Introduction Recent results from a number of laboratories have revealed that the normal expression of some eukaryotic genes can be altered dramatically by the perturbation of intracellular Ca 2+ . In different cell types, a variety of processes associ- ated with protein production appear to be regulated by Ca 2+ , including certain transcriptional and post-transcriptional events (Preston et al . 1990), translation (Kumar et al . 1989; Brostrom and Brostrom, 1990), glycosylation (Chang et al. 1987; Poruchynsky et al . 1991), protein degradation (Wile- man et al. 1991a), and secretion (Rudolph et al. 1989; Booth and Koch, 1989; Lodish and Kong, 1990; Sambrook, 1990). During early Dictyostelium discoideum development, the cells synthesize and secrete the enzyme cyclic nucleotide phosphodiesterase (also termed cAMP phosphodiesterase) (ePD) to regulate extracellular levels of the morphogen, cyclic AMP, which is released in a periodic fashion by the cells (Orlow et al. 1981; Devreotes, 1982). In response to repeated stimulation by pulses of cyclic AMP, the cells also produce a form of the enzyme (mPD) that becomes asso- ciated with the external surface of the plasma membrane (Yeh et al. 1978). Both enzymes are glycoproteins and are transcribed from the same gene (Shapiro et al . 1983; Faure et al . 1990). ePD activity in the cell population is regulated in turn by the synthesis and secretion of a high-affinity, gly- coprotein inhibitor of the enzyme (PDI) (Kessin et al . 1979; Franke and Kessin, 1981). High, constant levels of exo- genous cyclic AMP induce ePD (but not mPD) synthesis and repress PDI production (Tsang and Coukell, 1977; Yeh et al . 1978; Franke et al. 1987; Wu and Franke, 1990). During recent studies on the regulation of the PD-PDI system of Dictyostelium, it was observed that low mil- limolar concentrations of EGTA partially inhibited produc- tion of ePD, iPD (the intracellular precursor of the ePD) and PDI activities, and delayed the appearance of mPD activity. Furthermore, these inhibitory effects of EGTA could be overcome completely by the presence of 1 μM free extracellular Ca 2+ (Coukell and Cameron, 1988). Sub- sequent experiments revealed that the degree of inhibition of PD production by EGTA was enhanced greatly by addition of the Ca 2+ ionophore A23187 and that the kinet- ics of inhibition correlated closely with the loss of Ca 2+ from the cells. In addition, despite the fact that Ca 2+ deple- tion by EGTA and A23187 treatment had little effect on total protein synthesis, total protein glycosylation or PD transcription, PD-associated polypeptides could not be 371 Journal of Cell Science 103, 371-380 (1992) Printed in Great Britain © The Company of Biologists Limited 1992 We reported previously that Ca 2+ depletion of Dic - tyostelium discoideum cells severely inhibits extracellu- lar cyclic nucleotide phosphodiesterase (PD) synthesis at a post-transcriptional step. In this study, further exper- iments were performed to learn more about the nature of this phenomenon. Examination of the polysomal dis- tribution of PD transcripts in control cells and in cells depleted of Ca 2+ by incubation with EGTA and A23187 (EA) suggested that inhibition of PD production does not involve translational control. Kinetic analysis of this inhibitory process revealed that soluble, intracellular PD activity, synthesized from either the 2.4 or 1.9 kb PD mRNA, decreased very rapidly upon addition of EA. Furthermore, this decrease in activity was accompanied by the preferential loss of PD-related polypeptides, indi- cating a proteolytic event. EA-induced PD degradation required cellular energy and concomitant protein syn- thesis but was unaffected by most of the lysosomotropic agents tested. Therefore, PD proteolysis might not occur in the lysosome. In cell fractionation experiments, the EA-sensitive, intracellular PD activity comigrated with a rough ER marker in Percoll/KCl gradients. In addition to its effect on the PD, EA were also observed to inhibit production and rapidly lower the intracellu- lar levels of another secreted glycoprotein, the PD inhibitor. Together, these results suggest that depletion of some intracellular Ca 2+ store(s) in Dictyostelium, possibly the ER, disrupts the normal function of the secretory pathway, resulting in selective degradation of certain proteins. Key words: Ca 2+ depletion, protein secretion, protein degradation, Dictyostelium. Summary Involvement of intracellular calcium in protein secretion in Dictyostelium discoideum M. B. COUKELL, A. M. CAMERON and N. R. ADAMES Department of Biology, York University, 4700 Keele St, North York, Ontario, Canada M3J 1P3