Plant annexins form calcium-independent oligomers in solution

Abstract

Despite the identification of annexin proteins in higher plants as early as 1989 (Boustead et al. 1989), it was only in the past 5 years that the field of plant annexins emerged to become a separate, intensely studied area. So far, annexins have been found in abundance in every plant where a search was initiated (for reviews, see Clark and Roux 1995; Delmer and Potikha 1997). Unlike in the animal kingdom, where a varying "bouquet" of up to 13 different annexins is expressed, the plants seem to possess a smaller number of annexin proteins, two of which are found most frequently and show very high sequence similarity throughout different plants. Among others (Blackbourn et al. 1991, 1992; Battey et al. 1996; Seals and Randall 1997; Proust et al. 1999), in bell pepper Anx23(Ca38) and Anx24(Ca32) (Proust et al. 1996) have been identified, as well as Anx(Le34) and Anx(Le35) in tomato (Smallwood et al. 1990) and Anx(Gh1) and Anx(Gh2) in cotton (Andrawis et al. 1993). All these proteins migrate within the range of 33 and 35 kD on SDS-PAGE; however, both proteins from one species clearly migrate differently from each other despite having similar molecular weights. However, in Arabidopsis seven annexin homologs have been identified so far (Clark et al. 2001), thereby giving rise to the speculation that annexins in other plants might also appear within a diverse multigene family. Additionally, annexin proteins have been reported that apparently are different from the ones mentioned above. The annexin-like proteins from celery (Seals et al. 1994) and tobacco (Seals and Randall 1997) associated with the vacuolar membrane show an apparent mass of 42 kD, and have been termed VCaB42. It seems very likely, though, that the latter protein is, in fact, the tobacco homolog of Anx23(Ca38) and Anx(Le35). Another type was reported with the fern annexin, having an apparent molecular weight of 70 kD, and therefore, could resemble the topology of annexin A6 with eight homologous repeats, although the authors could not rule out the possibility that this species is a dimer of 35 kD polypeptides (Clark et al. 1995). Due to their high sequence similarity, it was recently proposed that three annexins from tobacco, tomato, and bell pepper, namely Anx(Nt32), Anx(Le34), and Anx24(Ca32), might constitute a distinct type of Sp32 annexins (Proust et al. 1999).

Some mammalian annexins have been reported to self-associate in solution. Creutz et al. (1979) described this phenomenon for annexin A7 in solution, where the protein formed rods, bundles of rods, and paracrystalline arrays in a calcium-dependent fashion. A similar self-association event was seen with isolated annexin A4 from the ray Torpedo marmorata (Walker et al. 1983). Other members of the mammalian annexin subfamily, however, are claimed not to self-associate (Shadle et al. 1985).

The oligomerization states of mammalian annexins A1, A4, A5, A6, and the heterotetramer [AnxA2 p11]₂ (Ahn et al. 1988), of annexin A7 (Creutz et al. 1979), as well as annexin C1-core from Dictyostelium discoideum (Liemann et al. 1997), have been investigated in solution using ultracentrifugation techniques. For all of these proteins a calcium-dependent monomer–dimer equilibrium has been observed with weak association constants in the range of 10 ^{M; these studies were carried out using sedimentation equilibrium experiments in the presence of 10 mM CaCl}₂. The association constant for the heterotetramer constituted by annexin A2 and p11 shows a much higher affinity (k_tetramer = 1.9 × 10 ^{M) and indefinite isodesmic self-association of the tetramer was observed with an association constant of} k_iso = 2.8 × 10 ^{M (}Ahn et al. 1988). The C-terminal core of annexin C1 was subjected to ultracentrifugation in the absence of calcium and found to be monomeric only. The half-maximal calcium concentrations for dimerization of mammalian annexins are in the range of 200 μM (annexin A7) (Creutz et al. 1979) and about 1 mM for other annexins (Südhof et al. 1982; Walker et al. 1983; Zaks and Creutz 1991). However, these oligomers of mammalian annexins are reported to be labile, and seem to gain stability only in the presence of membranes (Zaks and Creutz 1991).

The first report on the oligomerization state of plant annexins described an annexin from Capsicum annuum, Anx(Ca35), purified from a natural source; only a partial amino acid sequence of this protein has been reported to date by Hoshino et al. (1995). When crosslinking the protein bound to phosphatidylinositol vesicles at calcium concentrations higher than 0.75 mM, these authors found a small fraction of annexin homodimers. This result allows only limited conclusions about the oligomerization state of the protein in solution, because the concentration of this annexin on the membrane surface and in its immediate vicinity may be very high under the calcium conditions used. Accidental crosslinking of two monomers can therefore occur without a real dimer being present.

In the course of our ongoing studies on structural investigation of plant annexins, in particular their calcium-bound forms, we encountered severe difficulties in obtaining protein crystals in the presence of calcium. To obtain a clearer picture about the effects of calcium on these proteins we elucidate in the current study the oligomerization state of four plant annexins: Anx23(Ca38), Anx24(Ca32), Anx(Gh1), and Anx(Gh2). Based on a strict amino acid sequence comparison and according to the respective homologies it is tempting to assume that Anx(Gh1) belongs to the class of Sp32 annexins (Proust et al. 1999) while Anx(Gh2) rather seems to be the homolog of Anx23(Ca38), and thus belongs to the class of Sp38 annexins (see Fig. 1 ▶). All four proteins were subcloned as N-terminal His-tag fusions, and the recombinant proteins were subjected to equilibrium sedimentation analysis as well as gel filtration to characterize their oligomerization behavior.

Fig. 1.

Alignment of amino acid sequences of Anx(Gh1) [acc. number u73746], Anx24(Ca32) [acc. number x93308], Anx(Gh2) [acc. number u73747], and Anx23(Ca38) [acc. number aj130956]. The alignment was generated with the programs PILEUP from the GCG suite (Genetics Computer Group 1996) and ALSCRIPT (Barton 1993).

We report for the first time the presence of plant annexin trimers in solution, as observed with annexins 23(Ca38), 24(Ca32), and Anx(Gh1). For Anx(Gh2), analytical gel filtration indicates the presence of a dimeric species in solution.

Acknowledgments

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Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.4770102.