Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA
* Author for correspondence (e-mail: hgoodson{at}nd.edu )
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Summary |
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The actin family |
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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.
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Phylogenetic analysis |
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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
. 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.
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Functional predictions |
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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.
Methods
The public protein and nucleic acid databases (November 2001) were scanned
for actin-related proteins using either PSI-BLAST (protein databases) or
tBLASTn (nucleotide databases) (Altschul et
al., 1990; Altschul et al.,
1997
). After initial sequence collection, databases were probed
with individual yeast ARP sequences to enhance the chances of finding
sequences related to these divergent proteins; all `orphan' ARPs were also
used to individually probe the databases. A final set of sequences was
obtained by choosing only those sequences <95% identical and including only
one sequence from organisms with multiple conventional actins. Sequences were
aligned using ClustalX (Thompson et al.,
1997
) with default alignment parameters. Adjustments were made in
the resulting initial alignment by asking ClustalX to realign specified
sequences (across the entire length) or regions (all sequences were realigned
in the specified region), resulting in an otherwise good alignment that
contained unnecessary gaps. The final alignment was obtained by realigning the
adjusted alignment after resetting the gaps (in this procedure the guide tree
is calculated before the gaps are removed).
Phylogenetic analysis was performed on the conserved core of this alignment
(corresponding to residues of human ß-actin) by the neighbor-joining
algorithm of ClustalX (Thompson et al.,
1997) using default parameters (gapped regions were included).
Bootstrap analysis (1000 trials) provided a measure of confidence for the
detected relationships as described above. The resulting tree was graphed by
the program `Unrooted' provided with ClustalX, and was prepared for
presentation by Adobe Illustrator 7.0 (it should be noted that Illustrator 7.0
handles the pict file output from the tree graphing program much better than
does Illustrator 9.0). Phylogenetic analysis was also performed using the
neighbor-joining algorithm as implemented by the PHYLIP package [J.
Felsenstein, PHYLIP (Phylogeny Inference Package) version 3.5c, Department of
Genetics, University of Washington, Seattle, 1993]. Distance measurements were
based on the PAM250 matrix instead of an identity matrix, and sequence
addition was randomized to control for additional order bias. No significant
changes in the topology of the tree or bootstrap values were observed.
Sequence references: GenBank gi numbers for the protein sequences used are as follows and are listed by subfamily. It should be noted that unannotated ARP sequences are designated by their chromosomal locus and/or gi number both in this list and on the tree. Conventional actin: Gl Actin, gi1703155; DmArp53d, gi7302881; Tg Actin, gi1703160; Pf Actin, gi5911379; Eh Actin, gi113294; Sp Actin, gi113303; Sc Actin, gi170986; AtActin, gi6598382 (one of several At actin genes); Dd actin, gi4093161 (one of several Dd actin genes). Arp1: SpArp1, gi7490069; ScArp1, gi6321921; NcArp1, gi728797; AnArp1, gi4731565; CeY53F4B.22, gi17537473; DmArp87C, gi1168334; MmArp1b, gi18606465; HsArp1b, gi11342680; MmArp1a, gi8392847; HsArp1a, gi625520. Arp2: At Arp2, gi3818624; Sp Arp2, gi6650375; Sc Arp2, gi6320175; Ac Arp2, gi1703144; Dd Arp2, gi4093161; Ce K07C5.1, gi7505422; DmArp14D, gi1168330; GgArp2, gi806554; HsArp2, gi5031571. Arp3: AtArp3, gi4850401; ScArp3, gi6322525; NcArp3, gi11276973; SpArp3, gi416581; AcArp3, gi703143; DdArp3, gi1168328; CeY71F9AL.16, gi7105615; Dmactin66b, gi168329; HsArp3b, gi9966913; Mm 12835802, gi12835802; Hs Arp3a, gi5031573; Arp4: Sc Arp4, gi6322380; Sp P23A10.08, gi11276974; Sp C23D3.09, gi1351610; At 18394608, gi18394608; Ce ZK616.4, gi7332261; Dm CG6546, gi7302793; Hs BAF53b (also called `Arp6'), gi 7705294; Mm BAF53a, gi4001805; Hs BAF53a, gi4757718. Arp5: At 12321978, gi12321978; Os 13486900, gi13486900; HsArp5, gi13396318; Dm CG7940, gi7300345; ScArp5, gi6324269; SpBC365.10, gi7490072. Arp6: Scarp6, gi6323114; At 6091748, gi6091748; CeARP6, gi14916971; SpCC550.12, gi7490073; Dmactin 13E, gi1168327; GgArpX, gi12082091; Mm 12842577, gi12842577; Hs ArpX, gi12082089. Arp8: At 8843903, gi8843903; Sc Arp8, gi6324715; Sp C664.02, gi692009; Dm CG7846, gi7293397; Mm 12857259, gi12857259; Hs 10434709, gi10434709. Arp10: Dm CG12235, gi7293622; Hs Arp11, gi8923712; Mm Arp11, gi6176554; Ce C49H3.8, gi7497696; Sp C56F2, gi3116133; Sc Arp10, gi6320311; NcRo7, gi8347739. Orphans (listed by group): Hs 13383265, gi13383265; Mm 12840619, gi12840619; Mm 12840134, gi12840134; Mm 13386316, gi13386316; /Hs 11137605, gi11137605; /At 11276982, gi11276982; /Mm 12838437, gi12838437; Hs 10178893, gi10178893; Mm Actlike7a, gi6752956; Hs Actlike7a, gi5729720; Mm Actlike7b, gi6580806; Hs Actlike7b, gi5729722; /ScArp7, gi6325291; /ScArp9, gi6323676; Sp C1071.06, 7490070; /CeF42C5.9, 17540400.
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Acknowledgments |
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Footnotes |
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Nomenclature of actin-related proteins (ARPs) was originally defined by the
degree of relatedness to actin itself, with increasing numbers referring to
increasingly divergent actins (Schroer et
al., 1994
). However, this approach leads to ambiguities since many
ARPs have similar levels of divergence. We propose that ARP subfamilies be
named by the S. cerevisiae genes included in them, and that otherwise
uncharacterized ARPs be given names based on the subfamily to which they
belong. This approach is consistent with most of the established nomenclature
and allows unambiguous naming of most uncharacterized sequences. To avoid
future ambiguity, we suggest that ARPs that do not yet group into one of the
defined subfamilies be given alternative ARP names (for example, based on
functional characteristics or chromosomal loci) until further analyses clarify
the evolutionary relationships. The poster uses previously established gene
names where they exist, and either chromosomal loci/gi numbers where they do
not.
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