Biochemical Society Transactions


Arf family GTPases: roles in membrane traffic and microtubule dynamics

R.A. Kahn, L. Volpicelli-Daley, B. Bowzard, P. Shrivastava-Ranjan, Y. Li, C. Zhou, L. Cunningham


Database mining and phylogenetic analysis of the Arf (ADP-ribosylation factor) superfamily revealed the presence in mammals of at least 22 members, including the six Arfs, two Sars and 14 Arl (Arf-like) proteins. At least six Arf family members were found in very early eukaryotes, including orthologues of Arf, Sar, Arl2, Arl3, Arl6 and Arl8. While roles for Arfs in membrane traffic are well known, those for most of the Arls remain unknown. Depletion in cells of the most closely related human Arf proteins, Arf1–Arf5, reveals specificities among their cellular roles and suggests that they may function in pairs at different steps in endocytic and secretory membrane traffic. In addition, recent results from a number of laboratories suggest that several of the Arl proteins may be involved in different aspects of microtubule-dependent functions. Thus, a second major role for Arf family GTPases, that of regulating microtubules, is emerging. Because membrane traffic is often dependent upon movement of vesicles along microtubules this raises the possibility that these two fundamental functions of Arf family members, regulation of vesicle traffic and microtubule dynamics, diverged from one function of Arfs in the earliest cells that has continued to branch and allow additional levels of regulation.

  • ADP-ribosylation factor (Arf)
  • Arf-like (Arl)
  • cytokinesis
  • Golgi
  • membrane traffic
  • microtubule

Recent phylogenetic analyses of the Arf (ADP-ribosylation factor) family of regulatory GTPases identified 21 members in humans (see Figure 1), with orthologues identified in every eukaryote, including the earliest ones examined [1,2]. With six Arf family members and no orthologues of Ras or the heterotrimeric G-proteins in Giardia lamblia, the Arfs could have been the progenitor of the entire superfamily of regulatory GTPases [13]. It has even been suggested that the emergence of eukaryotes, coincident with the need for intracellular membranes, was intimately tied to the emergence of the Arf and Rab families [4]. Several members of the Arf family are essential [2,5,6] and very highly conserved proteins. The Arf family has continued to expand, with several members found only in metazoans. What are the functions of the Arf family members and to what extent have their biochemical mechanisms been conserved? What drives the expansion of the Arf family? Has the expansion of the Arf family resulted in an increasing number of cell functions regulated by this family or has it allowed an increasingly complex level of regulation around one or two fundamental cell function(s)? These are all questions that will be the focus of studies in the coming years. In this short review, we will summarize recent results that address issues of specificity among the Arf group of proteins and a second central role for Arf family GTPases, that as regulators of microtubule-dependent processes by several Arl (Arf-like) proteins.

Figure 1 Phylogram of the 21 human members (plus mouse Arf2) of the Arf family GTPases

Check Table 1 for issues regarding nomenclature. This Figure was constructed with the help of the CLUSTAL W program.

View this table:
Table 1 The Arf family GTPases: summary of names, identifiers and N-terminal sequences

Information is given for the human members of the Arf family, as well as four likely pseudogenes. Efforts are under way to clean up the nomenclature in the Arf family and should soon result in a consensus. HGNC, HUGO Gene Nomenclature Committee.

Specificity among the Arfs

The Arfs (human Arf1, Arf3–Arf6; Arf2 has been lost in humans but not other mammals) were the first members of the family discovered [710] and they all share the ability to regulate the budding and formation of vesicles in the endocytic and exocytic pathways. They are thought to accomplish this through the recruitment to membranes of adaptor proteins or complexes and through localized changes in lipid metabolism (including the activation of phospholipase D and phosphoinositide kinases [1114]). The number of coat proteins or complexes that are recruited by the soluble Arfs (Arf1–Arf5) to membranes of the early secretory pathway in a GTP-dependent manner has grown to at least 10 {COPI [coatamer protein I], the adaptin complexes AP-1, AP-3 and AP-4, GGA1–3 (Golgi-associated γ-adaptin ear homology domain Arf-interacting protein 1–3) and MINT1–3 [Munc18-interacting protein 1–3]}. Because there are already more coats than Arfs, there is uncertainty as to the source of specificity among the five soluble Arfs, which are thought to exist in soluble pools that cycle on and off membranes in concert with the binding and hydrolysis of GTP respectively. In addition, in vitro assays of coat protein recruitment to membranes or two-hybrid assays of Arf–coat protein interactions have displayed little or no specificity between the Arfs.

In efforts to determine specific roles for the different Arf isoforms in membrane traffic, we constructed at least two non-overlapping siRNA (small interfering RNA) plasmids that effectively depleted human cells of Arf1, Arf3, Arf4 or Arf5. We found no effect of any of the single knockdowns [15]. However, double knockdowns yielded a number of phenotypes that reveals specificity between the Arfs in effects on Golgi and endosome morphology and secretory traffic and endosome recycling. Specifically, Arf1 and Arf4 were required for the integrity of the Golgi structure, association of COPI with the Golgi, and exit of secreted proteins from the ER/VTCs (where ER stands for endoplasmic reticulum and VTC for tubulo-vesicular cluster). Arf1 and Arf3 in combination were required for traffic from VTCs/ERGIC (ER–Golgi intermediate compartment) to the cis-Golgi. The combined depletions of Arf1+Arf3 or Arf1+Arf5 also impaired retrograde traffic from VTCs back to the ER, while decreased expression of Arf4+Arf5 or, to a lesser extent, Arf3+Arf4 impaired retrograde traffic at the level of the cis-Golgi. In addition, each paired combination of Arf knockdowns, with the exception of Arf1+Arf4, impaired recycling of the TfnR (transferrin receptor) from early endosomes back to the plasma membrane. The most important observation from this study could turn out to be the apparent need to knock down two Arfs before functional consequences ensue. This could relate mechanistically to recent results suggesting functions for Arl1 and Arfrp in a common pathway [16,17]. It is possibly a common theme in Arf family signalling to couple two or more Arf family GTPases in one pathway.

New functions for old Arls

As indicated above, at least six Arf family members were present at very early points in eukaryotic evolution: Arf, Sar, Arl2, Arl3, Arl6 and Arl8. Arfs and Sars have well-defined roles in vesicle budding and membrane traffic. Arl2 is a regulator of tubulin folding and microtubule integrity and perhaps dynamics [1823], suggesting that the Arf family includes proteins with functions different from membrane traffic. We are almost completely ignorant of the functions and activities associated with the other oldest Arls: Arl3, Arl6 and Arl8. However, emerging results suggest that perhaps each of these GTPases are involved in the regulation of some aspect of microtubule function: including potential roles in mitosis, fidelity of chromosome segregation and cytokinesis.

Faithful segregation of the genome and essential cellular components into daughter cells is of paramount importance to every cell and in metazoans defects in this process may lead to apoptosis, aneuploidy, oncogenesis or other life-threatening diseases [24]. The centrosome is a key regulator of several processes that are intimately interconnected and are required for cell division [25]. Duplication and segregation of centrosomes is the prelude to the formation of the mitotic spindle. Segregation of the chromosomes is followed by cytokinesis and the collapse of the spindle into a mid-body, a dense structure consisting of microtubules from the spindle midzone with associated material that has been condensed by the cytokinetic furrow.

In addition, a centriole from a centrosome is required to form a basal body, from which cilia develop [26]. Cilia are thought to function predominantly in sensory transduction; in retinal rod cells they are the sight of photon capture, in cells of the renal collecting duct they are flow sensors, and in olfactory epithelial cells they concentrate olfactory receptors. But there is increasing evidence that the sensory roles for cilia, and in particular the primary cilium, may be linked to the regulation of cell division [27]. Mutations in a number of genes are now linked to the family of ‘cilia-related diseases’ that include polycystic kidney disease, primary cilia dyskinesia, Jeune syndrome and BBS (Bardet–Biedl syndrome) [28]. That functional interconnections between cilia, centrosomes, microtubule organization and cell division exist has become clear in the last few years, but we lack a lot of mechanistic details. Additional evidence for signalling between cilia/flagella and the cell cycle is summarized in recent reviews [28,29].

Arl2 and Arl3

Each arose very early in eukaryotic evolution and they have maintained a closer relationship with each other compared with other Arls in that they share structural, biochemical and functional (e.g. some shared effectors) characteristics [30,31]. Despite these similarities, Arl2 has emerged from several genetic screens for microtubule-related genes [1921,32] but Arl3 never has. Recent results from my laboratory revealed that Arl2, Arl3, and their shared effector Bart each localize to centrosomes, Arl3 and Bart are also found at mid-bodies, and depletion of Arl3 by siRNA in HeLa cells results in failure of cytokinesis (C. Zhou and R.A. Kahn, unpublished work). Thus Arl3 also appears functionally close to microtubule-related processes, although we do not yet understand its role or mechanism.


Was first identified in a search for proteins involved in erythropoietin-induced differentiation of erythroid cells [33], although it is very widely expressed in human tissues. More recently, Arl6 was identified as the protein encoded by one of the eight genes linked to BBS, specifically BBS3 [34,35]. BBS is a genetically heterogeneous, pleiotrophic disorder characterized by obesity, mental retardation, blindness, polydactyly, and malformation of the kidney and heart [36]. Fan et al. [34] also showed that the worm orthologue of Arl6 is expressed in sensory neurons and is proposed to traffic along cilia by intraflagellar transport (IFT). A role for Arl6 in ciliary functions has also been suggested by two comparative genomic searches [37,38]. These searches identified a small set (14 genes in one case) of genes implicated in ciliogenesis or ciliary function that included both Arl3 and Arl6. Together, these results point to a role for Arl6 in cilia or flagella function. The presence of Arl6 in flagella was confirmed in a study of the flagellar proteome of the biflagellated green alga Chlamydomonas, which found Arl6 and Arl3 [39].


The two human Arl8 paralogues share 92% identity and were cloned as a result of large-scale sequencing, either as ESTs (expressed sequence tags) or as random clones [40,41]. Arl8 orthologues are very highly conserved in eukaryotic evolution, e.g. with single orthologues in flies, worms and G. lamblia [2,41]. Okai et al. [42] found the two mammalian paralogues by homology searches of databases and named them Gie1 (Arl8B) and Gie 2 (Arl8A). They argue that Gie/Arl8 plays roles in microtubule-related functions based upon co-immunoprecipitation of Arl8 with β-tubulin, staining of mid-bodies with affinity-purified Arl8 peptide antibodies, and defects in chromosome segregation in cultured fly cells upon knockdown of Gie/Arl8 by siRNA.

Together, these results argue strongly for roles for Arf family members (at least Arl2, Arl3, Arl6 and Arl8) in these essential and microtubule-dependent cellular processes. Interestingly, there is also a growing appreciation of the fact that vesicle traffic is also required for cytokinesis [43,44] and many Golgi and traffic proteins are found in mid-bodies [43]. Thus whether these newly proposed roles for Arf family members at centrosomes and in cytokinesis are truly independent of the better-known roles for Arfs and Arl1 in membrane traffic remains to be discovered.

I propose that members of the Arf family are involved in at least two central cellular functions: (i) membrane traffic (Arf1-6, Arl1, Arl5, Arfrp and Sars) and (ii) microtubule-related functions (Arl2, Arl3, Arl6 and Arl8). Although each member of the Arf family appears to display unique properties and functions, it is also proposed that the use of two or more Arf family members acting in common pathways may be common and would provide exquisite control of these primordial and essential processes.


  • Cell Architecture: from Structure to Function: A Focus Topic at BioScience2005, held at SECC Glasgow, U.K., 17–21 July 2005. Edited by S. Cockroft (University College London, U.K.), Y. Goda (University College London, U.K.), R. Insall (Birmingham, U.K.) and M. Wakelam (Birmingham, U.K.).

Abbreviations: Arf, ADP-ribosylation factor; Arl, Arf-like; BBS, Bardet–Biedl syndrome; ER, endoplasmic reticulum; EST, expressed sequence tag; siRNA, small interfering RNA; VTC, tubulo-vesicular cluster


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