The actin family

The actin family is a diverse and evolutionarily ancient group of proteins. Conventional actin appears to be ubiquitous in eukaryotes and, recently, cell-division proteins bearing a striking structural similarity to actin have been identified in eubacteria ( van den Ent et al., 2001). Conventional actin is one of the principal components of the eukaryotic cytoskeleton, and it has a central role in cellular processes ranging from cell motility to intracellular transport and cell organization. In the early 1990s, researchers realized that most, if not all, eukaryotic cells also contain actin-related proteins, or `ARPs'. Some of these ARPs have well-characterized roles in cytoskeletal functions, including actin polymerization (ARP2/3) and dynein motor activity (ARP1) (reviewed by Machesky and May, 2001; Schafer and Schroer, 1999). In addition, both conventional actin and specific ARPs have been strongly implicated in the initially surprising functions of chromatin remodeling and/or transcription regulation ( Machesky and May, 2001; Schafer and Schroer, 1999; Sheterline et al., 1998). ⇓

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The ARP proteins have been named on the basis of their similarity to conventional actin ( Schroer et al., 1994). Members of the ARP1, ARP2, ARP3, and conventional actin subfamilies have been found in organisms ranging from humans to fungi. Additional ARPs that do not fall into these subfamilies have been identified in a number of organisms, but the relationship between these proteins has thus far been unclear ( Machesky and May, 2001; Schafer and Schroer, 1999). Moreover, genomesequencing projects have produced a large number of new ARP sequences. Some are closely related to characterized proteins, but many are not. What are the functions of these novel ARPs? One of the best ways to provide a necessary set of initial hypotheses is phylogenetic analysis.

Phylogenetic analysis

Phylogenetic analysis attempts to reconstruct evolutionary relationships between proteins by studying sequence relationships. These evolutionary relationships contain a wealth of functional information. For example, protein subfamilies that predate the divergence of animals and fungi probably act in fundamental cellular processes, given that they apparently existed in the common ancestor and were important enough to be retained for the billion or so years separating these organisms. By contrast, proteins specific to particular phyla, orders or species appeared relatively recently and are likely to have more specialized functions. Defining evolutionary relationships also allows a researcher to gauge the likelihood that an uncharacterized protein has functions similar to those of a particular characterized protein. For example, proteins that are `orthologs' (proteins related by species divergence) are likely to have similar functions, while `paralogs' (proteins related by gene duplication) are more likely to have functions that have themselves diverged.

To address these questions as they relate to the actin superfamily, we have performed a systematic phylogenetic analysis of actin-related proteins in all fully sequenced organisms, specifically Homo sapiens, Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae and Arabidopsis thaliana. Sequences from mouse, Schizosaccharomyces pombe and selected additional organisms were included to help define branch points. The poster shows the resulting unrooted phylogenetic tree (neighbor-joining method as implemented by ClustalX, bootstrapped 1000-times; to avoid crowding, some closely related ARPs were not included).

Examination of the topology of this tree and the confidence estimates provided by the bootstrapping analysis ^† reveals that the actin superfamily contains at least eight subfamilies that have been conserved from humans to yeast (see poster and Table 1; we define groups as `subfamilies' if they contain sequences from divergent organisms and are found in >90% of bootstrap trials). Consistent with previous studies ( Poch and Winsor, 1997; Schroer et al., 1994), we propose that these subfamilies be named by the yeast ARP contained in them <sup>‡</sup>. By this convention, the conserved actin subfamilies are: conventional actin, Arp1, Arp2, Arp3, Arp4, Arp5, Arp6 and Arp8. Experimental evidence supports the existence of an additional subfamily conserved from humans to fungi (Arp10), although bootstrapping support for this group is weak (<50%). Five of the subfamilies (Arp4, Arp5, Arp6, Arp8, Arp10) have not been rigorously defined before, although homologies between some Drosophila, mammalian and fungal proteins have previously been recognized ( Eckley et al., 1999; Kato et al., 2001; Lee et al., 2001). Most subfamilies contain at least one protein that has been at least partially characterized in mammalian cells, but no members of the Arp5 or Arp8 subfamilies have been identified outside of yeast, except as `hypothetical proteins'. All subfamilies except Arp1 and Arp10 have recognizable members in the Arabidopsis genome. A number of organisms, particularly mammals, possess additional `orphan' ARPs that did not group into any of these subfamilies.

Functional predictions

The existence of members of ARP subfamilies in widely divergent organisms indicates that these proteins are ancient and suggests that they have conserved roles in fundamental aspects of cell biology. It is interesting and perhaps surprising to note that the existing experimental evidence implicates the characterized members of most of the novel ARP subfamilies in nuclear functions ( Table 1). The sum of this data suggests that two of these conserved ARP subfamilies have a role in actin polymerization (Arp2, Arp3), two have a role in dynein motor function (Arp1 and the tentative subfamily Arp10), and four appear to act in the nucleus, particularly in chromatin remodeling (Arp4, Arp5, Arp6, Arp8). Actin itself has been found to be a stable stoichiometric component of several chromatin remodeling complexes (see references in Table 1), which suggests that actin's participation in chromatin remodeling predates the gene duplications leading to the chromatin remodeling ARP subfamilies. While most of the `orphan' ARPs have unknown functions, the S. cerevisiae proteins ARP7 and ARP9 are well-characterized members of chromatin remodeling complexes ( Cairns et al., 1998; Peterson et al., 1998). It is possible that some of the orphan proteins are pseudogenes, but all of the human orphans have at least five `hits' in the human expressed sequence tag (EST) database, and some have more than 100 hits (H.V.G. and W.F.H., unpublished). As is true for actin, it is possible that some ARPs have multiple functions. For example, recent proteomic analysis of the nucleolus has suggested that Arp2, Arp3 and actin itself may be part of this structure ( Andersen et al., 2002).

It is interesting to note that Arp1 and Arp10 are not obviously present in the Arabidopsis genome. Are members of these subfamilies missing, unsequenced, or just unrecognizable in flowering plants? Given that Arabidopsis also appears to lack cytoplasmic dynein ( Lawrence et al., 2001), and that dynactin is an activator of cytoplasmic dynein, it is tempting to speculate that plants lack dynactin ( Lawrence et al., 2001) and therefore lack the dynactin-associated ARPs (Arp1, Arp10).

Both actin and ARPs have previously been implicated in nuclear activities (reviewed by Machesky and May, 2001; Schafer and Schroer, 1999; Sheterline et al., 1998), although most attention by the cell biology community has focused on cytoskeletal functions of actin and ARPs. Our phylogenetic analysis shows that representatives of four apparently nuclear ARP subfamilies exist in organisms as divergent as humans, yeast and plants, and suggests that these ARPs and actin itself play ancient, fundamental and under-appreciated roles in the nucleus.