Annexin 2 is a Ca(2+) binding protein that binds to and aggregates secretory vesicles at physiological Ca(2+) levels [1] and that also associates Ca(2+) independently with early endosomes [2, 3]. These properties suggest roles in both exocytosis and endocytosis, but little is known of the dynamics of Annexin 2 distribution in live cells during these processes. We have used evanescent field microscopy to image Annexin 2-GFP in live, secreting rat basophilic leukemia cells and in cells performing pinocytosis. Although we found no evidence of Annexin 2 involvement in exocytosis, we observed an enrichment of Annexin 2-GFP in actin tails propeling macropinosomes. The association of Annexin 2-GFP with rocketing macropinosomes was specific because Annexin 2-GFP was absent from the actin tails of rocketing Listeria. This finding suggests that the association of Annexin 2 with macropinocytic rockets requires native pinosomal membrane. Annexin 2 is necessary for the formation of macropinocytic rockets since overexpression of a dominant-negative Annexin 2 construct abolished the formation of these structures. The same construct did not prevent the movement of Listeria in infected cells. These results show that recruitment of Annexin 2 to nascent macropinosome membranes 16656is an essential prerequisite for actin polymerization-dependent vesicle locomotion.

Annexin 2 is a Ca 2؉ -binding protein that has an essential role in actin-dependent macropinosome motility. We show here that macropinosome rocketing can be induced by hyperosmotic shock, either alone or synergistically when combined with phorbol ester or pervanadate. Rocketing was blocked by inhibitors of phosphatidylinositol-3-kinase(s), p38 mitogen-activated protein (MAP) kinase, and calcium, suggesting the involvement of phosphoinositide signaling. Since various phosphoinositides are enriched on inwardly mobile vesicles, we examined whether or not annexin 2 binds to any of this class of phospholipid. In liposome sedimentation assays, we show that recombinant annexin 2 binds to phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5P 2 ) but not to other poly-and mono-phosphoinositides. The affinity of annexin 2 for PtdIns-4,5P 2 (K D ϳ5 M) is comparable with those reported for a variety of PtdIns-4,5P 2 -binding proteins and is enhanced in the presence of Ca 2؉ . Although annexin 1 also bound to PtdIns-4,5P 2 , annexin 5 did not, indicating that this is not a generic annexin property. To test whether annexin 2 binds to PtdIns-4,5P 2 in vivo, we microinjected rat basophilic leukemia cells stably expressing annexin 2-green fluorescent protein (GFP) with fluorescently tagged antibodies to PtdIns-4,5P 2 . Annexin 2-GFP and anti-PtdIns-4,5P 2 IgG co-localize at sites of pinosome formation, and annexin 2-GFP relocalizes to intracellular membranes in Ptk cells microinjected with Arf6Q67L, which has been shown to stimulate PtdIns-4,5P 2 synthesis on pinosomes through activation of phosphatidylinositol 5 kinase. These results establish a novel phospholipidbinding specificity for annexin 2 consistent with a role in mediating the interaction between the macropinosome surface and the polymerized actin tail.

Several lines of evidence indicate that annexins, as calcium-dependent pbospbolipid-binding proteins, are involved in a variety of plant cellular processes. We were interested in determining if annexins are implicated in the highly regulated fruit development of bell pepper. By differential screening of several cDNA libraries, we isolated a full-length cDNA of 1180 bp encoding an annexin. Northern blot analyses show a differential expression pattern of the transcripts during the early stages of development and during ripening. Immunoblots using antiserum raised against p33/p35 from maize reveal that crossreactive polypeptides of about 30 kDa are present at each stage of fruit development in bell pepper. We partially purified the annexins from seedlings and green fruits. At least one annexin of 32 kDa is present in these plant tissues.

This work provides the first three-dimensional structure of a member of the plant annexin family and correlates these findings with biochemical properties of this protein. Annexin 24(Ca32) from Capsicum annuum was purified as a native protein from bell pepper and was also prepared by recombinant techniques. To overcome the problem of precipitation of the recombinant wildtype protein in crystallization trials, two mutants were designed. Whereas an N-terminal truncation mutant turned out to be an unstable protein, the N-terminal His-tagged annexin 24(Ca32) was crystallized, and the three-dimensional structure was determined by x-ray diffraction at 2.8 Å resolution. The structure refined to an R-factor of 0.216 adopts the typical annexin fold; the detailed structure, however, is different from non-plant annexins, especially in domains I and III and in the membrane binding loops on the convex side. Within the unit cell there are two molecules per asymmetric unit, which differ in conformation of the IAB-loop. Both conformers show Trp-35 on the surface. The loop-out conformation is stabilized by tight interactions of this tryptophan with residue side chains of a symmetry-related molecule and enforced by a bound sulfate. Characterization of this plant annexin using biophysical methods revealed calcium-dependent binding to phospholipid vesicles with preference for phosphatidylcholine over phosphatidylserine and magnesium-dependent phosphodiesterase activity in vitro as shown with adenosine triphosphate as the substrate. A comparative unfolding study of recombinant annexin 24(Ca32) wild type and of the His-tag fusion protein indicates higher stability of the latter. The effect of this N-terminal modification is also visible from CD spectra. Both proteins were subjected to a FURA-2-based calcium influx assay, which gave high influx rates for the wild-type but greatly reduced influx rates for the fusion protein. We therefore conclude that the N-terminal domain is indeed a major regulatory element modulating different annexin properties by allosteric mechanisms.Annexins have been a focus of research for nearly 20 years, and a large amount of data has accumulated especially for mammalian members of this protein family although annexin proteins are abundant throughout all species but yeast. In 1989, the first annexin-like proteins in plants were identified (1), and it is now well established that annexins are just as ubiquitous in the plant kingdom as elsewhere. The amount of annexin in plant cells makes up to 0.1% of the total protein content (2). As deduced from amino acid sequences, plant-type and vertebrate annexins show similarities up to 40%, whereas the plant-type members share up to 97% similarity with each other (see Table I). A more detailed analysis of the phylogenetic aspects led to the conclusion that within the overall annexin family a unique subset is made up by the plant-type members. In contrast to mammalian and vertebrate tissues, where varying amounts of different annexin proteins are expressed, plants poss...

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