© 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: dderetic@salud.unm.edu

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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