The tag genes of Dictyostelium are predicted to encode multi-domain proteins consisting of serine protease and ATP-binding cassette transporter domains. We have identified a novel tag gene, tagA, which is involved in cell type differentiation. The tagA mRNA accumulates during the first four hours of development,whereas TagA protein accumulates between two and ten hours of development and decreases thereafter. Wild-type cells express tagA in prespore cells and mature spores, defining tagA expression as prespore specific. However, tagA mutant cells that activate the tagA promoter do not sporulate, but instead form part of the outer basal disc and lower cup of the fruiting body. tagA mutant aggregates elaborate multiple prestalk cell regions during development and produce spores asynchronously and with low viability. tagA mutants produce about twice as many prestalk cells as the wild type as judged by a prestalk cell reporter construct. When mixed with wild-type cells, tagA- cells become overrepresented in the prestalk cell population, suggesting that this phenotype is cell-autonomous. These results suggest that TagA is required for the specification of an initial population of prespore cells in which tagA is expressed. Expression profiling uncovered a delay in the transcriptional program between 2 and 6 hours, coincident with TagA expression, revealing an early function for TagA. TagA also appears to play a general role in cell fate determination since tagA mutants express a spore coat protein gene (cotB) within vacuolated cells that form part of the stalk and they express a prestalk/stalk-specific gene (ecmB)within cells that become spores. The expression of TagA at two hours of development, the observed coincident delay in the transcriptional program and the subsequent mis-expression of cell-type specific genes provide evidence for cell fate determination beginning in some cells much earlier than previously believed.

We have identified a cellular efflux pump, RhT, with the properties of an MDR transporter-a type of ATP-binding cassette transporter whose substrates include small hydrophobic molecules. RhT transports rhodamine 123 (Rh123) and is inhibited by low temperature, energy poisons, and several MDR transport inhibitors, such as verapamil. All vegetative cells have RhT activity, but during development prestalk cells lose RhT activity while prespore cells retain it. We also identified several RhT inhibitors. The most effective inhibitor is the stalk cell-inducing chlorinated alkyl phenone, DIF-1. The RhT inhibitors disrupted development, to varying degrees, and induced stalk cell formation in submerged culture. The inhibitors displayed the same rank order of pharmacological efficacy for stalk cell induction as they did for Rh123 transport inhibition. We also found that cerulenin, a specific inhibitor of DIF-1 biosynthesis (R. R. Kay, 1998, J. Biol. Chem. 273, 2669-2675), abolished the induction of stalk cells by each of the RhT inhibitors, and this effect could be reversed by DIF-1. Thus, DIF-1 synthesis appears to be required for the induction of stalk cells by the RhT inhibitors. Since DIF-1 is the most potent inhibitor of RhT activity, and thus a likely transport substrate itself, we propose that RhT inhibitors induce stalk cell differentiation by blocking DIF-1 export, causing DIF-1 to build up within cells. Our results provide evidence for a prespore-specific efflux pump that regulates cell fate determination, perhaps by regulating the cellular concentration of DIF-1.

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