advantage of the biology of Chlamydomonas to isolate TZs. Proteomic analysis identified 115 proteins, ten of which were known TZ proteins related to ciliopathies, indicating that the preparation was highly enriched for TZs. Interestingly, six proteins of the endosomal sorting complexes required for transport (ESCRT) were also associated with the TZs. Identification of these and other proteins in the TZ will provide new insights into functions of the TZ, as well as candidate ciliopathy genes. Copyright © 2015 Elsevier Ltd. All rights reserved.Current Biology. 01/2015;
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ABSTRACT: Cilia dysfunction underlies a class of human diseases with variable penetrance in different organ systems. Across eukaryotes, intraflagellar transport (IFT) facilitates cilia biogenesis and cargo trafficking, but our understanding of mammalian IFT is insufficient. Here we perform live analysis of cilia ultrastructure, composition and cargo transport in native mammalian tissue using olfactory sensory neurons. Proximal and distal axonemes of these neurons show no bias towards IFT kinesin-2 choice, and Kif17 homodimer is dispensable for distal segment IFT. We identify Bardet-Biedl syndrome proteins (BBSome) as bona fide constituents of IFT in olfactory sensory neurons, and show that they exist in 1:1 stoichiometry with IFT particles. Conversely, subpopulations of peripheral membrane proteins, as well as transmembrane olfactory signalling pathway components, are capable of IFT but with significantly less frequency and/or duration. Our results yield a model for IFT and cargo trafficking in native mammalian cilia and may explain the penetrance of specific ciliopathy phenotypes in olfactory neurons.Nature Communications 01/2014; 5:5813. · 10.74 Impact Factor
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ABSTRACT: Proper functioning of cilia, hair-like structures responsible for sensation and locomotion, requires nephrocystin-5 (NPHP5) and a multi-subunit complex called the Bardet-Biedl syndrome (BBS)ome, but their precise relationship is not understood. The BBSome is involved in the trafficking of membrane cargos to cilia. While it is known that a loss of any single subunit prevents ciliary trafficking of the BBSome and its cargos, the mechanisms underlying ciliary entry of this complex are not well characterized. Here, we report that a transition zone protein, NPHP5 contains two separate BBS-binding sites and interacts with the BBSome to mediate its integrity. Depletion of NPHP5, or expression of NPHP5 mutant missing one binding site, specifically leads to dissociation of BBS2 and BBS5 from the BBSome and loss of ciliary BBS2 and BBS5 without compromising the ability of the other subunits to traffic into cilia. Depletion of Cep290, another transition zone protein that directly binds to NPHP5, causes additional dissociation of BBS8 and loss of ciliary BBS8. Furthermore, delivery of BBSome cargos, smoothened, VPAC2 and Rab8a, to the ciliary compartment is completely disabled in the absence of single BBS subunits, but is selectively impaired in the absence of NPHP5 or Cep290. These findings define a new role of NPHP5 and Cep290 in controlling integrity and ciliary trafficking of the BBSome, which in turn impinge on the delivery of ciliary cargo. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: firstname.lastname@example.org.Human Molecular Genetics 12/2014; · 6.68 Impact Factor
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J. Cell Biol. Vol. 187 No. 7 1117–1132
Correspondence to Karl-Ferdinand Lechtreck: Karl.Lechtreck@umassmed.edu; or
George B. Witman: George.Witman@umassmed.edu
Abbreviations used in this paper: AP, aqueous phase; BAC, bacterial artificial
chromosome; BBS, Bardet-Biedl syndrome; DIC, differential interference con-
trast; DP, detergent phase; IFM, immunofluorescence microscopy; IFT, intra-
flagellar transport; KAP, kinesin-associated protein; MS, mass spectrometry; PLD,
phospholipase D; PLDc, PLD type c; STPK, serine/threonine protein kinase; TIRF,
total internal reflection fluorescence; TIRFM, TIRF microscopy.
Bardet-Biedl syndrome (BBS; NCBI OMIM #209900) is a rare
human disorder causing hypogonadism, rod–cone dystrophy,
polydactyly, truncal obesity, and renal abnormalities (Katsanis
et al., 2001; Tobin and Beales, 2007; Zaghloul and Katsanis, 2009).
This combination of features partially overlaps with the pleio-
tropic phenotype of disorders caused by defective cilia, which is
consistent with the hypothesis that cilia are involved in the etiol-
ogy of BBS (Rosenbaum and Witman, 2002; Beales, 2005;
Blacque and Leroux, 2006). In humans, 12 genes (BBS1–12)
have been linked to BBS (Blacque and Leroux, 2006; Stoetzel
et al., 2006; Stoetzel et al., 2007); the proteins (BBS1/2/4/5/7/8/9)
encoded by 7 of these genes plus BBIP10 form a complex termed
the BBSome (Nachury et al., 2007; Loktev et al., 2008).
Experimental data from various species support a role for
the BBSome in cilia (Tobin and Beales, 2007). Several BBSome
proteins have been localized to basal bodies and cilia in mam-
malian cells (Ansley et al., 2003; Mykytyn and Sheffield, 2004)
and have been shown to undergo intraflagellar transport (IFT) in
Caenorhabditis elegans (Blacque et al., 2004). IFT is the bidi-
rectional movement of protein particles consisting of IFT com-
plex A and complex B driven by kinesin 2 (anterograde IFT)
and cytoplasmic dynein 1b/2 (retrograde IFT) along the axo-
neme (Cole et al., 1998; Rosenbaum and Witman, 2002). IFT is
required for flagellar assembly, maintenance, and signaling, and
the loss of bona fide IFT components results in a failure to as-
semble cilia, which causes embryonic lethality in mice (Nonaka
et al., 1998; Jonassen et al., 2008). In contrast, BBS1/2/4 knock-
out mice are viable, and cilia are at least initially assembled in
the majority of tissues, with sperm flagella being an exception.
Thus, the BBSome is dispensable for ciliary assembly in most
cell types (Blacque et al., 2004; Kulaga et al., 2004; Mykytyn
et al., 2004; Yen et al., 2006; Nachury et al., 2007; Loktev et al.,
2008; Mukhopadhyay et al., 2008).
BBSome. The function of the BBSome in the cilium is not
well understood. We purified a BBSome-like complex
from Chlamydomonas reinhardtii flagella and found that
it contains at least BBS1, -4, -5, -7, and -8 and undergoes
intraflagellar transport (IFT) in association with a subset of
IFT particles. C. reinhardtii insertional mutants defective in
n humans, seven evolutionarily conserved genes that
cause the cilia-related disorder Bardet-Biedl syndrome
(BBS) encode proteins that form a complex termed the
BBS1, -4, and -7 assemble motile, full-length flagella but
lack the ability to phototax. In the bbs4 mutant, the as-
sembly and transport of IFT particles are unaffected, but
the flagella abnormally accumulate several signaling pro-
teins that may disrupt phototaxis. We conclude that the
BBSome is carried by IFT but is an adapter rather than an
integral component of the IFT machinery. C. reinhardtii
BBS4 may be required for the export of signaling proteins
from the flagellum via IFT.
The Chlamydomonas reinhardtii BBSome is an
IFT cargo required for export of specific signaling
proteins from flagella
Karl-Ferdinand Lechtreck,1 Eric C. Johnson,1 Tsuyoshi Sakai,2 Deborah Cochran,1 Bryan A. Ballif,4,5 John Rush,6
Gregory J. Pazour,3 Mitsuo Ikebe,2 and George B. Witman1
1Department of Cell Biology, 2Department of Physiology, and 3Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01655
4Department of Biology and 5Vermont Genetics Network Proteomics Facility, University of Vermont, Burlington, VT 05405
6Cell Signaling Technology, Beverly, MA 01915
© 2009 Lechtreck et al. This article is distributed under the terms of an Attribution–
Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publica-
tion date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a
Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
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 187 • NUMBER 7 • 2009 1118
represents the novel allele ptx5-4. Two of these strains (ptx5-1
and -3) also failed to amplify with primer pair BBS4C. The
results indicate that all four mutant strains have defects in the
Mapping of the deleted region in these strains showed
that BBS4 and two other potential genes were deleted in
ptx5-1 (Fig. 1 A). Transformation of this mutant with a
6,797-bp genomic fragment encompassing only the BBS4
gene (Fig. 1 A) restored the strong negative phototaxis char-
acteristic of the parental strain g1 (Fig. 2 A; Pazour et al.,
1995). Therefore, the deletion of the BBS4 gene is responsi-
ble for the nonphototactic phenotype of the ptx5-1 mutant
(Videos 1–3). For the sake of clarity, the ptx5-1 mutant will
be called bbs4-1 in this study.
To determine whether mutations in other BBS genes
might also cause a nonphototactic phenotype in C. reinhardtii,
we selected 17 nonphototactic mutants with good motility
from our collection and analyzed them by quantitative RT-
PCR using 40 primer pairs designed to amplify parts of the
BBS genes. Primer pair BBS7-1, amplifying a 230-bp region
in the first exon of BBS7, failed to amplify in the previously
described strain ptx6-1 (Fig. 1 B; Pazour et al., 1995). Further
analysis identified a footprint of the retrotransposon TOC1
(Day et al., 1988) in codon 51 of BBS7 in ptx6-1; this insertion
resulted in a premature stop, deleting 77% of the predicted
protein (Fig. 1 B). This mutant will be referred to as bbs7-1 in
this study. In another nonphototactic strain, RIR7-2, the 5
half of the BBS1 gene and a gene upstream of BBS1 are de-
leted; we named this strain bbs1-1 (Fig. 1 C). The results
strongly suggest that defects in BBS1 and -7 also impair
phototaxis in C. reinhardtii.
Flagellar assembly and IFT are normal
without BBS4 in C. reinhardtii
Because defects in BBS proteins have been reported to affect
ciliary assembly, IFT particle integrity, and the rate of IFT
in other organisms (Blacque et al., 2004; Ou et al., 2005;
Nachury et al., 2007; Loktev et al., 2008), we investigated
whether the BBSome proteins are required for flagellar as-
sembly and IFT in C. reinhardtii. Swimming velocity is
The BBSome subunits are highly conserved and widely
distributed among organisms with cilia, suggesting that the
function of the BBSome has also been conserved. BBS proteins
have been implicated in intracellular transport (Kim et al., 2004;
Yen et al., 2006; Gerdes et al., 2007) and protein transport to
the cilium and ciliary membrane (Nachury et al., 2007; Berbari
et al., 2008; Shah et al., 2008). In C. elegans, BBS7 and -8 are
thought to function in controlling IFT by maintaining an associ-
ation of IFT complexes A and B (Blacque et al., 2004; Ou et al.,
2005; Pan et al., 2006). Despite these advances, the functions
of the BBSome in cilia remain unclear. This has been caused,
in part, by the lack of a genetic model from which BBSome-
defective cilia can be isolated and biochemically analyzed.
In this study, we explore the function of the BBSome in
Chlamydomonas reinhardtii, which has advantages that allowed
us to determine the structural, biochemical, and physiological
consequences for cilia when BBSome function is impaired. We
show that C. reinhardtii flagella contain a complex, including
at least BBS1, -4, -5, -7, and -8, that is orthologous to the
mammalian BBSome. Simultaneous tracking of BBS4-GFP
and IFT20-mCherry in living cells by total internal reflection
fluorescence (TIRF) microscopy (TIRFM) revealed that the
BBSome is moved anterogradely and retrogradely in the flagel-
lum in association with a subset of IFT particles. Consistent
with this, we find that BBSome proteins are present in flagella
at substoichiometric amounts relative to IFT proteins. We iden-
tified C. reinhardtii mutants for BBS1, -4, and -7; they have full-
length flagella, and IFT is unaffected. Collectively, the results
indicate that the BBSome is carried by IFT but is not an integral
component of the IFT machinery.
All three BBS mutants are nonphototactic. Biochemical
analyses of flagellar fractions of the BBS mutants revealed that
several proteins associated with the flagellar membrane and ma-
trix and likely to be involved in signal transduction are abnor-
mally accumulated in the flagella; this apparently impairs the
ability of the organelle to carry out phototactic steering because
phototaxis is transiently restored to bbs4 cells after the regener-
ation of new flagella. We propose that the C. reinhardtii BBSome
is an IFT adapter required for the turnover or removal of certain
proteins from the flagellum via IFT. BBS may be a degenerative
disease of the cilium in which certain proteins accumulate over
time, leading to progressive ciliary dysfunction.
Defects in BBS1, -4, and -7 impair
phototaxis in C. reinhardtii
All eight known subunits of the human BBSome (BBS1/
2/4/5/7/8/9) are conserved in C. reinhardtii (Table I). We
searched for C. reinhardtii BBSome mutants starting with the
BBS4 gene. Genomic DNA from a collection of 350 motil-
ity and phototaxis mutants was screened by quantitative
RT-PCR using two primer pairs (BBS4N and BBS4C) spe-
cific for the C. reinhardtii BBS4 gene (Fig. 1 A). Primer pair
BBS4N failed to amplify in four strains, namely the previ-
ously characterized nonphototactic mutants ptx5-1, -2, and -3
(Pazour et al., 1995) and the uncharacterized strain F36, which
Table I. C. reinhardtii homologues of BBSome proteins
Protein Predicted mass
in C. reinhardtii
The predicted molecular masses of human and C. reinhardtii BBSome proteins
are shown. BLASTP values are shown for the best match to the C. reinhardtii
protein in the human reference protein database. All pairs were reciprocal best
hits in BLASTP searches. Percent identity is based on the BLASTP alignment.
1119BBSome function in C. reinhardtii flagella • Lechtreck et al.
Axonemes of bbs4-1 lack a proper
Phototactic steering of C. reinhardtii is thought to involve a
differential sensitivity of the two flagella to intraflagellar Ca2+
(Witman, 1993). At very low (e.g., 109 M) Ca2+, the beating
of the cis flagellum (the one closest to the eyespot; Fig. S1 B)
will dominate over the trans flagellum, causing the cell to
turn away from its eyespot. At higher (e.g., 106 M) Ca2+, the
trans flagellum is dominant over the cis flagellum, causing the
cell to turn toward its eyespot. This can be demonstrated in
demembranated cells reactivated in ATP-containing solutions
having various concentrations of Ca2+ (Kamiya and Witman,
1984). Pazour et al. (1995) reported that intact bbs4-1 (ptx5-1)
cells lacked the ability to respond like wild type in Ca2+-free
medium. We confirmed this (Fig. S1 A) and further found that
demembranated models of bbs4-1 did not undergo the Ca2+-
induced switch in axonemal dominance at high (106 M) Ca2+
reduced by 25% in bbs4-1 and bbs7-1 (Pazour et al., 1995).
Flagellar length was unaffected in the bbs mutants (Fig. 2 B),
and flagellar regeneration appeared to be normal (not depicted).
The ultrastructure of mutant bbs4-1 flagella and IFT particles
was indistinguishable from that of wild type by thin-section
transmission EM (unpublished data). The velocity of IFT in
bbs4-1 was unaffected, as determined by differential inter-
ference contrast (DIC; Fig. 2 C) microscopy and by TIRFM of the
IFT kinesin motor subunit kinesin-associated protein (KAP)–
GFP expressed in bbs4-1/kapts or BBS4/kapts backgrounds
(Fig. 2 D; Mueller et al., 2005). In immunofluorescence micros-
copy (IFM), IFT complex B, visualized by anti-IFT46, and IFT
complex A, visualized by anti-IFT139, showed a near perfect
colocalization in bbs4-1 flagella (Fig. 2 E, d–f), as in wild-type
flagella (Fig. 2 E, a–c). We conclude that flagellar assembly and
the integrity and movement of IFT particles in C. reinhardtii are
independent of BBS4.
Figure 1. Identification of C. reinhardtii bbs1, -4, and -7 mutants. (A) Map showing the genomic region near the BBS4 locus. Black arrows indicate PCR
primer pairs; plus and minus signs indicate whether or not these primers gave PCR products. bbs4-1 lacks the entire BBS4 gene. The 6,797-bp fragment
used to rescue strain bbs4-1 is indicated. JGI protein IDs of the predicted gene products are shown. (B) Map showing the genomic region near the BBS7
locus. Only one of the tested primer pairs failed to amplify in the ptx6-1 strain. Sequencing of this region revealed the insertion of a retrotransposal footprint
into the BBS7 gene of ptx6-1, causing a premature stop after 52 codons. (C) Map showing the genomic region near the BBS1 locus. Approximately half
of BBS1 is deleted in bbs1-1. Gene orientation is marked by white arrows. wt, wild type.
JCB • VOLUME 187 • NUMBER 7 • 2009 1120
Figure 2. BBS4 is a flagellar protein required for phototaxis but not IFT. (A) Motion analysis of wild type, bbs4-1, and R26, a strain rescued with a ge-
nomic fragment encompassing the BBS4 gene. The direction of light (arrow) is indicated. The radial histograms show the percentage of cells moving in a
particular direction relative to the light (24 bins, each 15°). (B) Flagellar length of g1, bbs4-1, bbs7-1, and bbs1-1, as determined by DIC. (C) The velocity
of anterograde (a.) and retrograde (r.) IFT particles in wild-type (g1) and bbs4-1 flagella, as determined by DIC. (D) Velocity of KAP-GFP in BBS4 and
bbs4-1 flagella, as determined by TIRFM. (C and D) SDs are indicated. (E) Detached flagella from wild type (a–c) or the bbs4-1 mutant (d–f) were stained
with anti-IFT46 (a and d) and anti-IFT139 (b and e). (F) Western blot probing proteins of whole cells (a) or isolated flagella (b) with anti-BBS4 (a and b)
and anti-IFT139 (a) or anti-IC2, which is specific for an axonemal protein (b), as loading controls. Anti-BBS4 stained a band of 41 kD (arrowhead) in
whole-cell and flagella samples of wild type and the rescued strain R26; BBS4 was undetectable in bbs4-1. Molecular masses are indicated in kilodal-
tons. (G) Detached flagella from wild type and the bbs4-1 mutant were stained with anti–acetylated tubulin (ac-tubulin; a and c) and anti-BBS4 (b and d).
Bars, 5 µm.
1121BBSome function in C. reinhardtii flagella • Lechtreck et al.
acted with a few other proteins present in whole-cell extracts
(Fig. 2 F, a). Therefore, to determine the subcellular distribution
of BBS4, bbs4-1 was rescued by expression of BBS4-3xHA
(Fig. 3, A and B). Double staining of BBS4HA21 cells (bbs4-1
rescued by BBS4-3xHA) with anti-HA and antitubulin showed
that BBS4-3xHA was localized near the two basal bodies and in
a spotted fashion along both flagella (Fig. 3 C). This distribution
is very similar to that described for IFT particle proteins in
C. reinhardtii. Approximately 90% of BBS4-3xHA was present
in the cell body, as determined by Western blots comparing
deflagellated cell bodies and isolated flagella from BBS4HA21
(Fig. 3 D); similar results were obtained for wild-type BBS4 in
wild-type cells (not depicted). A similar distribution was ob-
served for the IFT particle proteins IFT81 and -57. In contrast,
only a small cytoplasmic pool was detected for the axonemal
protein IC2. Like the IFT particle proteins, BBS4 and BBS4-
3xHA were predominately present in the membrane-plus-matrix
fraction obtained by extraction of isolated flagella from wild type
or BBS4HA21 with 1% NP-40 (Fig. 3 E). Temperature-induced
(Fig. S1 C), indicating that the loss of BBS4 alters the Ca2+
sensitivity of the axoneme.
BBS4 is a component of the flagellar
matrix in C. reinhardtii
An antibody (anti-BBS4) was raised against the C-terminal 248
aa of BBS4. Western blots of whole-cell extracts from wild type,
bbs4-1, and R26, a strain rescued using genomic BBS4, were
probed using anti-BBS4 (Fig. 2 F, a). A band of 41 kD, which is
close to the expected size of C. reinhardtii BBS4 (46,360 D), was
detected in wild type and R26 but not in bbs4-1. When Western
blots of isolated flagella of these strains were probed with anti-
BBS4, a single band of 41 kD was present in wild type and the
rescued strain R26; the band was absent in bbs4-1, confirming the
loss of BBS4 in this mutant (Fig. 2 F, b). In IFM of detached wild-
type flagella, anti-BBS4 gave a punctate staining; as expected, the
staining was absent from flagella of bbs4-1 (Fig. 2 G).
Anti-BBS4 was not suitable for localization of BBS4
in whole cells by IFM because in Western blots, it cross re-
Figure 3. BBS4 has an IFT-like distribution
within the cell. (A) Dish phototaxis assay of
wild type, bbs4-1, and BBS4HA21 rescued by
BBS4-3xHA. (B) Western blot of flagella from
the strains shown in C probed with monoclonal
anti-HA. (C) BBS4HA21 cells labeled by anti–
-tubulin and anti-HA. BBS4-3xHA is located
near the basal bodies (arrowhead) and in
dots along both flagella. (D) Western blot of
BBS4HA21 to compare the amounts of various
proteins in cell bodies (CB) versus isolated fla-
gella (FLA). 1× indicates that approximately two
flagella were loaded per cell body, etc. The blot
was probed for BBS4-HA, IC2, and IFT57 and
-81. BBS4-3xHA is about eight times more abun-
dant in cell bodies than in flagella. (E) Western
blot analyzing equivalent amounts of whole
flagella, axonemes (AXO), and NP-40–soluble
membrane-plus-matrix (M&M) of flagella from
BBS4HA21 and wild-type cells. Antibodies were
as indicated; those to IC2 and various IFT par-
ticle proteins were used as controls. (F) Western
blot comparing equivalent amounts of wild-type
flagella, axonemes, AP, and DP resulting from
Triton X-114 phase partitioning. The blot was
probed with the antibodies indicated. (B and
D–F) The positions of standard proteins and their
molecular masses in kilodaltons are indicated.
Bar, 5 µm.
JCB • VOLUME 187 • NUMBER 7 • 2009 1122
centrifugation and analyzed by Western blotting (Fig. 4 A).
Both BBS4-3xHA and BBS4 sedimented at 12S, suggesting
that each was present in a complex. BBS4-3xHA did not co-
sediment with IFT46 (IFT complex B) or IFT139 (IFT complex A),
indicating that it is present in a complex distinct from the IFT
complexes. We affinity purified the BBS4-3xHA complex from
the BBS4HA21 flagellar membrane-plus-matrix fraction. At
least six bands were detected in silver-stained gels of the eluate
from BBS4HA21; these proteins were absent in a correspond-
ing eluate from wild type (Fig. 4 B). Analysis by mass spec-
trometry (MS) identified five of these bands as BBS4-3xHA
and BBS1, -5, -7, and -8 (Fig. S2); the sixth band was not identi-
fied. We conclude that (a) C. reinhardtii BBS4 is part of a
BBSome complex similar to that previously described for verte-
brates (Nachury et al., 2007) and (b) the C. reinhardtii BBSome
is present in the flagellum.
phase partitioning with 1% Triton X-114 (Everberg et al., 2008)
was used to separate the membrane-plus-matrix fraction into an
aqueous phase (AP) containing proteins of the flagellar matrix
and a detergent phase (DP; Fig. S5 B); the latter contained nearly
all of the transmembrane protein 183515 (not depicted), indicating
that it was enriched in proteins of the flagellar membrane. BBS4
and various IFT particle proteins were enriched in the AP and were
not detected in the DP (Fig. 3 F). Thus, both IFM and Western
blotting showed that BBS4 is present in the flagella of C. rein-
hardtii and has a distribution similar to that of IFT particles.
C. reinhardtii BBS4 forms a complex with
other BBS proteins
To determine whether a BBSome-like complex is present in the
flagellum of C. reinhardtii, detergent extracts from BBS4HA21
and wild-type flagella were fractionated by sucrose gradient
Figure 4. BBS4 forms a complex with other BBS proteins in the flagellum. (A) Proteins in the flagellar membrane-plus-matrix from BBS4HA21 (top) or wild type
(bottom) were separated by sucrose gradient centrifugation, and fractions were analyzed by Western blotting using antibodies to the HA tag or BBS4 and IFT172,
-139, and -46. White lines seperate images of the membranes used for this blot. (B) Silver-stained SDS gel of protein complexes affinity purified from the membrane-
plus-matrix fraction of BBS4HA21 and wild-type (wt) flagella using anti-HA beads. Proteins identified by MS are indicated. (C) Western blot analysis of isolated
flagella or whole-cell extracts from the strains indicated. The blots were probed with antibodies to BBS4 and, as loading controls, IC2 (flagella) or IFT139 (cells).
ptx1-1 and ptx7-1 are control nonphototactic strains. (A–C) The positions of standard proteins and their molecular masses in kilodaltons are indicated.
1123BBSome function in C. reinhardtii flagella • Lechtreck et al.
mulates at the tips of growing flagella, as previously described
for IFT particle proteins (Fig. 5 B; Deane et al., 2001). To de-
termine the extent to which BBS4 and IFT proteins colocalize,
we performed double IFM with anti-HA and antibodies to vari-
ous IFT particle proteins. In BBS4HA21 flagella, BBS4-3xHA
largely colocalized with IFT139 (Fig. 5 A) and IFT46 (Fig. 5 B),
but the number of particles stained by anti-IFT139 or anti-
IFT46 exceeded those stained by anti-HA. In wild-type flagella,
a majority of the BBS4 particles (95% of 242 BBS4 particles
from 35 flagella) colocalized with IFT139 particles, whereas
27% of 314 IFT139 particles lacked a BBS4 signal (Fig. 5 C).
In contrast, virtually all IFT B complexes labeled by anti-IFT46
were associated with A complexes labeled by anti-IFT139 and
To test for genetic interactions between BBS4 and other
BBS genes, whole-cell extracts or isolated flagella from bbs7-1
and bbs1-1 were probed with anti-BBS4 (Fig. 4 C). BBS4 was
lost from bbs1 cells, suggesting that the expression or stability
of BBS4 depends on BBS1. Normal amounts of BBS4 were
present in whole cells of bbs7-1, but BBS4 was strongly re-
duced in bbs7-1 flagella, indicating that the transport of BBS4
into the flagellum depends on BBS7.
BBS proteins are substoichiometric to IFT
proteins in C. reinhardtii flagella
The distribution of BBS4 in C. reinhardtii is very similar to
that described for IFT particle proteins, and BBS4-3xHA accu-
Figure 5. BBSomes are less abundant than IFT particles in C. reinhardtii flagella. (A and B) Double immunolabeling of a BBS4HA21 cell with polyclonal
anti-HA and anti-IFT139 (A) or anti-IFT46 (B). In B, cells were deflagellated by pH shock and allowed to regrow flagella for 10 (a–c), 20 (d–f), and
45 min (g–i). BBS4-HA and the IFT proteins colocalize at the flagellar base, and the signals partially overlap in the flagella. Arrowheads indicate flagel-
lar tips. (C) Detached wild-type flagella were double labeled with anti-BBS4 (a) and anti-IFT139 (b). Closed arrowheads indicate anti-IFT139 signals that
did not colocalize with anti-BBS4 signals; the open arrowhead indicates anti-BBS4 signal that did not colocalize with anti-IFT139 staining. (D) Relative
abundance of the native and the AQUA peptide LLETLNEDVK of IFT81. The AQUA peptide ion (590.831) and a peptide ion of monoisotopic m/z =
587.323, corresponding to the native LLETLNEDVK peptide ion (theoretical m/z value of 587.322), are marked. For details see Fig. S4. (E) As in D but
for the LYVEQTQR peptide of BBS1. Bars: (A and B) 5 µm; (C) 1 µm.
JCB • VOLUME 187 • NUMBER 7 • 2009 1124
Figure 6. BBS4 is moved by IFT. (A) BBS4-GFP undergoes IFT. (a) Cell attached to the glass surface by its flagella (arrowheads). (b) TIRFM image of the
two flagella of a BBS4-GFP1 cell. (c) Kymogram revealing the anterograde and retrograde movement of BBS4-GFP in the flagella. (B) Western blot of
flagella isolated from wild-type control (g1), bbs4-1, IFT20-mCherry, and strain MxG1.3 expressing BBS4-GFP and IFT20-mCherry. The blots were probed
as indicated; the positions of the immunoreactive proteins and standard proteins and their molecular masses in kilodaltons are marked. (C) The velocity
of fluorescent protein–tagged IFT20 and BBS4 in flagella, as determined by TIRFM. In the BBS4-GFP/IFT20-mCherry strain, only particles with both tags
were scored. n, number of particles analyzed. (D) Frequency of BBS4-GFP and fluorescent protein–tagged IFT20, as determined by TIRFM. n, number of
flagella analyzed. (C and D) SDs are indicated. (E) Single frame from simultaneous recording of BBS4-GFP and IFT20-mCherry. Flagella are marked by
arrowheads, and the cell body is marked by an arrow. (F) Kymograms showing the movement of BBS4-GFP (a) and IFT20-mCherry (b) in one flagellum. The
image in c is a merger of a and b. Arrowheads and arrows indicate anterograde and retrograde cotransport of both proteins, respectively. Several tracks
representing IFT particles are devoid of BBS4-GFP. Note the fission of a BBS4-GFP signal (arrowhead 1), a part of which is later picked up by a different
IFT particle (arrowhead 2). (G) A BBS4-GFP particle falls off (arrowheads) a moving IFT20-mCherry particle (arrows). See Video 7. Bar, 10 µm.
BBSome function in C. reinhardtii flagella • Lechtreck et al.
did not carry BBS4-GFP (111 of 192 particles). Occasionally, a
BBS4-GFP particle was observed to detach from an IFT20-
mCherry particle and cease moving (Fig. 6, F and G; and Video 7);
conversely, nonmoving BBS4-GFP particles were occasionally
picked up and transported by moving IFT20-mCherry particles
(Fig. 6 F). The data reveal that a subset of IFT particles trans-
ports BBS4-GFP in C. reinhardtii flagella.
bbs4 flagella accumulate putative
To examine the levels of selected proteins in wild-type and
bbs4-1 flagella, Western blots were probed with antibodies to IFT
particle proteins IFT20, -46, -172, and -139, IFT motor subunits
KAP and D1bLIC, and the flagellar membrane proteins poly-
cystin 2 (PKD2), FMG-1 (flagellar membrane glycoprotein-1),
FMG-3, and mastigoneme protein. Wild-type and bbs4-1 fla-
gella showed similar amounts of these proteins (Fig. S5 A),
suggesting that bbs4-1 has no general defect in the transport of
IFT or flagellar membrane proteins.
To determine more generally whether the lack of BBS4
affects the protein composition of bbs4-1 flagella, we compared
wild-type and mutant flagella and flagellar fractions by 1D and
2D PAGE. No differences were observed between axonemes
from wild type and bbs4-1 (Fig. 7 A and Fig. S5 C, a and b).
However, SDS-PAGE revealed at least three bands (no. 1, 3,
and 4) in the DP and one band (no. 2) in the AP of bbs4-1 that were
not detected in the corresponding wild-type fractions (Fig. 7 A).
MS identified band 1 as a phospholipase D (PLD) type c (PLDc;
Joint Genome Institute [JGI] protein ID 190403), band 2 as a
single-domain hemoglobin (THB1; JGI protein ID 81856),
band 3 as a serine/threonine protein kinase (STPK; JGI protein
ID 193039), and band 4 as JGI protein ID 191821 (a protein of
unknown function with putative pleckstrin homology–like do-
mains; Fig. S3). PLDc, STPK, and 191821 are predicted to lack
transmembrane domains and to be myristoylated on the second
glycine. 2D PAGE (Fig. 7 B) of the AP followed by MS of se-
lected protein spots confirmed the accumulation of THB1 and
STPK in bbs4-1 and revealed that OEE3 (oxygen-evolving en-
hancer 3/PSBQ; JGI protein ID 153656) was reduced in the
bbs4-1 mutant flagella. Bands corresponding in size to PLDc
(Fig. 7 C), THB1, STPK, and 191821 (not depicted) were also
vice versa, as shown in Fig. 2 E (a–c). Therefore, BBS4 is de-
tectable on only a subset of IFT particles in the flagellum. The
occasional BBS4 label not colocalized with an IFT label prob-
ably represents a BBS particle that transiently dissociated from
an IFT particle (Fig. 6, F and G).
The absence of BBS4 from some IFT particles in flagella
suggested that BBS proteins are substoichiometric to IFT pro-
teins. To test this, we used the absolute quantification (AQUA)
technique (Kirkpatrick et al., 2005) to determine the ratio be-
tween BBS1 and IFT81 by MS. Three AQUA peptides contain-
ing stable isotopes of carbon (13C) and nitrogen (15N) were
synthesized for each protein and used as internal standards to
determine the amount of their native peptide counterparts in the
wild-type membrane-plus-matrix fraction, which contains vir-
tually all of the flagellar IFT and BBSome proteins (Fig. 3 E).
The analyses revealed a ratio of 1:6 between BBS1 and IFT81
(Fig. 5, D and E; Fig. S4; and Table II). This result suggests that
BBSomes are considerably less abundant than IFT particles in
C. reinhardtii flagella.
BBS4 is transported by a subset
of IFT particles
We used TIRFM to analyze the movement of BBS4-GFP in
flagella of BBS4-GFP1 cells (bbs4-1 rescued by expression of
BBS4-GFP; Fig. 6 A). Speckles of BBS4-GFP moved up and
down the flagella at velocities typical for IFT in C. reinhardtii
(Fig. 6 A, b and c; and Video 4; Kozminski et al., 1993). Antero-
grade movement of BBS4-GFP progressed at 2.3 µm/s; retro-
grade transport occurred at 4 µm/s (Fig. 6 C). As expected, the
frequency of BBS4-GFP speckles in BBS4-GFP1 flagella was
lower than that of IFT complex B protein IFT20 speckles in
ift20:IFT20-GFP and ift20:IFT20-mCherry flagella (Fig. 6 D;
in ift20, the IFT20 gene is entirely deleted [not depicted]).
To test whether BBS4 is cotransported with IFT particles,
we generated a strain (MxG1.3) coexpressing BBS4-GFP and
IFT20-mCherry. This strain lacks wild-type BBS4 and IFT20
(Fig. 6 B). Simultaneous observation of both tagged proteins
using TIRFM revealed that BBS4-GFP mostly co-migrated
with IFT20-mCherry (91% of BBS4-GFP particles; n = 89 par-
ticles in 27 flagella; Fig. 6, E and F; and Videos 5 and 6). In
contrast, 58% of the IFT particles visualized by IFT20-mCherry
Table II. AQUA of IFT81:BBS1 stoichiometry in the flagellar membrane-plus-matrix by MS
ProteinPeptide Femtomole synthetic AQUA peptide/
femtomole native peptide
Analysis 1Analysis 2
Membrane-plus-matrix was prepared from isolated wild-type flagella and subjected to AQUA analysis. Data represent duplicate MS analyses, each containing
50 fmol of all six of the indicated synthetic AQUA peptides. # follows residues with 13C- or 15N-stable isotope mass tags. The IFT81 mean ratios for analyses 1 and 2
are 50:18.7 and 50:18.17, respectively. The BBS1 mean ratios for analyses 1 and 2 are 50:3 and 50:2.67, respectively. The ratios of IFT81 to BBS1 for analyses
1 and 2 are 6.23:1 and 6.81:1, respectively. The mean (±SD) of the two IFT81:BBS1 ratio values is 6.52:1 (±0.41).
JCB • VOLUME 187 • NUMBER 7 • 2009 1126
gest that at least THB1 normally moves into the flagellum and is
removed by retrograde IFT via the BBSome.
Newly assembled flagella of bbs4-1 have
transiently restored phototactic steering
The results suggest that the abnormal accumulation of signaling
proteins in bbs4-1 flagella somehow impairs the cells’ ability to
phototax. If so, freshly formed bbs4-1 flagella should be less
severely affected. To test this, the flagella were removed from
bbs4-1 by pH shock and, after the cells had regenerated new fla-
gella and recovered motility, their phototactic capability was
assessed (Fig. 8). bbs4-1 cells showed improved phototaxis for
up to 1 h after the beginning of flagellar regeneration. The re-
sults demonstrate that newly formed flagella of bbs4-1 cells are
capable of phototactic steering but then lose that ability.
Several C. reinhardtii BBS proteins
form an evolutionarily conserved
Nachury et al. (2007) purified, from RPE1 cells and mouse
testes, a 12S complex containing seven highly conserved BBS
present in bbs1-1 and bbs7-1 flagellar fractions. The enrichment
of PLDc and STPK in the DP of bbs7 was confirmed by MS
(unpublished data). In contrast, PLDc and 191821 (and THB1
for ptx1-1) were not detected in comparable fractions from the
nonphototactic mutants ptx1-1 and ptx7-1 (Fig. 7 C and not de-
picted; STPK was not tested), which have normal levels of
BBS4. Therefore, the accumulation of PLDc, THB1, and STPK
is linked to the loss of BBS proteins and is not caused simply by
the loss of phototaxis. We conclude that a set of proteins is ab-
normally enriched in the flagella and specifically in the flagellar
membrane-plus-matrix of the bbs mutants.
The abnormal accumulation of proteins in the bbs mutants
could be caused by failure of a mechanism for excluding nonflagel-
lar proteins from the flagellum or to a failure to remove certain pro-
teins by retrograde IFT. To investigate this, flagellar fractions from
the hypomorphic retrograde IFT mutant dhc1bts (unpublished data)
were analyzed by SDS-PAGE. Flagella of this strain were assem-
bled at the nonrestrictive temperature used in this study but con-
tained reduced amounts of the retrograde IFT motor dynein 1b
(Fig. 7 D). MS showed that THB1 was present in the AP of dhc1bts;
PLDc was not detected in the DP of this mutant (unpublished data).
Differences in the banding patterns of the mutant versus wild-type
DPs precluded assessment of STPK and 191821. The results sug-
Figure 7. Biochemical defects in bbs4-1 flagella.
(A) Silver-stained 5–15% SDS–polyacrylamide gel
of flagella (FLA), axonemes (AXO), AP, and DP from
wild type (g1) and bbs4-1. Proteins present in the AP
and DP of bbs4-1 (arrows 1–4) but not in the corre-
sponding fractions from wild type were identified by
MS analysis as PLDc (band 1), THB1 (band 2), STPK
(band 3), and JGI protein ID 191821 (band 4; see
Fig. S3). (B) Details of 2D gels (from Fig. S5 C) sepa-
rating matrix proteins from wild type (a and c) and
bbs4-1 flagella (b and d). Protein spots 1 (b) and 3 (d)
were enriched in the matrix of bbs4-1 and revealed to
be THB1 and STPK, respectively, by MS. Protein spot
2 (a), which was reduced in quantity in the bbs4-1
matrix, was identified as OEE3. (C) Detail of a silver-
stained SDS gel of the DP from the strains indicated.
The arrow indicates the position of PLDc. (D) Detail of
a silver-stained SDS gel and Western blots of the AP
of g1, bbs4-1, and the retrograde IFT mutant dhc1bts
probed with antibodies to the indicated proteins. The
arrow indicates the position of THB1. (A–D) The posi-
tions of standard proteins and their molecular masses
in kilodaltons are indicated.
1127BBSome function in C. reinhardtii flagella • Lechtreck et al.
We found that BBS4 was strikingly reduced in the cell
body of the bbs1 mutant, indicating that BBS1 is required for
the expression or stability of BBS4 and probably of the BBSome.
This is consistent with genetic analysis in zebrafish showing
that BBS1 has a central role in the interactions between the
genes encoding different BBSome proteins (Tayeh et al., 2008).
BBS4 was present in the cell body of the C. reinhardtii bbs7
mutant, but only small amounts were transported into the fla-
gella. Thus, flagellar transport of BBS4 requires BBS7. These
data provide independent biochemical and genetic evidence that
the BBSome proteins interact to form a complex in C. rein-
C. reinhardtii BBS4 is moved by IFT
but is not an integral component of
the IFT machinery
During IFT, particles consisting of 20 proteins organized into
complex A and complex B are transported anterogradely and
retrogradely in the flagellum (Rosenbaum and Witman, 2002;
Scholey, 2008). In C. elegans cilia, GFP-tagged BBS1, -2, -7,
and -8 undergo bidirectional transport similar to that of IFT
particles (Blacque et al., 2004). In this study, we report that
C. reinhardtii BBS4-GFP is also transported anterogradely and
retrogradely in the flagellum at rates identical to that of IFT
particles. Using double TIRFM with IFT20-mCherry, we found
that BBS4-GFP is indeed carried by IFT particles. This raises
the question of whether BBS4, and by extension the BBSome,
is an IFT cargo or an integral component of the IFT machinery.
In our C. reinhardtii bbs mutants, the assembly of flagella, the
amounts of IFT particle and motor proteins in flagella, and the
velocity of IFT are normal, and IFT complex A and B colocalize
as they do in wild-type flagella. Thus, BBS4 is not an essential
component of IFT in C. reinhardtii.
The situation is different in C. elegans. In channel cilia of
C. elegans bbs-7 and -8 mutants, IFT particles break down into
complex A and B, which then move at distinct velocities, giving
rise to the hypothesis that these BBS proteins are required to
stabilize the physical interaction between IFT complex A and B
(Ou et al., 2005). C. elegans deploys two distinct motors for an-
terograde transport, kinesin 2 attached to IFT complex A and
OSM-3 attached to complex B and solely responsible for IFT in
proteins (BBS1/2/4/5/7/8/9); this complex was termed the
BBSome. In this study, we demonstrate that C. reinhardtii
BBS4 extracted from isolated flagella sediments at 12S in su-
crose gradients, indicating that it also occurs in a complex. We
purified this complex from the membrane-plus-matrix fraction
of C. reinhardtii flagella and found it to contain BBS4 and at
least four other BBS proteins (BBS1/5/7/8). The presence of a
similar complex in C. reinhardtii and mammalian cells indi-
cates that the BBSome is evolutionarily conserved. Although
individual BBS proteins previously have been reported to be in
cilia (Blacque et al., 2004; Ou et al., 2005; Nachury et al., 2007),
this is the first direct evidence that the BBSome complex by it-
self enters and functions in the flagellum.
C. reinhardtii BBS1, -4, and -7 are not
required for flagellar assembly
We identified C. reinhardtii mutants defective in BBS1, -4, and -7.
These bbs mutants develop full-length flagella with appar-
ently normal ultrastructure, indicating that these BBSome pro-
teins are not required for flagellar assembly in C. reinhardtii.
The loss of BBS proteins has various effects on ciliary assem-
bly in different species and cell types. For example, zebrafish
bbs morphants have fewer and shorter cilia in the Kupffer’s
vesicle (Yen et al., 2006), and Bbs1, -4, and -6 knockout mice
are sterile because of a failure to assemble sperm flagella, al-
though other cilia develop more or less normally (Mykytyn
et al., 2004; Nishimura et al., 2004). RNAi targeting of BBS5
in C. reinhardtii resulted in the partial or complete loss of fla-
gella (Li et al., 2004). Similarly, the number of ciliated retinal
pigment epithelial cells is reduced by siRNAs targeting BBS1
and -5 (Nachury et al., 2007; Loktev et al., 2008). However,
cilia are present in human BBS patients and BBS knockout
mice as well as in the C. reinhardtii bbs mutants examined in
this study, suggesting that loss of the BBS proteins does not
cause a general failure of the ciliary assembly machinery. This
distinguishes the BBS mutants from IFT loss-of-function mu-
tants, which lack cilia/flagella and are embryonic lethal in mam-
mals (Brazelton et al., 2001; Rosenbaum and Witman, 2002; Hou
et al., 2007; Jonassen et al., 2008). It is possible that the effects
on ciliary assembly may be an indirect effect of loss of the
Figure 8. Phototaxis is partially restored in bbs4-1
during flagellar regeneration. Dish phototaxis assays
of wild type (g1) and bbs4-1 during flagellar regen-
eration. Cells were deflagellated by pH shock and
allowed to regenerate flagella. Samples were trans-
ferred to culture dishes and exposed to directional
light (direction indicated by arrows) for 10 min. T43,
T52, T69, and T120 refer to the time in minutes since
deflagellation. Controls were treated identically except
that the pH shock was omitted.
JCB • VOLUME 187 • NUMBER 7 • 2009 1128
and the G protein–coupled receptors Sstr3 and Mchr1 fail to lo-
calize to primary cilia of cultured neurons in Bbs2/ and
Bbs4/ mice (Berbari et al., 2008). This has been interpreted as
evidence that the BBSome is involved in transport of at least
some flagellar membrane proteins into the cilium. Consistent
with this, Nachury et al. (2007) have proposed that the mamma-
lian BBSome interacts with the Rab8 GDP/GTP exchange fac-
tor Rabin 8 to promote recruitment of specific post-Golgi
membrane vesicles to the base of the cilium followed by entry
of membrane proteins into the cilium. Because flagella can be
isolated from C. reinhardtii, we were able to assess specific pro-
teins as well as a broad ensemble of flagellar proteins for
changes caused by defects in the BBS proteins. In the C. rein-
hardtii bbs4-1 mutant, the amounts of four selected flagellar
membrane proteins were unaltered, as determined by Western
blotting, suggesting that bbs mutants do not have a general de-
fect in the delivery of membrane proteins to the flagellum. 1D
and 2D PAGE showed that the vast majority of proteins, like-
wise, were similar in their amounts in wild-type and mutant fla-
gella. However, at least four proteins were abnormally present
in bbs flagella, and three of these were in the Triton X-114 DP,
which is enriched in proteins of the flagellar membrane. The
four proteins together were represented by only one peptide in
the flagellar proteome, suggesting that they are not present or
present in only small amounts in wild-type flagella.
One possible explanation for the accumulation of specific
proteins in the flagella of C. reinhardtii bbs mutants is that
the BBSome is required for the export of these proteins from the
flagella. To test this hypothesis, we examined the flagella of the
retrograde IFT motor mutant dhc1bts and observed an accumu-
lation of THB1 similar to that observed in bbs4 flagella. An ac-
cumulation of PLDc was not observed, but this may be because
PLDc outcompetes THB1 for binding to the small number of
BBSomes presumed to still be trafficking in this hypomorphic
mutant or because the mutant is abnormal in other aspects of
IFT. Further studies will be necessary to determine whether
STPK and protein 191821 accumulate in the flagella of this or
other retrograde IFT mutants.
In agreement with a function for the BBSome in the ex-
port of flagellar proteins, Shah et al. (2008) observed that mem-
brane vesicles accumulated near the tips of cilia of Bbs1, -2, -4,
and -6 knockout mice; they suggested that this phenotype might
be caused by a defect in retrograde IFT or loss of cargo from the
IFT complex. It also may be relevant that dynein-dependent
retrograde transport of melanosomes is defective in zebrafish
bbs morphants (Yen et al., 2006).
At least three of the four proteins abnormally accumulated
in C. reinhardtii bbs mutants (an STPK, a phospholipase, and a
truncated hemoglobin) are potentially involved in intracellular
signaling. This raises the possibility that a specific function of the
BBSome is to transport signaling proteins. Cilia are involved in
various signaling pathways (e.g., phototransduction, olfaction,
PDGF-, hedgehog, Wnt, etc.), and IFT is thought to be in-
volved in moving receptors within flagella and transmitting some
signals (Pazour and Witman, 2003; Pan et al., 2005; Qin et al.,
2005; Wang et al., 2006). IFT is also likely to be involved in the
turnover of these proteins. For example, polycystin 2 abnormally
the distal segment of the cilium (Scholey, 2008). Because the
two motors have inherently different velocities, their coopera-
tive actions could create tension between the two IFT com-
plexes, and the BBS proteins might be required to prevent
complex A and B from separating from each other. In contrast,
C. reinhardtii deploys only one anterograde IFT motor and
therefore may not require the stabilizing effect of the BBSome.
The C. elegans model for BBS protein function would
predict that all IFT particles are associated with a BBSome.
However, our double IFM localization of BBS4 and IFT46
(complex B) or IFT139 (complex A) revealed that not all IFT
particles carry BBS4 in C. reinhardtii. Consistent with this,
double TIRFM showed that many IFT20-mCherry particles
were not associated with BBS4-GFP as they moved along the
flagellum. We further observed that some BBS4-GFP–labeled
particles separated from IFT particles and remained stationary
while the IFT particles continued to move along the flagellum.
Therefore, in vivo studies indicate that IFT–BBSome inter-
action differs in C. reinhardtii (this study) versus C. elegans
(Blacque et al., 2004).
The C. elegans model would also predict that the IFT par-
ticle proteins and BBSome proteins should be present in similar
amounts. To test this, we used quantitative MS (Kirkpatrick
et al., 2005) to assess the relative amounts of an IFT protein and
a BBS protein in C. reinhardtii flagella. The results indicated
that IFT81 is approximately six times more abundant than BBS1.
Assuming one BBS1 subunit per BBSome (Nachury et al.,
2007) and two IFT81 subunits per IFT particle (Lucker et al.,
2005), there are at least three times as many IFT particles as
BBSomes in the flagellum. Data from the C. reinhardtii flagel-
lar proteome analysis (Pazour et al., 2005) extend this conclu-
sion qualitatively to virtually all of the IFT and BBSome
proteins: two (BBS5 and -9) of the eight conserved BBSome
proteins were identified by only a single unique peptide each
(0.2 peptides/10 kD of BBS5 and -9), and the rest were not
found at all; in contrast, numerous peptides were found that
originated from IFT complex B proteins and IFT motor proteins
(2.4 peptides/10 kD and 2.0 peptides/10 kD, respectively), sug-
gesting an 1:1 stoichiometry between the motor and complex
B proteins and an 1:10 stoichiometry between BBS5 and -9
compared with the IFT complex B proteins: Table S4).
In summary, in C. reinhardtii, BBS1, -4, and -7 are not re-
quired for IFT, the BBSome proteins are considerably less abun-
dant in flagella than are IFT proteins, and only a subset of IFT
particles are involved in BBSome transport. We conclude that the
C. reinhardtii BBSome is not an integral part of the IFT machin-
ery. It is more likely to be an adapter that couples specific car-
goes to IFT or an IFT cargo that carries a signal. These
possibilities are not mutually exclusive.
The BBSome is required for the export of
specific proteins from the flagellum
If the BBSome is involved in transport of proteins, cilia and fla-
gella defective in BBSome proteins should have an altered pro-
tein composition. Studies of mouse models defective in Bbs
genes have revealed a mislocalization of rhodopsin in photore-
ceptor cells (Nishimura et al., 2004; Abd-El-Barr et al., 2007),
1129BBSome function in C. reinhardtii flagella • Lechtreck et al.
Importantly, we found that phototaxis is restored in
bbs4-1 cells for a short period of time after the formation of new
flagella. This is consistent with a model in which the BBSome
continually removes certain proteins from the flagellum and that
in the absence of the BBSome, an accumulation of these pro-
teins impairs flagellar function. This experiment also indicates
that the functional defect in bbs mutant flagella is not caused by
a failure to transport certain proteins into the flagellum; if this
were the case, the cells would not have regained the ability to
phototax immediately after flagellar regeneration.
The aberrant accumulation of proteins
caused by defects in BBSome proteins
may lead to progressive degeneration of
Because C. reinhardtii disassembles its flagella before mitosis
(Cavalier-Smith, 1974), there is only a limited time for proteins
to aberrantly accumulate in the flagella and only a limited time
for them to damage the flagella. In contrast, many cilia in verte-
brates persist for long periods of time, and a failure to remove
certain proteins from the cilia could progressively impair their
functionality and maintenance. Indeed, the outer segments of
rod cells in Bbs knockout mice are initially formed but later de-
generate (Nishimura et al., 2004; Abd-El-Barr et al., 2007).
Similarly, cilia are progressively lost in the Kupffer’s vesicle of
zebrafish bbs morphants (Yen et al., 2006). We propose that de-
fects in BBSome proteins cause an aberrant accumulation of
specific proteins in the cilia, which, over time, lead to increas-
ingly dysfunctional cilia, ciliary damage, and ciliary degeneration.
Therefore, BBS may be a degenerative disease of the cilium.
Materials and methods
Strains and culture conditions
C. reinhardtii strains used in this study are listed in Table S1. Cells were
grown in modified minimal medium (Pazour et al., 1995) in 24-well plates,
250-ml Erlenmeyer flasks, or aerated 5-liter diphtheria toxin flasks with a
light/dark cycle of 14:10 h at 23°C.
Identification and characterization of C. reinhardtii bbs mutants
A genomic DNA library of 350 mutant strains was screened by quantita-
tive RT-PCR using primer pairs BBS4N and BBS4C (see Table S2 for a list of
primers used in this work). A total of 40 primer pairs against various parts
of the C. reinhardtii BBIP10 and BBS1/2/3/5/7/8/9 genes were tested
on DNA isolated from 17 nonphototactic mutants. Primer pair BBS7-1
failed to amplify in strain ptx6-1, and primer pair BBS1-1 failed to am-
plify in the uncharacterized nonphototactic strain RIR7-2 (provided by
C. Dieckmann, University of Arizona, Tucson, AZ). PCR was performed
using Quantitect Syber green master mix (QIAGEN), a thermal cycler (Op-
ticon; MJ Research), and the following cycle conditions: 15 min at 94°C;
35 cycles of 30 s at 94°C, 30 s at 54°C, and 30 s at 72°C; followed by
10 min at 72°C. Primer pairs 173322, 148017, 173319, and 190839
were used to map the deletions in the bbs4 mutants. To determine the
genetic defect in bbs7/ptx6, primer pairs BBS7FIN1F/BBS7FIN2R and
BBS7FIN2F/BBS7FIN1R were used for nested PCR amplification of the
affected region from wild type and ptx6; primer BBS7SEQU1F was used
for direct sequencing of the PCR products.
Rescue of bbs4-1/ptx5-1
Screening of a bacterial artificial chromosome (BAC) library of C. rein-
hardtii (Nguyen et al., 2005) using a labeled PCR product generated with
primer pair BBS4N identified BAC 1H15 (BAC library CRCCBa, Clemson
University Genomics Institute), which contained the entire BBS4 sequence.
A 6,797-bp HindIII–BsmI fragment of BAC 1H15 covering BBS4 was sub-
cloned into pBR322. To engineer plasmids encoding epitope-tagged BBS4,
accumulates in the cilia of mice with defects in Ift88 (Pazour
et al., 2002). Similarly, the C. reinhardtii homologue of poly-
cystin 2, PKD2, is transported by IFT and accumulates in the fla-
gella of fla10 mutants when IFT is turned off at the restrictive
temperature (Huang et al., 2007). Because no accumulation of
PKD2 was observed in flagella of the C. reinhardtii bbs4 mutant,
PKD2 may be coupled directly to the IFT particle or to another
adapter for its removal from the flagella, whereas the protein
kinase, the phospholipase, and the truncated hemoglobin are linked
to retrograde IFT via the BBSome. Because the latter proteins are
absent or present only in low concentrations in wild-type flagella,
it is likely that normally they continually enter and exit the fla-
gella, possibly as part of a signaling system that monitors the fla-
gellum, and build up in the flagella only when export is blocked.
The abnormal accumulation of proteins
in bbs flagella is the likely cause of the
The defects in BBS1, -4, and -7 all cause loss of phototaxis in
C. reinhardtii. Phototaxis requires the ability to precisely control
the two flagellar axonemes in response to Ca2+-mediated signals
induced by photostimulation. bbs4 cells are nonphototactic be-
cause their axonemes lack the ability to respond normally to
changes in intraflagellar Ca2+. However, BBS proteins are com-
ponents of the flagellar matrix, and our biochemical analyses
have so far failed to identify differences in the axonemes.
The explanation for this conundrum may be that the pro-
teins that accumulate in the bbs mutant flagellar matrix directly
or indirectly modify the axoneme and thereby impair a proper
Ca2+ response. For example, PLDs generate phosphatidic acid,
which acts as a second messenger and can activate or inactivate
other proteins (Hancock, 2007). In Chlamydomonas eugametos,
a PLD is activated by Ca2+ influx induced by membrane
depolarization (Munnik et al., 2000); Ca2+ influx and mem-
brane depolarization are both involved in the photobehavior of
C. reinhardtii. The accumulated protein kinase could abnormally
phosphorylate axonemal proteins. The I1 inner arm dynein
intermediate chain IC138, for example, is hyperphosphorylated
in axonemes of nonphototactic mia (modifier of inner arms)
mutants (King and Dutcher, 1997; Wirschell et al., 2007). The
accumulated THB1 may also disrupt normal signaling. Trun-
cated hemoglobins have been implicated in NO metabolism and
are capable of altering redox states (Smagghe et al., 2008). The
C. reinhardtii flagellum is rich in flavoproteins and other redox
proteins, and at least two, AGG2 and -3, are involved in the reg-
ulation of phototaxis (Iomini et al., 2006). Light-dependent
changes in redox poise alter the disulfide-based interactions
of various components of the outer dynein arms, changing the
flagellar beat frequency and the duration of the photophobic
response (Wakabayashi and King, 2006). Therefore, the accu-
mulation of one or more of these proteins could easily be
responsible for the nonphototactic phenotype of C. reinhardtii
bbs mutants. Given the potentially far-reaching effects of these
signaling pathways, it is tempting to speculate that a similar
disruption of ciliary signaling could be responsible for the di-
verse and complex phenotypes observed when BBS proteins are
defective in other organisms.
JCB • VOLUME 187 • NUMBER 7 • 2009 1130
antibodies were linked to Alexa Fluor 488, 568, or 594 (Invitrogen).
Images were acquired using Axiovision software (Carl Zeiss, Inc.) and a
camera (AxioCam MRm; Carl Zeiss, Inc.) on a microscope (Axioskop 2
plus; Carl Zeiss, Inc.) equipped with a 100× NA 1.4 oil DIC Plan-Apochromat
objective (Carl Zeiss, Inc.) and epifluorescence. Image brightness and con-
trast were adjusted using Photoshop 6.0 (Adobe), and figures were assem-
bled using Illustrator 8.0 (Adobe). Capture times and adjustments were
similar for images mounted together.
Observation of IFT
IFT in the flagella was observed using DIC or TIRFM at RT. Cells in medium
were placed onto poly-l-lysine–treated coverslips and allowed to adhere for
0.25–1 min. Coverslips were washed carefully with medium and covered
with a second coverslip to form a chamber enclosed by a Vaseline ring. The
fluorescence images were captured using a custom-built TIRF microscope
based on an inverted microscope (IX71; Olympus) equipped with a Plan-
Apochromat 60× NA 1.4 objective (Olympus). Multiline argon/krypton lasers
(CVI Melles Griot) provided excitation light at 488 and 568 nm. Both lasers
were cleaned up with appropriate MaxLine Laser-line filters (Semrock Inc.).
A 488-nm RazorEdge beam splitter (Semrock, Inc.) was used for the GFP signal.
An FF498/581 beam splitter (Semrock Inc.) was used for the GFP/mCherry
signals. The resulting two-color emission signals were separated using a
custom-built dual-view system equipped with an FF562-Di01 dichroic mirror
(Semrock Inc.) and 525/50-nm and 630/69-nm emission filters (Semrock
Inc.). Signals were recorded using a back-illuminated electron-multiplying
charge-coupled device camera (iXon DV860; Andor Technology). Data were
analyzed using ImageJ (National Institutes of Health) and Photoshop.
Protein identification by MS
Silver-stained gel bands were excised and digested in gel with trypsin (pro-
teomics grade; Sigma-Aldrich) at 37°C overnight. For purification, eluted
peptides were loaded on a µC18 ZipTip (Millipore) equilibrated in 0.1%
trifluoroacetic acid. Peptides were deposited directly onto the sample target
and allowed to air dry before insertion into the mass spectrometer. Analy-
sis was performed on a matrix–assisted laser desorption/ionization time of
flight (MALDI-TOF) mass spectrometer (Kratos Axima QIT; Shimadzu Instru-
ments). Peptides were analyzed in positive ion mode in mid–mass range
(700–3,000 D). The instrument was externally calibrated with Angiotensin II
(MH+ of 1046.54), P14R (MH+ of 1533.86) and ACTH clip 18–39 (MH+
of 2465.20). Precursors were selected based on signal intensity at a mass
resolution width of 250 for collision-induced dissociation fragmentation
(MS/MS) using argon as the collision gas. All spectra were peak processed
with Mascot Distiller (Matrix Sciences, Ltd.) before database searching.
Database searches were performed in house with the Mascot search en-
gine (Matrix Sciences, Ltd.). NMT–The MYR Predictor (http://mendel.imp
.ac.at/myristate/SUPLpredictor.htm) and Myristoylator (http://www.expasy
.ch/tools/myristoylator/) were used to predict myristoylation sites.
The membrane-plus-matrix fraction isolated from wild-type flagella was
subjected to SDS-PAGE, and the region from 40 to 95 kD (Fig. S4 A)
was diced and subjected to in-gel digestion with 6 ng/µl sequencing-
grade modified trypsin (Promega) in 50 mM ammonium bicarbonate
overnight at 37°C. Peptides were extracted first with 50% MeCN (aceto-
nitrile) and 2.5% formic acid and then with 100% MeCN. Peptides
were then dried using a SpeedVac. Peptides were resuspended in 8.5 µl
of 2.5% MeCN and 2.5% formic acid containing 12.5 fmol/µl each of
the three BBS1 and three IFT81 synthetic AQUA peptides shown in Table II.
AQUA peptides with 13C-, 15N-labels were synthesized at Cell Signaling
Technology. 4 µl of the samples were shot in duplicate. Mass measure-
ments were made in a mass spectrometer (LTQ-Orbitrap; Thermo Fisher
Scientific), which was set up with a liquid chromatography interface essen-
tially as described previously (Ballif et al., 2008). Quantification was per-
formed as outlined in Fig. 5 (D and E) and Fig. S4. Before collection of the
measurements reported in Table II, liquid chromatography–MS/MS anal-
yses were conducted on the mixture of six AQUA peptides. For each pep-
tide, the retention time on the reverse-phase C18 column was determined,
and MS and MS/MS spectra were collected. Importantly, similar results
to those reported in Table II were obtained with a fourfold increase in the
concentration of trypsin used in the in-gel digestion, a fourfold increase in
the amount of AQUA peptide standards used, or a fivefold increase in the
amount of purified flagellar protein.
Online supplemental material
Fig. S1 shows the defective Ca2+ response of bbs4-1. Figs. S2 and S3
show peptides identified by MS. Fig. S4 shows MS data related to the
the protein-encoding part of the message was amplified from cDNA using
primer pair CBBS4 and cloned into FLA14 gene–based expression vector
pKL3-3xHA or pKL3-GFP; the former additionally contained the ble select-
able marker gene. After transformation or cotransformation together with
pSP124S containing the ble resistance gene, transformants were selected
on Zeocin Tris-acetate-phosphate plates (Stevens et al., 1996), picked into
liquid M medium, and screened for negative phototaxis (Pazour et al.,
1995). DNA was isolated from four selected strains rescued with pBR322-
BBS4 and tested for the absence of the regions flanking the BBS4 gene and
for the presence of the BBS4 gene to verify that they represented trans-
formed bbs4-1/ptx5-1 cells. BBS4HA21 rescued by BBS4-3xHA was
mated to strain g2, and random progeny were analyzed for phototactic
behavior and for the expression of BBS4-3xHA by indirect IFM. Of 96
strains tested, 50 expressed BBS4-3xHA, and all of these showed photo-
To analyze phototactic behavior, cells in a 35 × 10–mm culture dish were
illuminated from one side and scored for the ability to accumulate on the
other side (Hegemann and Berthold, 2009). Phototaxis was quantitated as
described by Moss et al. (1995). Measurements of the Ca2 response of re-
activated cells models were performed as described by Kamiya and
Witman (1984). All phototaxis experiments were performed 4–8 h after
the beginning of the light phase.
Antibody production and epitope tagging
Primers BBS4S3F and BBS4A1R were used to amplify a partial cDNA en-
coding the C-terminal 248 residues of BBS4; after digestion with EcoRI, the
fragment was inserted into the EcoRI site of the bacterial expression vector
pMAL-cR1,and the purified fusion protein was used for antibody produc-
tion in rabbits (Covance). A full-length cDNA encoding BBS4 was cloned
into the bacterial expression vector pGEX (GE Healthcare), and the immo-
bilized fusion protein was used to affinity purify the immune serum.
A full-length BBS4 cDNA was cloned into the FLA14-based expres-
sion vectors pKL3-3xHA+ble and pKL3-GFP, adding three HA tags or a
GFP tag to the C terminus of BBS4 (Lechtreck and Witman, 2007). After
transformation into bbs4-1, 2 (BBS4HA21 and BBS4HA24) out of 98
transformants showed restored phototactic behavior and expression of
BBS4-3xHA (50 kD); BBS4-3xHA was not expressed in 10 other transfor-
mants randomly selected for testing. To test whether BBS4 undergoes IFT,
we cloned the BBS4 cDNA into a GFP expression vector and used this con-
struct to rescue bbs4-1. 1 out of 78 transformants tested showed restored
phototaxis and expression of BBS4-GFP, as detected by Western blots
using polyclonal anti-GFP.
Flagellar isolation and fractionation
Flagellar isolation using the dibucaine method and flagellar amputation by
pH shock were performed as previously described by Witman (1986) and
Lefebvre (1995), respectively. Isolated flagella were extracted with 1%
NP-40 Alternative (EMD) for 20 min on ice, and the suspension was separated
by centrifugation into a pellet containing the axonemes and a supernatant
consisting of the membrane-plus-matrix. For MS quantitation of BBS1 and
IFT81 in the membrane-plus-matrix, the AQUA peptide technique was used
(see Quantitative MS and Fig. S4). The membrane-plus-matrix was further
separated by sucrose gradient centrifugation (36,000 rpm for 12.5 h at
4°C on an SW41 rotor [Beckman Coulter]), and fractions were analyzed
by SDS-PAGE and Western blotting. For phase partitioning, isolated fla-
gella were extracted using 1% Triton X-114 for 20 min on ice; after re-
moval of the axonemes by centrifugation (Witman, 1986), the supernatant
was incubated briefly at 37°C, and phases were separated by centrifuga-
tion (3,300 g for 10 min at RT). The AP was treated with 1% Triton X-114,
the DP was diluted with buffer, and the phase separation was repeated to
yield the final APs and DPs.
Immunoprecipitation of complexes containing BBS4-3xHA
The membrane-plus-matrix fractions of isolated wild-type and BBS4HA21 fla-
gella were incubated with anti-HA affinity matrix (Roche) in 125 mM NaCl,
30 mM Hepes, 5 mM MgSO4, 0.5 mM EGTA, 25 mM KCl, pH 7.4, and
Complete Protease Inhibitor Cocktail (Roche) and incubated overnight at 4°C
with agitation. After repeated washes, 1 mg/ml HA peptide (Roche) was
added to elute bound proteins. Proteins were separated by 5–15% SDS-PAGE
(Bio-Rad Laboratories), excised after silver staining, and analyzed by MS.
Indirect IFM was performed as described by Lechtreck and Witman (2007).
The primary antibodies used in this study are listed in Table S3; secondary
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culture dish assay. Video 2 shows phototaxis of wild-type C. reinhardtii
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flagella. Online supplemental material is available at http://www.jcb
We thank Dr. C. Dieckmann for sharing phototaxis mutants, Drs. D. Cole (Uni-
versity of Idaho, Moscow, ID), R. Bloodgood (University of Virginia, Charlottes-
ville, VA), and J. Rosenbaum (Yale University, New Haven, CT) for sharing
antibodies, and Dr. J. Brown (University of Massachusetts Medical School
[UMMS], Worcester, MA) for analyzing the sedimentation of wild-type BBS4.
We are grateful to Drs. G. Hendricks and J. Leszyk of UMMS for expert help
with EM and MS, respectively.
Core facilities used in this research were supported by a Diabetes Endo-
crinology Research Center grant (DK32520). This work was supported by
National Institutes of Health grants (GM030626 to G.B. Witman; DC006103,
AR048898, and AR048526 to M. Ikebe; and GM060992 to G.J. Pazour),
the Vermont Genetics Network through a National Institutes of Health grant
(P20 RR16462) from the IDeA Network of Biomedical Research Excellence
Program of the National Center for Research Resources (to B.A. Ballif), and the
Robert W. Booth Endowment (G.B. Witman).
Submitted: 30 September 2009
Accepted: 24 November 2009
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