Membrane recruitment of effector proteins by Arf and Rab GTPases Masato Kawasaki1, Kazuhisa Nakayama2 and Soichi Wakatsuki1 In their GTP-bound form, Arf and Rab family GTPases associate with distinct organelle membranes, to which they recruit specific sets of effector proteins that regulate vesicular transport. The Arf GTPases are involved in the formation of coated carrier vesicles by recruiting coat proteins. On the other hand, the Rab GTPases are involved in the tethering, docking and fusion of transport vesicles with target organelles, acting in concert with the tethering and fusion machineries. Recent structural studies of the Arf1���GGA and Rab5���Rabaptin-5 complexes, as well as other effector structures in complex with the Arf and Rab GTPases, have shed light on the mechanisms underlying the GTP-dependent membrane recruitment of these effector proteins. Addresses 1 Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), Tsukuba, Ibaraki 305-0801, Japan 2 Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshida-shimoadachi, Sakyo-ku, Kyoto 606-8501, Japan Corresponding author: Wakatsuki, Soichi (soichi.wakatsuki@kek.jp) Current Opinion in Structural Biology 2005, 15:681���689 This review comes from a themed issue on Proteins Edited by Edward N Baker and Guy G Dodson Available online 9th November 2005 0959-440X/$ ��� see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2005.10.015 Introduction Intracellular transport of proteins and lipids between membrane-bound organelles is mediated mainly by coated carrier vesicles. Three types of coated vesicles have been well characterized to date: COPI-, COPII- and clathrin-coated vesicles [1]. COPI- and COPII-coated vesicles are involved in retrograde and anterograde trans- port processes, respectively, between the endoplasmic reticulum (ER) and the Golgi apparatus. Clathrin-coated vesicles (CCVs) mediate diverse transport steps between the trans-Golgi network (TGN) and the plasma mem- brane. The coat of CCVs is composed of clathrin and adaptor proteins [2]. Adaptor protein (AP) complexes (AP-1, AP-2, AP-3 and AP-4) are heterotetrameric, whereas GGAs (Golgi-localizing, g-adaptin ear domain homology, Arf-binding proteins) are monomeric adaptors. ADP-ribosylation factor (Arf) family GTPases are crucial for assembling coat proteins during vesicle formation [3]. Arf1 recruits the COPI coat to the Golgi membranes, whereas a distantly related GTPase, Sar1, recruits the COPII coat to the ER membrane. Arfs also regulate the recruitment of clathrin adaptor proteins, AP-1, AP-3, AP-4 and GGAs, to the TGN and/or endosomes. Exception- ally, AP-2 is recruited to the plasma membrane through interaction with phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2]. Arf has a myristoylated amphipathic helix at its N terminus, which is folded into the molecule in its GDP-bound state. However, exchange of GDP for GTP on Arf, which is catalyzed by guanine nucleotide exchange factors (GEFs), exposes the myristoylated N- terminal helix for membrane anchoring. GDP/GTP exchange also causes a dramatic change in the conforma- tion of the switch 1 and switch 2 regions of the GTPases, enabling only GTP-bound GTPases to bind specific sets of effector proteins. Thus, Arf GTP recruits coat proteins to the membrane and, in turn, promotes vesicle budding. Upon GTP hydrolysis, which is stimulated by GTPase- activating proteins (GAPs), Arf then retracts its myristoy- lated N terminus and dissociates from the membrane. This step underlies the shedding of the coat from the vesicles before they fuse with target membranes. Rab GTPases, on the other hand, mediate the tethering of transport vesicles to target membranes [4]. The teth- ered vesicles are then docked by the specific pairing of the SNARE proteins on the vesicle and target mem- branes, and finally the vesicle membrane fuses with the target membrane. In contrast to Arfs, Rab proteins undergo isoprenylation at the C terminus for membrane anchoring. The GTP-bound Rab GTPases are also recruited to membranes and interact with their specific effectors, which in turn regulate the vesicular tethering/ fusion events. Membrane recruitment of effectors by these GTPases is therefore crucial to the regulation of vesicular transport events. We here review recent structural analyses of Arf and Rab GTPases in complex with their effectors, which revealed divergent but common mechanisms of their membrane recruitment. In most cases, the GTPase-bind- ing regions of the effectors are composed of two a helices, which are aligned along the interswitch b strands between switch 1 and switch 2 of the GTPases, allowing their strict interaction with the two switch regions only in the GTP- bound state. We also highlight the GGA���Rabaptin-5 interaction as an example of crosstalk between the Arf and Rab pathways. www.sciencedirect.com Current Opinion in Structural Biology 2005, 15:681���689

Arf/Arl���effector complexes Arf1���N-GAT complex GGAs (three isoforms in human and two in yeast) are monomeric clathrin adaptor proteins involved in the selective transport of lysosomal cargo receptors from the TGN to endosomes [5,6]. GGAs consist of four functional regions: an N-terminal VHS (Vps27/Hrs/Stam) domain a GAT (GGA and Tom1) domain: a hinge region and a C-terminal GAE (g-adaptin ear) domain. The GAT domain of GGAs has been shown to bind Arf1, which is responsible for targeting GGAs to the TGN membrane. In 2003, four groups, including our own, reported the structure of the GGA1-GAT domain in the unbound form [7 ,8,9,10 ]. Two of the reported GAT structures are composed of four helices [7 ,8], whereas the other two structures contain only three, lacking the N-terminal short helix of the other structures [9,10 ] (Figure 1). The four-helix GAT structures consist of two indepen- dent subdomains: N-terminal helix-loop-helix (N-GAT) and C-terminal three-helix bundle (C-GAT). In the three-helix crystal structures, the N-GAT subdomain is disordered, while the C-GAT subdomain is intact. Muta- tional studies suggested that the N-GAT subdomain solely participates in Arf binding. Although Tom1 (target of Myb1) and Tom1L1 (Tom1-like 1) also have a GAT domain, neither binds Arf because they do not have the N-GAT subdomain. Crystallization of the complex between Arf1 and the GAT domain was unsuccessful until the C-GAT subdo- main was removed from the GAT construct [10 ]. The Arf1���N-GAT complex structure revealed that N-GAT, which was unstructured in the three-helix structures of free GAT, adopts a helix-loop-helix structure similar to that seen in the four-helix structures of free GAT. These structures and circular dichroism (CD) data suggest that N-GAT is in equilibrium between unfolded and folded states, and is stabilized in the helix-loop-helix structure by Arf binding [10 ]. The Arf-binding surface of N-GAT is on one side of helices a0 and a1, and is predominantly hydrophobic with several polar residues. The two helices of N-GAT are positioned against the antiparallel b sheet of the Arf interswitch. Helix a0 of N-GAT interacts mainly with switch 2 of Arf1, whereas a1 interacts with switch 1 (Figure 2a). Ile197 of N-GAT, located near the well-conserved Gly50 of switch 1, is crucial to Arf1 binding, because it interacts with both switch regions (Figure 2a). Given that the N terminus of Arf1 GTP is membrane anchored and that helix a1 of N-GAT con- tinues to a1 of C-GAT (Figure 1), the long a1 helix of the GAT domain would protrude into the cytosol (Figure 3). The orientation of helix a1 would probably be perpen- dicular to rather than along the negatively charged mem- brane surface, because the C-GAT subdomain is predominantly negatively charged. Arf6���CTA1 complex Arf was first discovered as a cofactor of cholera-toxin- dependent ADP-ribosylation of the a subunit of the heterotrimeric Gs protein (hence designated ADP-ribo- sylation factor). The ADP-ribosyltransferase activity of the cholera toxin A1 subunit (CTA1) is allosterically increased by Arf binding. A very recent report describes an Arf6���CTA1 complex structure, in which the Arf-bind- ing site of CTA1 consists of loop regions with little secondary structure [11]. CTA1 interacts with the switch and interswitch regions of Arf6, with Asn93 located between the two switches (Figure 2b). This structure suggests that a rigid tertiary structure is not a prerequisite for recognizing Arf GTP thus, CTA1 can pretend to be an Arf effector. Although the Arf-binding site of CTA1 is distant from the enzymatic active site, the conformation of the activation loop changes from a coil to an amphi- pathic helix upon Arf binding, resulting in the opening of the active site to bind the substrate NAD+ [11]. Arl1���GRIP complex Arf-like (Arl) GTPases are related to Arfs, with 40���60% sequence identity [12]. Through its N-terminal myristoyl moiety, Arl1 is anchored to the Golgi membranes, to which it recruits golgins. Golgins are a family of Golgi- 682 Proteins Figure 1 Comparison of the four crystal structures of the GGA1-GAT domain. The C-GAT subdomains of the free GGA1-GAT structures are superimposed: blue [7 ], green [8], yellow [9] and red [10 ]. The Arf1���N-GAT complex [10 ] (N-GAT is shown as an orange line and Arf1 is drawn as a light-blue ribbon diagram, with switch 1 highlighted in light green and switch 2 in light red GTP and Mg2+ are shown as ball-and-stick models) is superimposed on the N-GAT subdomains of the two four-helix structures (blue [7 ] and green [8]). Current Opinion in Structural Biology 2005, 15:681���689 www.sciencedirect.com