© 2017. Published by The Company of Biologists Ltd.
The Arf GEF GBF1 and Arf4 synergize with the sensory receptor cargo, rhodopsin, to
regulate ciliary membrane trafficking
Jing Wang1, Theresa Fresquez1, Vasundhara Kandachar1 and Dusanka Deretic1,2
Departments: 1Surgery, Division of Ophthalmology and 2Cell Biology and Physiology,
University of New Mexico, Albuquerque, New Mexico 87131
Address Correspondence to:
Dusanka Deretic
University of New Mexico School of Medicine
Department of Surgery, Division of Ophthalmology
Basic Medical Sciences Building, Rm. 377
915 Camino de Salud, N. E.
Albuquerque, NM 87131
Tel: (505) 272-4968
Fax: (505) 272-6029
E-mail: [email protected]
Key words: Cilium, Arf GTPases, Sensory Receptors, Rhodopsin
The abbreviations used are: GEF, Guanine Nucleotide Exchange Factor; GAP, GTPase
Activating Protein; RIS, Rod Inner Segment(s); ROS, Rod Outer Segment(s); RTC(s),
Rhodopsin Transport Carrier(s); TGN, Trans-Golgi Network.
Summary Statement
Sensory receptor cargo promotes its intracellular progression by providing input to a specific
Arf GEF to activate a cognate Arf directing transport to the cilia.
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JCS Advance Online Article. Posted on 12 October 2017
Abstract
The small GTPase Arf4 and the Arf GTPase activating protein (GAP) ASAP1 cooperatively
sequester sensory receptor cargo into transport carriers targeted to primary cilia, but the input
that drives Arf4 activation in this process remains unknown. Here, we show that during the
carrier biogenesis from the photoreceptor Golgi/trans-Golgi network (TGN) a functional
complex is formed between Arf4, the Arf guanine nucleotide exchange factor (GEF) GBF1
and the light-sensing receptor, rhodopsin. Rhodopsin and Arf4 bind the regulatory N-terminal
DCB-HUS domain of GBF1. The complex is sensitive to Golgicide A (GCA), a selective
inhibitor of GBF1 that accordingly blocks rhodopsin delivery to the cilia, without disrupting
the photoreceptor Golgi. The emergence of newly synthesized rhodopsin in the
endomembrane system is essential for GBF1-Arf4 complex formation in vivo. Notably,
GBF1 interacts with the Arf GAP ASAP1 in a GCA-resistant manner. Our findings implicate
that converging signals on GBF1 from the influx of cargo into the Golgi/TGN and the
feedback from Arf4, combined with an input from ASAP1, control Arf4 activation during
sensory membrane trafficking to primary cilia.
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Introduction
The Arf family of small G-proteins constitutes a crucial component of the intracellular
membrane trafficking machinery. Through the control of lipid metabolism and the
recruitment of canonical coat complexes and protein adaptors that recognize and sequester
the appropriate membrane cargo, Arf GTPases play a central role in key processes such as the
maintenance of the Golgi architecture, progression of cargo through the Golgi complex, as
well as Golgi-to-plasma-membrane targeting that is responsible for the delivery of sensory
receptors, and their associated complexes, to primary cilia (Deretic, 2013; Donaldson and
Jackson, 2011; Ezratty et al., 2016; Hilgendorf et al., 2016; Humbert et al., 2012; Schou et
al., 2015; Schwarz et al., 2012; Wang and Deretic, 2014; Wright et al., 2011). The distal
ciliary membrane of vertebrate retinal rod photoreceptor cells elaborates a unique sensory
organelle, the rod outer segment (ROS), which is filled with several thousand membranous
disks containing as many as a billion copies of the light receptor rhodopsin (Besharse, 1986).
Rhodopsin is directed to cilia through the ciliary targeting signal (CTS) VxPx that directly
binds activated Arf4 at the Golgi/TGN (Deretic et al., 2005; Mazelova et al., 2009; Wang et
al., 2012). The importance of this trafficking pathway is underscored by autosomal dominant
retinitis pigmentosa (ADRP), a group of blinding diseases that result from mutations in more
than 25 genes. Mutations affecting the rhodopsin CTS VxPx are among the most severe
forms of ADRP (Berson et al., 2002). On the other hand, different targeting signals and
trafficking mechanisms direct other ROS membrane components to cilia. Cyclic nucleotide-
gated (CNG) channel transport relies on the cytoskeletal adaptor Ankyrin-G (Kizhatil et al.,
2009). Guanylyl cyclase 1 (GC1) and the Progressive Rod-Cone Degeneration (PRCD)
protein appear to require rhodopsin for their ciliary trafficking (Pearring et al., 2015; Spencer
et al., 2016), whereas targeting of the ROS disk rim protein Peripherin-2/rds (P/rds) requires
its C terminus and interactions with SNARE proteins (Salinas et al., 2013; Tam et al., 2004;
Zulliger et al., 2015). Defects in P/rds cause retinitis pigmentosa and macular dystrophies
(Goldberg et al., 2016), while defects in trafficking of prenylated ROS proteins cause retinitis
pigmentosa 2 (Zhang et al., 2014).
Broad dysfunction of ciliary trafficking causes human genetic diseases and syndromic
disorders collectively known as ciliopathies (Reiter and Leroux, 2017). The transition zone
and basal body multiprotein complexes NPHP-JBTS-MKS and BBS participate in ciliary
morphogenesis and gating. These processes are affected by mutations causing
nephronophthisis, as well as Joubert, Meckel and Bardet Biedel syndrome, which affect
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multiple organs, including the eyes (Craige et al., 2010; Datta et al., 2015; Garcia-Gonzalo et
al., 2011; Nachury et al., 2010; Sang et al., 2011; Shimada et al., 2017; van Reeuwijk et al.,
2011). Intraflagellar transport (IFT) regulates entrance and exit of regulatory components and
the progression of ciliary cargo, including rhodopsin, through the transition zone (Bhowmick
et al., 2009; Eguether et al., 2014; Keady et al., 2011; Krock et al., 2009; Liew et al., 2014;
Zhao and Malicki, 2011). These ciliary networks are directly linked to the small GTPase
Rab8 and its GEF Rabin8 (Bachmann-Gagescu et al., 2011; Chiba et al., 2013; Nachury et
al., 2007; Omori et al., 2008), which are crucial regulators of ciliary membrane trafficking
(Deretic et al., 1995; Feng et al., 2012; Moritz et al., 2001; Wang and Deretic, 2015a;
Westlake et al., 2011). Dysfunction of Arf GTPases and their regulators is also a known
cause of ciliopathies (Seixas et al., 2013; Wiens et al., 2010; Zhang et al., 2013).
Arf GTPases exert their regulatory function through the cycles of GTP binding and
hydrolysis that are regulated by Arf guanine nucleotide exchange factors (GEFs) and GTPase
activating proteins (GAPs), which control their membrane association and signaling
pathways through activation cascades and positive-feedback loops (Bui et al., 2009;
Casanova, 2007; Jackson and Casanova, 2000; Lowery et al., 2013; Stalder and Antonny,
2013). One of the outstanding questions in the regulation of Arf GTPases is the role of
protein cargo in their activation. It has been proposed that the cargo acts upstream of Arf
activation, in a manner analogous to the activation of heterotrimeric G-proteins by G-protein
coupled receptors (GPCRs) that serve as their GEFs, upon light or ligand stimulation (Caster
et al., 2013). Although multiple Arf GEFs activate Arfs in spatiotemporally restricted
manners, it is not clear what signals Arf GEFs recognize in order to activate Arfs. The
specific cargo has the capacity to regulate the Arf-dependent recruitment of the protein
adaptors (Caster et al., 2013), which suggests that a functional complex between the cargo,
the cognate Arf and an Arf GEF likely exists during membrane trafficking. However,
currently the evidence for such a complex is absent.
The BIG/GBF family of Golgi-localized large Arf GEFs contains the highly conserved Sec7
domain involved in the nucleotide-exchange activity, surrounded by several conserved
domains involved in functional interactions that regulate their activity and membrane-
association (Bui et al., 2009; Casanova, 2007; Wright et al., 2014). Golgi-localized large Arf
GEFs are autoinhibited in solution. Their catalytic activity and membrane association are
controlled by cooperative allosteric regulation via coincidence detection by DCB
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(dimerization and cyclophillin binding) and HDS (homology downstream of Sec7) domains,
which integrate direct inputs from membranes and multiple activated Arfs and Rabs (Alvarez
et al., 2003; Bouvet et al., 2013; McDonold and Fromme, 2014; Monetta et al., 2007;
Nawrotek et al., 2016; Richardson and Fromme, 2012; Richardson et al., 2012; Stalder and
Antonny, 2013). GBF1 and Arf4 function within the early Golgi, and at the TGN (Ben-
Tekaya et al., 2010; Chun et al., 2008; Garcia-Mata et al., 2003; Kawamoto et al., 2002;
Mazelova et al., 2009; Nakai et al., 2013; Szul et al., 2005; Szul et al., 2007; Wang et al.,
2012; Zhao et al., 2006). At the TGN, GBF1 initiates an Arf activation cascade through direct
interactions of Arf4 with the DCB domains of BIG1 and BIG2 (Lowery et al., 2013). It is
thus plausible to hypothesize that GBF1 may function as the Arf4 GEF that activates Arf4 in
ciliary receptor targeting.
Although the directed cargo delivery is tightly regulated in all cells, the limited quantity of a
specific ciliary cargo often necessitates its overexpression to analyze ciliary transport, thus
retinal rod photoreceptors provide a clear advantage for these studies (Pearring et al., 2013;
Wang and Deretic, 2014; Wensel et al., 2016). Because of their extensive ROS membrane
turnover, amphibian rods have consistently offered a unique model where biochemical and
morphological data can be correlated in a single experimental system for the study of
otherwise basic mechanisms underlying ciliary and photoreceptor membrane biogenesis
(Besharse, 1986; Hall et al., 1969; Papermaster et al., 1975; Papermaster et al., 1985; Young,
1967; Young, 1976). Although photoreceptors are highly specialized cells, a recombinant
rhodopsin-GFP fusion protein expressed in epithelial cells maintains the restricted ciliary
localization, indicating that certain aspects of ciliary transport are highly conserved
(Mazelova et al., 2009; Trivedi et al., 2012; Wang et al., 2012; Ward et al., 2011). In this
study, we take advantage of the photoreceptor paradigm because of the abundance of
rhodopsin transport carriers (RTCs) that mediate Golgi-to-cilia transport in photoreceptors
(Deretic and Mazelova, 2009; Wang and Deretic, 2014), and we examine the role of the
ciliary cargo, rhodopsin, in the recruitment of the Arf GEF GBF1 and the activation of Arf4.
We find that GBF1 interacts with rhodopsin at the Golgi/TGN and that the activity of GBF1
is essential for their interaction and for communication with Arf4, which, in turn, regulates
the formation of RTCs and the delivery of rhodopsin to sensory cilia.
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Results
The Arf GEF GBF1 is localized at the trans-Golgi where it interacts with Arf4, the Arf
GAP ASAP1 and the ciliary cargo, rhodopsin.
To determine which Arf GEF is responsible for the activation of Arf4 in retinal photoreceptor
cells, we examined the distribution of GBF1, a candidate Arf GEF that is reported to activate
Arf4 in HeLa cells (Lowery et al., 2013). By confocal microscopy, GBF1 exhibited a
distribution that differed from that of the cis-Golgi marker GM130 and closely resembled that
of the trans-Golgi marker Rab6 (Fig 1A-D, arrows). The pixel colocalization of Rab6 with
GBF1 was significantly higher than with GM130 (p=3.42E-5, n=5 cells) (Fig 1D). In
photoreceptors, Golgi is localized within the myoid region (M) of the rod inner segment
(RIS), as schematically presented in Fig 1E. To pinpoint the localization of GBF1 within the
Golgi, we performed in situ proximity ligation assay (PLA), a molecular technique suitable
for proteomic analysis, because a positive signal is possible only when the fluorescent PLA
probes are <40 nm apart (Raykova et al., 2016; Soderberg et al., 2006). We employed PLA
modified for studies of brain and retinal tissue (Blasic et al., 2012; Trifilieff et al., 2011;
Wang and Deretic, 2015b; Wang et al., 2012; Zulliger et al., 2015). GBF1-Rab6 interaction
sites (Fig 1F, red dots), aligned well with the trans-Golgi, which was identified post-PLA by
staining with anti-Rab6 conjugated to Alexa Fluor 488 (Fig 1F, green). No interaction sites
were detected between GBF1 and the cis-Golgi markers GM130 (Fig 1G) and p115 (Fig 1H),
despite the robust Golgi labeling with the antibody to p115 (Fig. 1I). Thus, in photoreceptor
cells, GBF1 does not associate with the cis-Golgi but is specifically localized at the trans-
Golgi.
Next, we determined that, in the RIS, GBF1 and Arf4 interact in close proximity to the trans-
Golgi (Fig 1J, red dots), identified by Rab6, as above (Fig 1J, green). Notably, within the
same area, GBF1 also interacted with rhodopsin (Fig 1K). The distribution of these
interaction sites was comparable to the distribution of rhodopsin-Arf4 interaction sites (Fig
1L), as noted before (Wang et al., 2012). Unexpectedly, GBF1 also interacted with the Arf
GAP ASAP1, which is known to form a complex with rhodopsin and Arf4 (Wang et al.,
2012). As shown in Fig 1M, GBF1-ASAP1 interactions were not restricted to the Golgi area,
but were distributed throughout the RIS. To quantify the number of protein-protein
interaction sites in these experiments, the red fluorescent signals detected by PLA in the
Golgi area of the myoid region (M) were assigned to the Golgi/TGN and those in the
ellipsoid region (E) to RTCs, as described (Wang et al., 2012). The quantitative analysis
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revealed that interactions of rhodopsin with GBF1 and Arf4 occur nearly exclusively at the
Golgi/TGN, whereas GBF1-ASAP1 interactions occur at the Golgi/TGN and on RTCs (Fig
1N).
To further characterize subcellular localization of GBF1, we performed retinal subcellular
fractionation by a standard procedure (Deretic and Mazelova, 2009), generating ROS and
retinal post-nuclear supernatant (PNS), highly enriched in photoreceptor biosynthetic
membranes (Deretic and Papermaster, 1991; Papermaster et al., 1975). PNS was separated
into three fractions designated as the Golgi/TGN/ER-enriched, RTC-enriched (T/G/E and
RTCs hereafter), and the cytosol, as reported before (Deretic, 2000; Deretic et al., 1995;
Deretic et al., 1996; Mazelova et al., 2009; Morel et al., 2000; Wang et al., 2012). In
agreement with microscopy data, GBF1 was present in T/G/E fraction, on RTCs and in the
cytosol (Fig 1O), paralleling the known distribution of the Arf GAP ASAP1 (Mazelova et al.,
2009) (Fig 1O). Arf4 was detected only in the T/G/E fraction and in the cytosol, as previously
determined (Mazelova et al., 2009; Wang et al., 2012) (Fig 1O). Subcellular fractionation
corroborated the PLA data and revealed that despite the lack of Arfs (Mazelova et al., 2009;
Wang et al., 2012), both the Arf GEF GBF1 and the Arf GAP ASAP1 also associate with
RTCs.
Golgicide A (GCA), a selective inhibitor of GBF1, significantly disrupts rhodopsin-
GBF1-Arf4-ASAP1 interactions.
To determine if the activity of GBF1 affects its interactions with the ciliary cargo, Arf4 and
ASAP1, we inactivated GBF1 in cultured eyecups with Golgicide A (GCA) for 3 hours. GCA
is a selective inhibitor of GBF1 that has no effect on other Arf GEFs due to the unique
conformation of the nucleotide-binding pocket of GBF1 (Saenz et al., 2009). In control
retinas, GBF1 interactions were detected around the Golgi/TGN (Fig 2A and C), as before.
Rhodopsin-Arf4 interactions were detected at the Golgi (Fig 2B, arrows). ASAP1 interactions
with rhodopsin and GBF1 were detected at the Golgi and on RTCs (Fig 2D and E, arrows).
GCA treatment greatly diminished rhodopsin-GBF1, rhodopsin-Arf4, Arf4-GBF1 and
rhodopsin-ASAP1 interactions (Fig 2F-I), but had minimal effect on GBF1-ASAP1
interactions (Fig 2J). In GCA-treated retinas rhodopsin-ASAP1 interactions were diminished
both at the Golgi and on RTCs, and the remaining interaction sites were observed along the
myoid-ellipsoid border (Fig 2I, arrows), at an unusual location not seen in the controls. These
data suggest that GCA affected both the formation of nascent RTCs at the Golgi/TGN, and
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the cilia-directed trafficking of RTCs formed before the addition of GCA. The activity of
GBF1 was essential for rhodopsin-GBF1-Arf4-ASAP1 communications, as GCA caused a
significant decrease in their interaction sites (p<2.2E-6) (Fig 2K-N). By contrast, GBF1-
ASAP1 interactions were unaffected (Fig 2O). From the comparable number and distribution
of rhodopsin interaction sites with GBF1 and Arf4 detected by PLA at the Golgi/TGN, we
conclude that the specific complex between the cargo, the cognate Arf and the Arf GEF may
be formed there.
GCA minimally affects the morphology of photoreceptor Golgi.
Given the reported disassembly of the Golgi by GCA (Lowery et al., 2013; Saenz et al.,
2009), we examined the state of the Golgi complex in GCA-treated retinas. Surprisingly, in
photoreceptor cells GCA had minimal effect on the Golgi morphology. Both cis-Golgi,
identified by GM130 staining, and trans-Golgi, identified by Rab6, were largely unchanged
in GCA-treated retinas (Fig 3A). By contrast, Brefeldin A (BFA), a non-competitive inhibitor
of Golgi Arf GEFs (Peyroche et al., 1999), caused substantial swelling and perturbation of
the photoreceptor Golgi, as previously described (Deretic and Papermaster, 1991; Mazelova
et al., 2009)(Fig 3A). Thus, in all probability, the Golgi organization in photoreceptors is not
controlled by GBF1, but by another BFA-sensitive Arf GEF, very likely BIG1 (Boal and
Stephens, 2010), which is detected in the mouse retinal transcriptome at a similar level as
GBF1 (Brooks et al., 2011).
The activity of GBF1 is necessary for the Golgi export of ciliary cargo.
Because the activity of GBF1 is important for its interactions with Arf4 and the ciliary cargo
at the Golgi, we asked whether it is also necessary for ciliary trafficking. We performed
pulse-chase experiments in isolated retinas using established methodology: following a one
hour pulse and a two hour chase, photoreceptor ER membranes are cleared of newly
synthesized proteins and radiolabeled rhodopsin localizes in the Golgi/TGN, RTCs and the
ROS, with the kinetics paralleling its trafficking in vivo (Deretic and Papermaster, 1991;
Mazelova et al., 2009). We followed the progression of radiolabeled proteins through the
biosynthetic membranes separated on sucrose density gradients in control retinas, or in the
continuous presence of GCA. At the GCA concentration tested, the majority of the newly
synthesized rhodopsin was arrested in the T/G/E fraction and its delivery to the ROS was
significantly inhibited (p<0.01) (Fig 3B-D). Based on its uniform molecular weight in all
fractions, we determined that, in the presence of GCA, newly synthesized rhodopsin had left
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the ER and reached the Golgi where it was terminally glycosylated. This is in contrast to
BFA-induced trafficking disruption, which also affects oligosaccharide trimming (Deretic
and Papermaster, 1991). Furthermore, GCA did not alter the Golgi localization of GBF1 (Fig
3E, arrows), in line with the report that in GCA-treated cells GBF1 remains on the Golgi
membranes (Lowery et al., 2013). Consistent with the preservation of interactions between
GBF1 and ASAP1 detected in Fig. 2, their colocalization was unaltered by GCA (Fig 3E,
arrows). By contrast, colocalization between Arf4 and GBF1 was disrupted by GCA
treatment (Fig 3E, arrows), in accord with the absence of a PLA signal observed in Fig. 2.
Finally, GCA treatment had minimal effect on the distribution of GBF1, Arf4 and ASAP1
among retinal subcellular fractions (Fig 3F and G). Because GCA significantly slowed down
the exit of rhodopsin while maintaining the Golgi structure and localization of key associated
proteins, we conclude that in retinal photoreceptors the activity of GBF1 is necessary for the
Golgi export of ciliary cargo.
GBF1 directly interacts with Arf4 and the ciliary cargo, rhodopsin.
In addition to the catalytic Sec7 domain, GBF1 contains a DCB domain, a HUS domain
(homology upstream of Sec7 domain), and three HDS domains (Bui et al., 2009; Mouratou et
al., 2005). To determine whether the binding of GBF1 to rhodopsin is direct, we employed
human DCB-HUS (AA 1-710) and Sec7-HDS1 (AA 695-1066), fused to GST (Bouvet et al.,
2013) (Fig 4A). GST fusion proteins were incubated with purified bovine rhodopsin, with or
without recombinant human Arf4 pre-loaded with GTPS or GDPS. GST DCB-HUS pulled
down rhodopsin significantly better than the GST Sec7-HDS1, or GST alone (P<0.005), both
in the presence and absence of Arf4, demonstrating a direct cargo-GBF1 interaction (Fig 4B).
GST DCB-HUS pulled down Arf4, bound to GTPS or GDPS, whereas Sec7-HDS1
preferably interacted with GDPS-bound Arf4. To ascertain that GBF1 DCB-HUS is
properly folded and binds rhodopsin specifically, we used Arl1 as a negative control, whose
binding to the DCB domain is conserved in BIG1 and BIG2, but not in GBF1 (Christis and
Munro, 2012; Galindo et al., 2016). We incubated purified bovine rhodopsin, or Arl1Q71L,
with increasing amounts of GBF1 DCB-HUS bound to glutathione beads. Rhodopsin binding
robustly increased with the increase of DCB-HUS beads, in contrast to the barely detectable
increase in non-specific Arl1 binding (Fig. 4C). To examine the specificity of GBF1
interaction with Arf GTPases, we compared the full-length Arf4 to 17Arf1, a truncated
construct in which the N-terminal -helix of Arf1 was removed to facilitate nucleotide
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loading in the absence of membranes (Randazzo et al., 1995). Unlike Arf1, Arf4 does not
require myristoylation and membranes for activation (Chun et al., 2008; Duijsings et al.,
2009), therefore full-length Arf4 was pre-loaded with GTPS or GDPS. GBF1 is known to
co-precipitate and activate both Arf1 and Arf4 in vivo (Szul et al., 2007). However, GST
DCB-HUS pulled down only Arf4 (Fig 4C), whereas 17Arf1 did not show above
background binding (Fig 4C). Although these data point to the specificity of Arf4 binding to
the DCB-HUS domain of GBF1, they could be attributed to the absence of the N-terminal
helix of Arf1, which, although different from Arf4 (Duijsings et al., 2009), may still have the
ability to interact with the DCB-HUS of GBF1. Lack of strong discrimination between the
nucleotide bound states of Arf4 by GST DCB-HUS indicates that the contact surface on Arf4
does not undergo conformational changes upon nucleotide binding.
Rhodopsin transiting the RIS provides a signal crucial to Arf4 interaction with GBF1.
A key step in the assembly of the ciliary targeting complex is the binding of rhodopsin to
activated Arf4 at the TGN (Mazelova et al., 2009). We thus wanted to test if the influx of
rhodopsin plays a role in the activation of Arf4. For this purpose, we treated retinas with
cycloheximide, which essentially abolished rhodopsin-GBF1, rhodopsin-Arf4 and Arf4-
GBF1 interactions (Fig. 5A-C and E-G), but, like GCA, had minimal effect on GBF1-ASAP1
interactions (Fig. 5D and H). A significant decrease in interaction sites detected by PLA in
the RIS (p<2.0E-13) indicates that not only rhodopsin interactions, but also the Arf4-GBF1
interaction, were contingent upon the presence of ciliary-targeted cargo in biosynthetic
membranes (Fig. 5I). A parallel pulse-chaise experiment confirmed a near complete
inhibition of protein synthesis by a 3-hour treatment with cycloheximide (Fig. 5J and L).
Subcellular fractionation showed that cycloheximide minimally affected intracellular
distribution of Arf4 and ASAP1 (Fig 5K). Cycloheximide did not alter the localization of
GBF1 and Arf4 (Fig 5M), but completely depleted rhodopsin from the RIS endomembrane
system (Fig. 5N and O). While it is formally possible that the depletion of proteins other than
rhodopsin might have contributed to the observed effects of cycloheximide on Arf4-GBF1
interactions, this is highly unlikely considering that rhodopsin represents as much as 90% of
the ROS membrane protein, has by far the fastest turnover in the retina and is the major
protein synthesized and transported to the cilia of photoreceptor cells (Brooks et al., 2011;
Deretic and Papermaster, 1991; Hall et al., 1969; Papermaster et al., 1975; Papermaster and
Dreyer, 1974; Papermaster et al., 1985). The dominance of rhodopsin is also evident from Fig
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5L, where only a couple of other radiolabeled proteins are clearly detected in the
autoradiogram. They most likely correspond to the subunits of the next most abundant
photoreceptor protein, the heterotrimeric G-protein transducin, which is activated by
rhodopsin in the ROS upon light stimulation. Figure 5P schematically represents molecular
interactions between the cargo, rhodopsin, the cognate Arf, Arf4, and the Arf GEF, GBF1,
during the formation of nascent RTCs from the TGN, consistent with the results of our study.
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Discussion
In this study, we provide evidence for the role of protein cargo in the regulation of Arf
GTPases in vertebrate rod photoreceptors by establishing the existence of a functional
complex between the ciliary cargo, rhodopsin, the cognate Arf, Arf4, and the Arf GEF GBF1.
Although ciliary transport is highly conserved, our present study reveals a particular
adaptation of photoreceptor cells that are synthesizing and transporting considerable amounts
of rhodopsin-containing membranes, through apparent concentration of the Arf GEF GBF1 at
the Golgi exit where it senses emergence of the cargo essential for ciliary biogenesis. Our
study also broadly implicates the protein cargo in promoting its progression through the
endomembrane system by providing input to a specific Arf GEF to activate a cognate Arf
directing cargo transport to its correct subcellular location.
The Arf GEF GBF1 activates Arf4, which was initially identified as an essential factor for the
generation of ciliary-targeted post-Golgi carriers (RTCs) via interaction with the VxPx CTS
of rhodopsin (Deretic et al., 2005). Recent in vivo studies of trafficking of rhodopsin fused to
the photoconvertible fluorescent protein Dendra2 in Xenopus photoreceptors showed that the
VxPx motif enhances ciliary targeting at least 10-fold and accelerates trafficking of post-
Golgi vesicular structures (Lodowski et al., 2013), most likely acting through Arf4. In further
support of Arf4 function in ciliary trafficking, the reduction in Arf4 also caused a delay in
delivery of ciliary sensory receptor fibrocystin from the Golgi to the cilium (Follit et al.,
2014). In photoreceptors, the Arf4-based complex forms in sequential order at the TGN and
includes the Arf GAP ASAP1, the Rab8 GEF Rabin8, Rab11, and the Arf/Rab11 effector
FIP3 (Mazelova et al., 2009; Wang and Deretic, 2015a; Wang et al., 2012). The notion that
membrane-targeting modules assemble through multiple weak interactions that create high-
avidity complexes was recently reinforced by crystallization and analysis of the Rab11-FIP3-
Rabin8 dual effector complex (Vetter et al., 2015).
A partially redundant role for Arf1 and Arf4 in intracellular trafficking was proposed based
on double knockouts, which caused tubulation and vesiculation of the Golgi and defects in
HeLa cells (Volpicelli-Daley et al., 2005). However, a recent study revealed not only
similarities but also many differences between Arf1 and Arf4 (Christis and Munro, 2012).
Arf1 and Arf4 showed similar interactions with COP I coat proteins and GM130 from insect
cell extracts, but Arf4 preferentially bound Rab6 and Rab11, the two Rabs known to be
involved in trafficking of both Drosophila and vertebrate rhodopsin (Deretic and
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Papermaster, 1993; Mazelova et al., 2009; Satoh et al., 2005; Shetty et al., 1998). A more
specific role for Arf4 in directing transport out of the Golgi complex is further substantiated
by its binding to GMAP210, which is implicated in ciliary trafficking in photoreceptors
(Follit et al., 2008; Keady et al., 2011), and a 9- fold higher interaction with Src, a kinase that
regulates Golgi exit (Pulvirenti et al., 2008), and phosphorylates the Arf4 GAP ASAP1
(Brown et al., 1998).
Our recent study in transgenic frogs showed that the Arf4I46D mutant, deficient in GTP
hydrolysis by ASAP1, caused dysfunctional rhodopsin trafficking and rapid retinal
degeneration (Mazelova et al., 2009). Nevertheless, the role of Arf4 in rhodopsin trafficking
in mouse retinas has been brought into question by monitoring morphology of photoreceptors
in a conditional knockout mouse (Pearring et al., 2017). Using a mouse model system with
low demands on membrane trafficking volumes, the authors reported that the absence of Arf4
caused no mislocalization of rhodopsin as evidenced by the morphological appearance of the
published data. However the data are difficult to interpret as no quantification of rhodopsin
localization was performed, although mild mislocalization was evident. Notably, in the same
mouse, in cells with high volumes of cargo and membrane transiting through the secretory
pathway, such as the exocrine pancreas, the absence of Arf4 caused a major phenotype. The
most likely explanation for the data showing that mouse photoreceptors lacking Arf4 appear
to deliver rhodopsin to the ROS is that the compensatory mechanisms, probably involving
Arf1, which interacts with many of the same proteins (Christis and Munro, 2012), allow the
process to proceed, perhaps at a suboptimal level. Over time, an Arf4 KO retina may prove to
be more susceptible to light damage and other stress leading to slow retinal degeneration, as
Arf4 is also implicated in the signaling pathway mediating Golgi stress response (Reiling et
al., 2013).
There are four issues of significance when comparing the data from the two published Arf4 in
vivo models (i) Absence of a gene vs. a dominant negative action often have different effects
both ex vivo (in cellular models) and in vivo e.g. even retinas of a rhodopsin hypomorph
mouse develop normally, rods elaborate ROS of normal size, and retinas look identical to
controls at P41, whereas comparable expression of a dominant negative mutant affecting
VxPx CTS of rhodopsin causes retinal degeneration modeling ADRP (Concepcion and Chen,
2010; Concepcion et al., 2002; Humphries et al., 1997; Lem et al., 1999; Li et al., 1996). (ii)
The volume of membrane trafficking in the frog eye exceeds by an order of magnitude that of
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the rodent rods. Xenopus and Rana photoreceptors synthesize and transport ~3 µm2 and ~1.5
µm2 of membrane per minute, respectively, vs. 0.1 µm2 synthesized by rodent photoreceptors
(Besharse, 1986). Additionally, due to their larger size, light-sensing membranes in
amphibians contain 6x104 molecules of rhodopsin vs. 2000 molecules of rhodopsin in rats
(iii) Mouse models do not always recapture retinal membrane trafficking disease phenotype
e.g. despite a relatively faithful manifestation of the hearing and balance disorders found in
Usher syndrome, none of the Usher 1 mouse models undergo retinal degeneration (Williams,
2008) (iv) Neither the frog nor mouse models are faithful representations of the human eye,
but are useful when dissecting disease-related processes. The frog, by magnifying the role of
trafficking through its high volumes of membrane and cargo synthesis and vectorial transport,
allows us to dissect the stages and molecular machineries involved. The mouse has its own
advantages, and it would be of interest to follow up on the absence of, or only very mild
morphological change as discussed above, by generating knock-in mouse with dominant
negative mutant to assess the role of Arf4 in this particular model.
The cognate activating Arf GEFs principally control membrane association of Arfs (Bui et
al., 2009; Casanova, 2007; Nawrotek et al., 2016; Stalder and Antonny, 2013), but their
membrane recruitment also involves protein interactions that include SNAREs and the ciliary
cargo (Honda et al., 2005; Mazelova et al., 2009). Initially, Arf1 weakly associates with
membranes through the N-terminal myristoyl group, but GEF activation and GTP binding
cause a conformational transition, termed the “myristoyl switch” that tightly couples Arf1
activation with stable membrane association (Antonny et al., 1997; Franco et al., 1996;
Goldberg, 1998; Pasqualato et al., 2002; Randazzo et al., 1995). Activation of Arf1 by Arf
GEF Sec7 is amplified by the DCB and HUS regulatory domains that form a single compact
helical structural unit, which facilitates membrane insertion of the Arf1 amphipathic N-
terminal helix (Richardson et al., 2016). By contrast, GDP-bound Arf4 and Arf5 stably
associate with membranes independently of GBF1 (Chun et al., 2008). The membrane-
binding properties of Arf4 and Arf5 that differ from those of Arf1 and Arf3 are mediated by
the N-terminal amphipathic helix and a class-specific residue in the conserved interswitch
domain (Duijsings et al., 2009). Our study suggests that unique properties of Arf4 and its N-
terminal amphipathic helix may be responsible for GBF1 DCB-HUS interactions that involve
both GDP and GTP-bound Arf4. If activation and membrane association are uncoupled in
class II Arfs, than multiple, random activation events at the Golgi/TGN can create a small
number of active Arf4 clusters, which, through the operation of an autocatalytic amplification
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mechanism (positive feedback) (Jackson, 2014), may serve to quickly build up levels of
active Arf4 that recognizes and directly binds to the VxPx C-terminal signal of the incoming
ciliary cargo, such as rhodopsin, which leads to the assembly of the ciliary targeting complex
(Mazelova et al., 2009). Similar molecular interactions may be involved in epidermal
differentiation, as Arf4 recognizes the VxPx motif of Presenilin-2 and regulates its
localization to basal bodies/cilia to modulate Notch signaling (Ezratty et al., 2016).
Further studies will be necessary to determine if the GBF1-ASAP1 interaction results in
reciprocal changes in their catalytic activity to modulate the location and the duration of Arf4
signaling. However, they will require a new paradigm: the comprehensive analysis of full-
length Arf-GEFs to reveal aspects of their regulation and functions that cannot be identified
by using isolated domains and truncated proteins as employed thus far. Nevertheless, given
the remarkably high conservation of the GEF and GAP cascades that regulate the ordered
recruitment and activation of small GTPases (Deretic, 2013; Mizuno-Yamasaki et al., 2012;
Stalder and Antonny, 2013), our finding that the prototypical ciliary membrane receptor
rhodopsin may promote its transport from the Golgi to the primary cilium through Arf GEF-
cognate-Arf interaction implies that other membrane cargo may also promote its progression
through the endomembrane system through hierarchical interactions with the highly
conserved functional GTPase networks.
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Materials and methods
Materials—GST DCB-HUS (AA 1-710) and GST Sec7-HDS1 (AA 695-1066) in the pGEX-
4T1 vector (Bouvet et al., 2013), as well as the purified human 17Arf1 were kind gifts of
Cathy Jackson (Institut Jacques Monod, CNRS, Paris). Purified bovine rhodopsin was a gift
of Kris Palczewski (Case Western Reserve University)(Palczewski et al., 2000). Purified Arl1
and anti-Arl1 were kind gifts of Antonio Galindo (MRC LMB, Cambridge)(Galindo et al.,
2016). Recombinant human Arf4 was expressed and purified as described previously (Wang
et al., 2012). Golgicide A (GCA) and cycloheximide were from Sigma-Aldrich. Antibodies
used in this study were: rabbit polyclonal anti-Arf4 (Mazelova et al., 2009); anti-rhodopsin
C-terminus mAb 11D5 (Deretic and Papermaster, 1991) and mAb 1D4 (ab5417, Abcam);
rabbit anti-GBF1 (ab105111, Abcam); mouse monoclonal anti-GBF1 (612116), anti-ASAP1
(612073) and anti-GM130 (610823, BD Biosciences); rabbit polyclonal anti-Rab6 (sc-310,
Santa Cruz Biotechnology); rabbit anti-p115 (13509-1-AP, Proteintech); rabbit anti-ASAP1 a
kind gift of Paul Randazzo (NCI/NIH)(Randazzo et al., 2000); mouse anti-Arf1
(ThermoFisher Scientific), mouse anti-GST (SAB4200237, Sigma); Cy3– and Cy5-
conjugated secondary antibodies (Jackson Immunoresearch) and To-Pro3 (Life
Technologies). Duolink II Rabbit/Mouse Red Kit (excitation: 598 nm; emission: 634 nm) was
from Sigma (DUO92101, Sigma). For some experiments rabbit anti-Rab6 antibody was
directly conjugated to Alexa Fluor 488 using Antibody Labeling Kit (Invitrogen), according
to manufacturers instructions.
Pulse-chase labeling, preparation of the photoreceptor-enriched PNS and retinal
subcellular fractionation—These experiments were performed according to established
procedures (Deretic, 2000; Deretic and Papermaster, 1991; Deretic et al., 1996; Mazelova et
al., 2009; Morel et al., 2000; Wang et al., 2012). Briefly, following 1 hour pulse labeling of 7
isolated frog retinas and 2 hour chase, ROS were removed and pelleted without further
purification (crude ROS); retinal pellets were homogenized and centrifuged to generate the
post-nuclear supernatant (PNS). PNS was centrifuged at 17,500 gav, for 10 min to obtain a
pellet enriched in Golgi/TGN/ER membranes. The supernatant was centrifuged at 336,000 gav
for 30 minutes to separate the RTCs from the cytosol. In some experiments retinas were
incubated with 10 µm GCA or 30 µg/ml cycloheximide for 3 hours, during pulse-chase
labeling.
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Confocal microscopy and Proximity ligation assay (PLA)—Confocal microscopy was
performed on dark-adapted frog retinas as described (Mazelova et al., 2009). In some
experiments isolated eyecups were incubated for 3 hours with 10 µm GCA or 30 µg/ml
cycloheximide. Eyecups were fixed with 4% paraformaldehyde overnight and embedded in
5% agarose. 100 μm sections were cut, permeabilized in 0.3% Triton X-100 and labeled with
specific antibodies as described in figure legends. Antibodies used for confocal microscopy
were: rabbit polyclonal anti-Arf4 (1:400); anti-rhodopsin C-terminus mAb 11D5 (1:400);
rabbit anti-GBF1 (1:200); mouse anti-GBF1 (1:200), mouse anti-ASAP1 and mouse anti-
GM130 (1:200); rabbit polyclonal anti-Rab6 (1:200); rabbit anti-p115 (1:200); rabbit anti-
ASAP1 (1:200). The staining with primary antibodies was followed by Cy3– and Cy5-
conjugated secondary antibodies (1:200). Nuclei were stained with To-Pro3 (1:1000).
Proximity ligation assay (PLA) was performed on fixed retinal sections using Duolink II
Rabbit/Mouse Red Kit, as described previously (Wang et al., 2012). In some experiments
retinal sections labeled with Duolink were stained overnight a 4°C with anti-Rab6 antibody
conjugated to Alexa Fluor 488 (1:100) to highlight Golgi localization. Confocal optical
sections were generated on a Zeiss 800 LSM (Carl Zeiss, Inc). Digital images were prepared
using Adobe Photoshop CS4 (Adobe Systems Inc). Co-localization analysis (Pearson’s
coefficient) was calculated using SlideBook Image Analysis software (Intelligent Imaging
Innovations). To quantify interaction sites detected by PLA in control, GCA or
cycloheximide treated retinas, three separate experiments were conducted, each including the
rhodopsin-Arf4 pair as a positive control (Wang et al., 2012), and over 10 Z-stacks
containing at least 10 confocal optical sections were generated for each PLA pair. From these
Z-stacks, two representative confocal sections, generally from the middle of the stack,
encompassing at least 5 photoreceptors with clearly visible RIS demarcations were selected
from each experiment. Interaction sites were counted (10 photoreceptors × 3 experiments) for
each PLA pair. In control retinas numerous interaction sites were detected in nearly every
photoreceptor, whereas in GCA and cycloheximide treated retinas occasional interaction sites
were detected in less than half of the cells counted, except in the GBF1-ASAP1 pairs.
GST-fusion protein pull-down assay— To analyze direct protein interactions, purified
human proteins Arf4 (Wang et al., 2012), 17Arf1, or Arl1 (5 µg each), were preincubated
with 100 µM GDPS or GTPS in 100 µl of nucleotide loading buffer (25 mM Hepes, pH
7.4, 100 mM NaCl, 0.5 mM MgCl2, 1 mM EDTA, 1 mM ATP and 1 mM DTT) at 30°C for 1
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hr. Purified bovine rhodopsin (functionally equivalent to frog rhodopsin (Deretic et al., 1998),
was in a 1.6 mg/ml solution in a buffer containing 0.5 mM n-dodecyl--maltoside (DDM).
GST and GST-fusion proteins were expressed in Rosetta 2 E.coli cells and direct protein
interactions were analyzed as described previously (Wang et al., 2012). Briefly, GST-fusion
proteins on glutathione-Sepharose 4B beads (50 µl per sample) were washed 2x with PBS.
Immobilized GST-fusion proteins were incubated at RT for 2 hr. in 500 µl reaction buffer (50
mM Hepes, pH 7.4, 150 mM NaCl, 5 mM MgCl2.6H2O, 0.1% Triton X-100, 0.1% BSA, and
1 mM PMSF) with 5 µg each of: Arf4, 17Arf1, Arl1 and/or rhodopsin, as indicated.
Glutathione-Sepharose 4B beads were then washed 8 times with the reaction buffer. Bound
proteins were eluted by 20 µl of 2X SDS-PAGE sample buffer. Protein-protein interactions
were analyzed by SDS–PAGE and Western blotting.
SDS-PAGE and Immunoblotting—Proteins were separated by SDS-PAGE on 4-15% TGX
gels (BioRad). Gels were either dried and exposed to autoradiography, or blotted onto
Immobilon-P membranes (BioRad) and probed with specific antibodies, as indicated. The
antibodies used for western blotting were: rabbit polyclonal anti-Arf4 (1:1000); anti-
rhodopsin C-terminus mAb 11D5 mAb 1D4, and anti-Arl1 (1:500); rabbit anti-GBF1, mouse
monoclonal anti-GBF1, and mouse anti-ASAP1; rabbit anti-ASAP1; mouse monoclonal anti-
GST (1:1000) and anti-Arf1 (1:100). Bound antibodies were detected using a
chemiluminescent Western Lightning immunodetection system (Perkin Elmer Life Sciences).
Because of high retinal tissue requirements, and for more accurate quantification obviating
the need for additional loading controls, immunoblots were cut into strips and multiple
antibodies were tested on the same blot. Before its use on the strips, each antibody was tested
on an entire Western blot of the frog retinal PNS to ascertain its specificity. The distribution
of radiolabeled rhodopsin, or antigens detected by immunobloting, was quantified using
Quantity One 1-D analysis software (BioRad) and expressed in arbitrary O.D. units.
Acknowledgments: We thank Drs. Antonio Galindo, Cathy Jackson, Kris Palczewski and
Paul Randazzo for their generous gifts of reagents. Supported by the NIH grant EY-12421.
UNM Fluorescence Microscopy Facility is supported by NCI and the UNM Cancer Center.
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Competing interests
No competing interests declared.
Funding
National Institutes of Health, National Eye Institute, EY-12421.
Data availability
N/A
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Figures
Figure 1. The Arf GEF GBF1 is localized at the trans-Golgi and interacts with Arf4, the
Arf GAP ASAP1 and the ciliary cargo, rhodopsin. (A-D) Retinas were labeled with rabbit
anti-GBF1 and mouse anti-GM130, followed by Cy3– and Cy5-conjugated secondary
antibodies, followed by rabbit anti-Rab6 conjugated to Alexa Fluor 488. Individual optical
section is shown. (G)=Golgi. Bar=3 µm. GBF1 (red) overlaps with the trans-Golgi marker
Rab6 (green) (arrows), significantly better than with the cis-Golgi marker GM130 (blue), as
per pixel colocalization analysis performed within the Golgi and expressed by the Pearson’s
coefficient (***, p=3.42E-5) (n=5 cells). (E) Scheme of a photoreceptor cell. ROS is an
elaborate primary cilium. Golgi and the TGN are localized in the myoid region (M) of the
RIS. RTCs bud from the TGN and travel to the cilium (arrow), through the ellipsoid region
(E) packed with mitochondria. Adherens junctions (AJ) form the outer limiting membrane
(OLM) throughout the retina. (F) GBF1+Rab6 interaction sites (red dots) detected by PLA
using mouse (m) anti-GBF1 and rabbit (r) anti-Rab6. Following the detection of interaction
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sites by PLA (arrows), sections were subsequently stained with antibody to Rab6 conjugated
to Alexa Fluor 488 (green). Nuclei were stained with TO-PRO-3 (blue). PLA for: (G)
GM130(m)+GBF1(r) and (H) P115(m)+GBF1(r). (I) Golgi staining with Rab6(Alexa Fluor
488), p115(r) and GM130(m). PLA for: (J) GBF1(m)+Arf4(r), (K) Rhodopsin(m)+GBF1(r),
(L) Rhodopsin(m)+Arf4(r), (M) ASAP1(m)+GBF1(r). Bar=5 µm. (N) Red dots were counted
for the PLA pairs shown in panels J-M (30 cells each) in three separate experiments. The
data from a representative experiment were expressed as a percent of total interaction sites
within the RIS, analyzed using Student’s t test (n=30) and presented as the means ± SEM.
(O) PNS (0.1 retina), or T/G/E, RTC and cytosolic fractions (0.25 retina each) were analyzed
by immunoblotting (IB), as indicated. All antibodies were tested on a single blot.
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Figure 2. Golgicide A (GCA), a selective inhibitor of GBF1, significantly disrupts
rhodopsin-GBF1-Arf4-ASAP1 interactions. (A) Rhodopsin(m)+GBF1(r) interaction sites
(red dots) detected by PLA in control retinas. Retinal sections were visualized by DIC. The
same was repeated for: (B) Rhodopsin(m)+Arf4(r), (C) GBF1(m)+Arf4(r), (D)
Rhodopsin(m)+ASAP1(r) and (E) ASAP1(m)+GBF1(r). PLA of GCA-treated retinas for: (F)
Rhodopsin(m)+GBF1(r), (G) Rhodopsin(m)+Arf4(r), (H) GBF1(m)+Arf4(r), (I)
Rhodopsin(m)+ASAP1(r) and (J) ASAP1(m) +GBF1(r). Bar=5 µm. (K-O) Experiments
were repeated three times and over 10 Z-stacks containing at least 10 confocal optical
sections were generated for each PLA pair. From these Z-stacks, two representative confocal
sections encompassing at least 5 photoreceptors with clearly visible RIS demarcations were
selected from each experiment. Interaction sites were counted (10 photoreceptors × 3
experiments) for each PLA pair, analyzed using Student’s t test (n=30) and presented as in
Fig. 1. (***, p<2.2E-6).
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Figure 3. GBF1 regulates Golgi-to-cilia transport of rhodopsin. (A) Control, GCA- and
BFA-treated retinas were labeled with antibody to Rab6(r) (green) and GM130(m) (red).
Bar=5 µm, and 1 µm in insets. (B) Isolated retinas were incubated in the presence or absence
of GCA during the pulse-chase experiment. Following treatment, T/G/E, RTC and ROS
fractions were analyzed by SDS PAGE and autoradiography. (C) Autoradiogram of the gel
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shown in B. (D) Radiolabeled rhodopsin was quantified in three separate experiments. The
data were analyzed using Student’s t test (n=3) and presented as the means ± SEM (*,
p=0.01). (E) Control and GCA-treated retinas were labeled with anti-Rab6 (green), GBF1
(red) and GM130 (blue), as in Fig.1. Rab6 and GBF1 colocalize (arrows) in control and
GCA-treated retinas. Labeling with anti-GBF1(m) and anti-ASAP1(r) in control and GCA-
treated retinas shows Golgi and RTC colocalization (arrows), whereas colocalization detected
with anti-GBF1(m) and anti-Arf4(r) in controls is lost upon GCA treatment (arrows). Bar=5
µm. (F) Following the pulse-chase experiment, subcellular fractions of control or GCA-
treated retinas were separated by SDS-PAGE and immunoblotted as indicated in the figure.
All antibodies were tested on a single blot. ASAP1 detected in the crude ROS fraction
originates from a minor contamination with RIS proteins. (G) The distribution of GBF1, Arf4
and ASAP1 in T/G/E fraction, RTCs and the cytosol was quantified in three separate
experiments and presented as the means ± SEM.
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Figure 4. GBF1 directly interacts with Arf4 and the ciliary cargo, rhodopsin. (A)
Schematic of GBF1. DCB-HUS (AA 1-710) and Sec7-HDS1 (AA 695-1066) are indicated.
(B) GST-DCB-HUS, GST- Sec7-HDS1, or GST were incubated with purified bovine
rhodopsin, with or without recombinant human Arf4 bound to GDPS or GTPS. Rhodopsin
and Arf4 were detected by immunoblotting. The GST fusion proteins were detected with anti-
GST antibody. Arrowheads point to the GST-fusion proteins used in pulldowns. Breakdown
products of GBF1 DCB-HUS were also observed by Galindo, et al., 2016. Sec7-HDS1 is
partially obscured by BSA, present in all samples. Rhodopsin and Arf4 were quantified in
three separate experiments. The data were analyzed using Student’s t test (n=3) and presented
as the means ± SEM (**, p<0.005). (C) Comparable amounts of bovine rhodopsin and human
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Arl1Q71L were subjected to pulldowns by GST-DCB-HUS. Bound proteins and GST-fusion
proteins were detected by specific antibodies. Rhodopsin and Arl1 were quantified in two
separate experiments and presented as the means ± range. (D) GST pulldown of human Arf4,
or 17Arf1, bound to GDPS or GTPS. Bound Arfs and GST-fusion proteins were detected
by immunoblotting, and Arf4 was quantified in three separate experiments and presented as
the means ± SEM.
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Figure 5. Influx of rhodopsin provides a signal crucial to Arf4 interaction with GBF1.
(A) Rhodopsin(m)+GBF1(r) interaction sites detected by PLA in control, or (E)
cycloheximide-treated retinas. PLA was repeated for: (B) and (F) Rhodopsin(m)+Arf4(r), (C)
and (G) GBF1(m)+Arf4(r), (D) and (H) ASAP1(m) +GBF1(r). Bar=5 µm. (I) Interaction
sites were analyzed and presented as in Fig. 2. (***, p<2.0E-13). (J) Isolated retinas were
incubated in the presence or absence of cycloheximide. Following a pulse-chase experiment,
T/G/E, RTCs, ROS and cytosol were analyzed by SDS PAGE. (K) Distribution of Arf4 and
ASAP1 among subcellular fractions in control and cycloheximide-treated cells was
determined by immunoblotting, as indicated. (L) Autoradiogram of the gel shown in J. (M)
localization of GBF1 (red) and Arf4 (green) in control and cycloheximide-treated retinas. (N)
and (O) retinas were labeled with antibody to Rab6(r) (green) and rhodopsin(m) (red).
Arrows indicate Golgi-localized rhodopsin in the control (N), but not in cycloheximide-
treated retinas (O). (Bar=8 µm in M-O; 5 µm in insets in N and O. (P) A diagram
summarizing the apparent sequence of events in ciliary trafficking leading to the formation of
the complex comprising the cargo, the cognate Arf and an Arf GEF: cytosolic Arf4 becomes
membrane-associated and activated, either through random activation events, or by an Arf-
GEF. Through interactions with Rab6, GBF1 is positioned at the trans-Golgi membranes,
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where it associates with rhodopsin and Arf4. This process is inhibited by cycloheximide,
which chiefly blocks the influx of rhodopsin into the endomembrane system. GBF1,
stimulated by the cargo and Arf4 binding to the regulatory DCB-HUS domain, quickly builds
up levels of active Arf4, a process inhibited by GCA. Activated Arf4 recognizes and directly
binds to the VxPx C-terminal signal of incoming rhodopsin, which leads to the assembly of
the ciliary targeting complex, starting with the Arf GAP ASAP1. GTP hydrolysis on Arf4,
catalyzed by ASAP1, releases inactive Arf4 into the cytosol and directs rhodopsin into the
nascent RTCs that contain both ASAP1 and GBF1.
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