Nuclear localization of annexin XI, a novel annexin

WS2-J-4-02 NUCLEAR LOCAI.IZATION OF ANNEXIN XI, A NOVEL ANNEXIN N. Mamiya, A. Mizutani, and H. Hidaka Department of Pharmacology, Nagoya University School of Medicine, Nagoya, Japan Annexin XI is a newly identified member of the annexin family. It was discovered from the rabbit lung as a calcium binding protein which binds in a calcium dependent manner to calcyclin, a member of the S-100 family. In the presence of calcium, annexin XI binds to acidic phospholipids and forms a complex with calcyclin. Molecular cloning of the annexin XI revealed the unique hydrophobic N-terminal region composed of 202 amino acids. Studies using a series of deletion mutants of annexin XI has shown that calcyclin-binding site locates on this long N-terminal domain, adjacent to or within the region from 27y to 52L. C-terminal region include four imperfect repeats. One of the conspicuous features of annexin XI is the nuclear localization in 3Y1 cells derived from rat embryonic fibroblasts. Subcellular fractionation and immunofluorescence studies of 3Y1 cells have shown that annexin XI is mainly localized in the nuclei. Moreover, we found that in 3Y1 cells transformed by Rous sarcoma virus oncogene (SR-3Y1 cells) phosphorylation of annexin XI was enhanced, compared with control 3Y1 cells. Phosphorylated annexin XI was recovered in the cytoplasmic fraction and did not bind to the ph0spholipids even in the presence of 1 mM or higher concentration of calcium. Therefore it is possible that phosphorylation regulates the subcellular localization of annexin XI and ultimately its functions. In spite of the obvious nuclear localization, no typical nuclear localization signals including a cluster of positively charged amino acids, reside in the annexin XI molecule. To define the regions responsible for the nuclear transport, we have constructed various truncated mutants and expressed them in COS-7 cells. Deletion of the residues 3-196 changed the distribution from the nucleus to the cytoplasm. In contrast, deletion of the residues 208-504 from the C-terminus domain resulted in nuclear localization. Three other mutants lacking 60-80 amino acids in the N-terminus regions (residues 3-61, 61-115, and 115-197) did not show predominantly nuclear localization any longer. These data suggest that the whole Nterminus region of annexin XI is important for nuclear transport of annexin XI, probably working by its tertiary structure. Furthermore, Western blot analysis showed ubiquitous distribution of annexin XI in adult rat tissues. However, we have found that nuclear localization of annexin XI is uncommon in the adult rat tissues. Examination of rat embryos revealed that nuclear localization of annexin XI is related to developmental stages and cell types.

WS2-J-4-03 EXPRESSION OF ANNEXINDURING SPERMATOGENESISIN THE NEWT, C Y N O P S P Y R R t I O G A S T E R T. Yamamoto and S. -I. Abe Department of Biological Science, Faculty of Science, Kumamoto University, Kumamoto 860, Japan To elucidate the molecular mechanism of meiosis, we have analyzed some genes which were differentially expressed in the secondary spermatogonia and primary spermatocytes. Using immno-differential screening(IDS) with anti-spermatogonia and antispermatocytes antisera, we have isolated a newt annexin eDNA clone from the testes ~ 1 1 eDNA library. The eDNA was 1,583 nucleotides long and predicteda 322 residue protein with a calculatedMr of 35,895. The tsedicted amino acid sequenceshowed 63% and 53% sequence identities with human annexin V and IV, respectivelyend the N-terminal region showed 40% and 33% identities with human annexin IV and V, respectively. Like annexin family proteins the newt annexin had a 4-fold internal repeat of approximately 70 residues. A single mRNA species was found to be expressedstrongly in the testes and brain, but weakly in the other organs. In sit,, hybridizationrevealedthat the annexin mRNA was expressed scarcely in the spermatogonia and Sertoli cells, but markedly in the germ cells from the leptotene-zygoteneprimary spermatocyles to round spermatids. To examine the localization of newt aunexin protein, we prepared the antiserum against newt annexin which was expressed in Escbcdchia coil Western blotting showedthat the annexin had a molecular weight of 35kDa, in accordancewith the predictedMr, and isoelectric point of 5.5. Imnmohistochemical analysis revealedthat the expression of the annexin increased after the initiation of meiosis concomittantly with the increaseof the mRNA on the plasma membrene of germ cells and Sertoli cells. In addition, it was revealedthat the annexin was localizedin the axial rod part of mature sperm. These findings indicatedthat the newt annexin was upregulated temporally and spatially during the spermatogenesis and suggested that the annexin plays an important role in the differentiation of sperm.

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