Journal of Experimental Botany, Vol. 53, No. 369, pp. 769-771,
April 1, 2002
© 2002 Oxford University Press
Gene Note |
Hamy3, a novel type 100 kDa myosin from sunflower
1 Max-Planck-Institute for Cell Biology, Rosenhof, 68526 Ladenburg, Germany
2 Abteilung Pharmakologie und Toxikologie, Universität Ulm, Oberer Eselsberg, 89081 Ulm, Germany
Received 10 October 2001; Accepted 23 November 2001
Abstract
Hamy3, a novel type myosin heavy chain from sunflower is the smallest myosin described so far, with only 900 amino acid residues. One interesting finding in Hamy3 is the glycine to glutamine alteration at residue 741, which corresponds to chicken skeletal muscle myosin glycine 699 (G699). G699 is found in 125 out of 129 myosin sequences and is interpreted in terms of its role as a pivot point for motion in the myosin ‘lever arm hypothesis’. Changes in this crucial part of myosin might indicate a role that is different from the generation of intracellular motility.
Key words: Glycine 699, lever arm hypothesis, phylogenetic analysis, sunflower, unconventional myosin.
Since the release of the first complete myosin gene sequence from Caenorhabditis (Karn et al., 1983
) a superfamily of myosins with at least 18 distinct classes showing fascinating variability in structure and function has emerged (Hodge and Cope, 2000). Based on the primary structure, all myosins have a modular organization in common. It consists of a conserved amino-terminal head region, known as the motor domain, followed by the neck region and a highly variable tail region (Sellers, 2000). The motor domain exhibits the ATPase activity and is able to bind F-actin (Homes and Geeves, 2000). The neck or regulatory region contains at least one repeat of a sequence known as the IQ motif, which could serve as a putative calmodulin binding site (Sellers, 2000). Finally, numerous sequence motifs in the tail region have been identified among different myosin classes. Most common are regions with heptat repeats, capable of forming alpha-helical coiled-coil structures, or motifs which may be able to bind to F-actin such as the tail homology region 2 (Sellers, 2000). Recently, regions involved in signalling processes such as kinase domains or GTPase-activating domains have been characterized assigning even more functions to unconventional myosins (Oliver et al., 1999).
In this report another new member of the myosin family, sunflower myosin Hamy3, is described that has only 900 amino acid residues.
A RT-PCR-based approach was used to generate a myosin-specific probe suitable for cDNA library screening. Conserved regions of myosin subfragment 1 (EAFGNAKT and QQHFNQHV) served as the target sequence for the synthesis of degenerated primers (GARGCITTYGGIGAYGCIAARAC; CAIRTGIYKRTTRAAITGYTGYTG). To avoid high degeneracy inosine was introduced as the universal base. Screening of a root-specific sunflower cDNA library led to the isolation of a full length cDNA of 3019 bp, designated Hamy3, with an open reading frame of 2700 bp encoding a 900 amino acid polypeptide and a predicted molecular mass of 101.2 kDa (Genbank accession number U94783). Accordingly, Hamy3 represents the smallest cloned myosin so far and might represent the first member of a new plant myosin size class of approximately 100 kDa.
Hamy3 exhibits a typical myosin modular domain structure, although there are several findings which are characteristic for this myosin. The amino-terminal region has an extension of the same size as Arabidopsis myosin ATM1 (Knight and Kendrick-Jones, 1993), but is unique in its sequence. So far no possible function can be deduced. The core motor domain of Hamy3 contains 651 amino acids as concluded by comparing the core motor domain defined for chicken muscle myosin II (693 aa; Cope et al., 1996).
The actin binding interface of Hamy3 as deduced from the actomyosin rigour complex and crystallographic data obtained from chicken and Dictyostelium myosin II (Schröder et al., 1993) consists of several regions (amino acid residues: region 1 [672–699]; region 2 [585–619]; region 3 [462–481]; secondary actin binding region [620–641]). Both amino acid composition and residue count in terms of charged/uncharged or hydrophobic/hydrophilic residues in the actin binding interface correlate with existing sequence data.
The region C-terminal to the motordomain contains one IQ-motif (LQSFIRGENAR), which resembles the consensus sequence (IQxxxRGxxxR) (Sellers, 2000). A short, unique tail region of 40 residues concludes the sequence. The short tail and the lack of any alpha-helical coiled-coil regions are indicating that Hamy3 could be a single-headed myosin. Interestingly, results from the phylogenetic analysis show (Fig. 2), that Hamy3 groups with class VIII myosins, which presumably are double-headed (Yamamoto et al., 1999). So far all myosins can be classified by single- and double-headed molecules, whereas no single-headed myosin is found in classes occupied by double-headed myosins and vice versa (Hodge and Cope, 2000). Hamy3 might be the first exemption of this rule.
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The most striking new feature in the motor domain of Hamy3 is an amino acid alteration at residue 741, which corresponds to chicken myosin II amino acid 699, designated G699 (Kinose et al., 1996
). This particular glycine is replaced by glutamine in Hamy3 (CSQVMEVMQIS). The only other exceptions out of 129 myosin sequences are found in Caenorhabditis myosin HUM-4 (G
L; NLLLAELLSFR) and two Toxoplasma myosins of the new class XIV (GS; myoA: ALSVLEALQLR; myoB: SLSILEALQLR). Kinose et al. proved that mutagenesis of chicken myosin glycine 699 to alanine altered the motor activity with tremendous impact on the capability on moving actin filaments (Kinose et al., 1996). This could imply that Hamy3 represents a motor with significantly changed characteristics of activity and force production comparing to other unconventional myosins. Other reasons for a putative altered motor activity of Hamy3 could be due to changes in conserved amino acids surrounding residue G699 (Consensus: CNGVLEGIRIC; Warrick and Spudich, 1987), a region which contains the most reactive thio groups SH1 and SH2 in the myosin molecule as well (Reisler, 1985). Hamy3 only possesses thio group SH2, which is common to other members of class VIII myosins. This region connects the postulated lever arm of myosin to the head domain, whereas G699 is interpreted as the pivot point for motion of the lever arm (Homes and Geeves, 2000). Other regions in the myosin molecule important for subdomain movement have been proposed by analysing the crystal structure of chicken and Dictyostelium myosin II (Homes and Geeves, 2000). Further, truncated myosin heads complexed with artificial nucleotides have been produced to identify residues involved in nucleotide binding (Homes and Geeves, 2000). Due to high sequence conservation in the ATP-binding pocket of Hamy3—motifs GESGAGKT and NWNSSRFGK include almost all residues involved in ATP-binding (Cope et al., 1996)—it can be expected that ATP-binding and hydrolysis is achieved by similar principles as suggested for chicken and Dictyostelium myosin (Homes and Geeves, 2000). On the other hand important functional domains in the myosin head such as the region starting with lysine 453 in Dictyostelium myosin II (Cope et al., 1996) show sequence variability in Hamy3 (Lys 523; Fig. 1). It is postulated that conformational changes occur in this region. Furthermore, it is presumed that these changes are transmitted to the actin-binding interface, where the energy transduction finally results in a power stroke producing relative movement along F-actin (Holmes and Geeves, 2000). Considering the high conservation of residues in the ATP-binding pocket and significant amino acid exchanges in crucial parts of myosin responsible for subdomain movement, one could speculate that Hamy3 has a different role than a classical molecular motor. Hypothetically, Hamy3 could just function as a molecular clamp powered by ATP and switching between two states, i.e. F-actin ‘hold’ or ‘release’. Switching could be regulated through binding of calmodulin-like proteins to the IQ-motif as suggested for other plant myosins (Yokota et al., 1999). Hints for possible roles of Hamy3 might be obtained by the generation of chimerical myosin molecules, where regions important for subdomain movement in well-characterized myosins are replaced by sunflower sequences. Other important features such as direction of motility have not been determined for any plant myosin and will also require further studies. Finally, the identification and biochemical characterization of new myosins will indicate whether the motor domain of Hamy3 contains the minimum number of residues necessary for force production.
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Acknowledgments
I had excellent advice from Kenneth Holmes, especially for bringing the G699 story to my attention. I am grateful to Bob Shoeman for providing all the necessary oligos and to Dominik Hepperle for help with the phylogenetic analysis. Special thanks go to Susanne Liebe for great support of all kinds and for making preliminary data available.
Notes
3 Present address and to whom correspondence should be sent: Kraljevec 81, 10000 Zagreb, Croatia. Fax: +38514551196. E-mail: vugrek{at}altavista.net 
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