Are plant formins integral membrane proteins?
© GenomeBiology.com 2000
Received: 2 November 1999
Accepted: 28 January 2000
Published: 27 April 2000
The formin family of proteins has been implicated in signaling pathways of cellular morphogenesis in both animals and fungi; in the latter case, at least, they participate in communication between the actin cytoskeleton and the cell surface. Nevertheless, they appear to be cytoplasmic or nuclear proteins, and it is not clear whether they communicate with the plasma membrane, and if so, how. Because nothing is known about formin function in plants, I performed a systematic search for putative Arabidopsis thaliana formin homologs.
I found eight putative formin-coding genes in the publicly available part of the Arabidopsis genome sequence and analyzed their predicted protein sequences. Surprisingly, some of them lack parts of the conserved formin-homology 2 (FH2) domain and the majority of them seem to have signal sequences and putative transmembrane segments that are not found in yeast or animals formins.
Plant formins define a distinct subfamily. The presence in most Arabidopsis formins of sequence motifs typical or transmembrane proteins suggests a mechanism of membrane attachment that may be specific to plant formins, and indicates an unexpected evolutionary flexibility of the conserved formin domain.
Some mechanisms involved in cell morphogenesis, such as membrane vesicle transport, are conserved at least among crown eukaryotes (metazoa, fungi and plants) [1,2], whereas others, such as those involving extracellular structures or the precise roles of different Rho-like GTPases , are not. Yet other cellular processes, such as cytokinesis, often recruit conserved proteins to accomplish superficially dissimilar tasks (for example, budding, cleavage or phragmoplast-based cell division of plant cells) . For many morphogenetic mechanisms, the question of evolutionary conservation remains unresolved because available information is limited to one or a few model organisms. For example, this is the case for the molecular mechanisms that ensure the communication between the cytoskeleton and the surface of the cell. However, the recent increase in the data available from a number of genome projects allows wide-ranging searches for homologs of known components of signaling and morphogenetic pathways. The results of such searches can lead both to experimentally testable hypotheses and to general conclusions regarding the evolution of morphogenetic processes.
Formins, also known as formin homology (FH) proteins, are proteins implicated in cellular and organismal morphogenesis of both metazoa and fungi. On the cellular level, they are involved in the establishment and maintenance of cell and/or tissue polarity [5,6], in cytokinesis  and in the positioning of the mitotic spindle . They interact directly or indirectly with actin, profilin, Rho-like GTPases [5,6,8,9,10,11], the yeast Spa2 protein and septins [12,13], proteins containing SH3 or WW domains [10,14], dynein and microtubules [7,15,16,17]. The yeast formin homolog encoded by BNI1 is localized to the cell periphery and participates in positioning cortical actin patches towards distinct regions of the plasma membrane [5,13,18]. Some kind of contact with the plasmalemma (in addition to that mediated by a Rho-like GTPase) might therefore be expected, although there is no evidence as yet for such a contact. Furthermore, metazoan formins are believed to be cytoplasmic or nuclear proteins [19,20].
Nothing is known about formin function in plants, although the existence of two Arabidopsis thaliana proteins containing the conserved formin-homology 2 (FH2) domain has been reported recently [6,10]. Given that all known formins represent a well-defined family, this class of proteins may be a good candidate for a systematic genome sequence search. Here, I present the results of such an approach, which has led to the identification of putative plant formin genes, as well as to the finding that the evolutionarily old formin domain may be used in a number of different ways and contexts ('modules' as defined by Hartwell et al. ) by recent eukaryotes.
Results and discussion
Putative formin-related genes of Arabidopsis thaliana
47 640...48 637;
AtORF2 in 
48 716...50 000
28 161...26 738;
AtORF1 in 
26 653...26 466;
26 314...26 061;
25 979...25 161
30 407... 30 285;
Sequencing error at
30 171... 29 688;
the 5' end leading to
29 608... 29 146;
29 075... 28 870;
28 800... 28 683;
28 602... 28 566;
28 485... 28 147
28 830... 29 848;
ORF extends 15 base
29 951... 30 296;
pairs upstream of the
30 542...31 218;
31 885... 31 320
67 574... 67 401;
66 710... 66 520;
66 171... 66 092;
66 004... 65 389;
65 298... 65 099;
64 637... 63 784;
6 001... 7 470;
7 550... 7 757;
8 244... 8 506;
8 587... 9 378
121 331... 120 011;
122 896...121 428;(R)
41 595... 39 722;
39 635... 39 430;
39 248... 39 004
38 919... 38 092
The other proline-rich domain of AtFORMINs 2, 6 and 8 is predicted to be exposed to a non-cytoplasmic compartment. Given that polyproline stretches are characteristic for a class of structural cell-wall proteins known as extensins , it is tempting to speculate about a possible role for this domain in communication between formins and structures within the cell wall. Apart from this, few predictions of function can be made on the basis of the sequence data. Although formins are well conserved with respect to their molecular structure, we do not know the extent of their conservation within signaling or structural modules . As the relationships between protein structure, module structure and biological function are far from straightforward , we can at present neither prove nor exclude the possibility that plant formins contribute to similar functional modules to their animal and fungal counterparts. The question of whether these proteins have a direct role in cytokinesis, in mitotic spindle localization, or in some other cellular process, possibly involving cytoskeleton rearrangement or cell-surface growth, will have to be answered experimentally.
A systematic search of the available Arabidopsis genomic and cDNA sequences revealed the presence of eight genes encoding proteins that define a novel subfamily of the formin family. At least six out of eight Arabidopsis formins appear to be integral membrane proteins. This indicates a mechanism of membrane localization that may be specific to plants and functionally related to a possible role for formins in the communication between the plant cell and extracellular structures.
Materials and methods
Identification of Arabidopsisformin homologs and protein sequence prediction
The initial search for formin homologues in the non-redundant Arabidopsis thaliana protein (NRAT) database, performed using the PatMatch program [31,32] with the query pattern L-x-x-G-N-x-M-N, yielded three potential formin homologs - AtFORMIN1 to AtFORMIN3. AtFORMINs 2 to 8 were found by a TBLASTN 2.0 search [33,34] in GenBank, using the predicted protein sequence of AtFORMIN 1 as query (P(N) values in the range of 5.8×10-227 to 1.3×10-11). Known members of the formin family (a human diaphanous homolog and Drosophila melanogaster cappucino) were found in the same search (P(N) values 1×10-21 and 1.3×10-13, respectively), verifying the statistical significance of the initial PatMatch results.
Intron positions in the genomic sequences were determined (or confirmed) using the NetGene2 server . Translation of the DNA sequences was performed on the SIB ExPASy WWW server [35,36]. Only the longest predicted ORFs were subjected to further analysis.
Sequence alignment and domain structure analysis
All sequence comparisons were done on a set of 20 metazoan, yeast and plant formin sequences. These were FUGU, Fugu rubripes formin homolog gb|AAC34395.1; LFORMIN, mouse lymphocyte-specific formin gb|AADo1273; BNR1, yeast Bnr1 protein sp|P40450; BNI1, yeast Bni1 protein sp|P4183; FHOS, human formin-like protein gb|AAD39906.1; CAENO, Caenorhabditis elegans formin homolog gb|AAB42354.1; CAPPU, D. melanogaster Cappuccino gb|AAC46925.1; P14oMDIA and P134MDIA2, mouse Diaphanous homologs gb|AAC53280 and gb|AAC71771.1; DIA-DROME, D. melanogaster Diaphanous sp|P48608; CYK1, C. elegans Cyk1 assembled from gb|AAA81161.1 and gb|AAC17501.1; MFORMIN, mouse formin sp|Qo5860; and AtFORMIN 1 to 8. Protein sequences were aligned with the aid of MACAW , using the Gibbs sampler and segment pair algorithms, BLOSUM45 matrix. Only blocks with P<10-7 were considered. No homology to FH3 as defined by Petersen et al.  or to the amino-terminal conserved region  was revealed by this tool, whereas the FH2 domain was readily identified. Non-aligned parts of the sequence within the FH2 domain were adjusted manually. Consensus of the resulting alignment of FH2 (deposited in the EMBL alignment database, accession number DS39866) has been calculated for each subdomain separately (see Figure 1) by the method of Brown and Lai [38,39].
The SMART program [26,27] was used to examine predicted protein sequences for the presence and location of known sequence domains, putative secretion signals, transmembrane segments, coiled-coil motifs and low sequence complexity regions (usually representing proline-rich FH1 domains whose location was confirmed by visual inspection). Prediction of signal peptides by the neural network (NN) method ) was independently verified by a hidden Markov model-based (HMM) method on the SignalP 2.0 server [40,41]). Results of both methods were in agreement, with the exception of AtFORMIN5, which was predicted to be membrane-anchored by NN but cytoplasmic by HMM.
Construction of the evolutionary tree
The tree (Figure 3) was calculated from the three FH2 subdomains present in all formins studied, using programs from the PHYLIP package [42,43] version 3.573. An input file was prepared by joining subdomains a, c and h and was used to produce a bootstrapped data set by SEQBOOT with 500 sampling cycles. Distances were calculated using PROTDIST with the PAM distance matrix, and the results were used for tree construction using the neighbor-joining method  by NEIGHBOR. The consensus tree was determined by CONSENSE and plotted using DRAWTREE.
This work has been supported by the Grant Agency of the Czech Republic Grant 204/98/0482 and by the Czech Ministry of Education Program J13/98:113100003. I thank J. Flegr for helpful discussion.
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