ABSTRACTcAMP has been implicated in the control of the expression of developmental genes in Dictyostelium discoi- We have recently characterized two cAMP binding proteins in developing D. discoideum cells, termed CABP1 and CABP2 (19). CABP1 is an unusual cAMP binding protein in that it consists oftwo subunits of different molecular weights. CABP2 has properties identical to the regulatory subunit of the cAMP-dependent protein kinase, which has been characterized extensively by other workers (20-30). The subcellular localization of the regulatory subunit of cAMP-dependent protein kinase has been examined before (22,30). In the present communication, we have examined the subcellular localization of CABP1 during development. CABP1 is found in the intracellular fraction as well as on the surface of developing cells. During development, CABP1 is translocated to the nucleus. This pattern of cellular localization is compatible with the idea that CABP1 plays a pivotal role in mediating the effect of cAMP on developmental gene expression. MATERIALS AND METHODSConditions of Growth and Development. The D. discoideum strain NC4 used in this study was originally isolated by K. Raper (Univ. of Wisconsin, Madison). The cells were grown in association with Enterobacter aerogenes on SM agar (31) in the dark at 220C for 40-44 hr until the bacterial lawn was just starting to clear. The vegetative amoebae were washed free of bacteria with 20 mM KH2PO4-K2HPO4 (pH 6.2) by centrifugation at 400 x g. The washed cells were used for analysis immediately if vegetative cells were required or allowed to develop at 5 x 106 cells per cm2 on 1.5% agar containing 40 mM KH2PO4-Na2HPO4 (pH 6.4), 20 mM KCl, and 2.5 mM MgCl2.Isolation of Nuclear and Postnuclear Supernatant Fractions. The isolation procedure was adapted from Cococci and Sussman (32). All steps were carried out at 40C unless otherwise specified. The cells were harvested at various times during development, washed twice with 0.2% NaCl, and resuspended at 108 cells per ml in NIB [50 mM HEPES, pH 7.5/5 mM magnesium acetate/10 mM benzamidine/50 ,ug of antipain per ml/50 pug of pepstatin per ml/2% Nonidet P-40/10% (wt/vol) sucrose]. An aliquot was removed and designated as total cellular extract.Abbreviation: CABP, cAMP binding protein.

Several lines of evidence indicate that cAMP modulates developmental gene activity via cell-surface receptors. We describe here a novel cAMP receptor, CABP1, whose properties are consistent with the idea that this protein is involved in gene regulation. Firstly, immunological techniques using anti-CABP1 antibodies as probes showed that this cAMP receptor can be detected on the surface of developing cells. Secondly, there is a steady migration of CABP1 to the nucleus during development. Thirdly, some genetic variants exhibiting an altered pattern of development are found to possess modified CABP1. We also showed that CABP1 co-purifies with at least seven other polypeptides which share common epitopes with CABP1. Interestingly, four of the CABP1-related polypeptides can be detected on the cell surface as well as in the nucleus.

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Acid phosphatase-2, as characterized by gel electrophoresis under non-denaturing conditions, is a convenient marker for prestalk cells in Dictyostelium discoideum. We have purified this prestalkspecific enzyme and have examined its regulation during development. Under denaturing conditions, the enzyme has a molecular weight of 50,000 and an isoelectric point of 4.0. On the other hand, acid phosphatase-1 have a Mr-55,000 polypeptide (AP1-55) and a minor Mr-50,000 polypeptide (AP1-50) and both have diffuse isoelectric point from 3.4 to 4.1. Using monoclonal antibodies directed against acid phosphatase-2 as probes, we showed that some acid phosphatase-2 are newly synthesized at slug stage and some are converted from AP1-50 which was synthesized during ealy development.During development, amoebae of Dictyostelium discoideum aggregate to form multicellular structures which are called psuedoplasmodia or slugs. Cells in the anterior region of the pseudoplasmodium give rise to stalk cells of the terminally differentiated fruiting body, while cells in the posterior region are precursors of the mature spores (1). A number of differences can be detected between the anterior, prestalk cells and the posterior, prespore cells (2-5). These differences have been used as markers to investigate the mechanisms which regulate cell differentiation. Thus, the appearance of prespore-specific characters such as the prespore vacuoles (2, 6 ) and UDP galactosyl polysaccharide transferase (3) after 10 hr of development suggests that developing cells enter the prespore pathway at about this time. The time of synthesis of prespore specific proteins analyzed by two-dimensional gel are consistent with this information (7, 8).Until recently, analysis of cell differentiation was confined to prespore cells because of the difficulty involved in obtaining large quantities of prestalk cells. The improvement on cell sepalation techniques (9,10) has alleviated this problem. However, the time of the differentiation of prestalk cells has produced a complicated picture. Analyses by two-dimensional gels have shown that some proteins which are specific to prestalk slug cells begin their synthesis during early development and that the synthesis of these proteins is shut off in prespore cells. Conclusion from these results is that prestalk slug cells are similar to preaggregative cells (7,8). Furthermore, some prestalk-specific mRNAs (1 1, 12) and prestalk specific antigen (13) are