As illustrated in Figure 2A, the H ATPases in both male and female thalli showed an identical response to FC. We performed further experiments using Tak 1. Mp14 3 3a Binds to the Phosphorylated H ATPase We detected endogenous 14 3 3 proteins in thalli having molecular masses of 31 and 32 kD using antibodies raised against Arabidopsis GF14phi . We then performed a BLAST search against M. polymorpha ESTs and found a typical 14 3 3 protein, designated M. polymorpha 14 3 3a . Expression of Mp14 3 3a in thalli was confirmed by RTPCR . Moreover, protein blot analysis using the recombinant Mp14 3 3a as a probe revealed that it bound to phosphorylated H ATPase in thalli . These results indicate that the pT H ATPases in M. polymorpha might be activated via phosphorylation of the penultimate Thr and subsequent binding of the endogenous 14 3 3 protein, as in vascular plants. Effects of Physiological Signals on the Phosphorylation Status of the pT H ATPases in M. polymorpha To clarify the physiological signals that regulate the phosphorylation status of the pT H ATPase in M.
polymorpha, we next examined the effects of putative physiological signals on the pT H ATPase in thalli. We treated thalli with white light , Suc as a photosynthesis product , and mannitol as an osmotic agent . Interestingly, all treatments induced phosphorylation of the H ATPase in thalli without altering the H ATPase amount. Sucinduced phosphorylation cannot be interpreted as custom peptide selleck chemicals a result of its osmotic pressure, because treatment with the same concentration of mannitol had no effect on phosphorylation level . Osmotic shock dependent phosphorylation required over 100 mM mannitol and was concentration dependent between 100 and 200 mM . Notably, light illumination had a drastic effect on the phosphorylation level of the H ATPase in thalli . Therefore, we examined the light induced phosphorylation of the H ATPase in more detail. As shown in Figure 5A, the phosphorylation level of the H ATPase reached a maximum within 15 min after the start of illumination.
Phosphorylated H ATPase was dephosphorylated gradually after the end of light illumination, and phosphorylation level reached the original level in around 60 min . We further determined the effects of light quality on phosphorylation and found that red light and blue light also induced phosphorylation of the H ATPase . Interestingly, both 3 1,1 Oligomycin A dimethylurea and 2,5 dibromo 3 methyl 6 isopropyl p benzoquinone , which are inhibitors of photosynthetic electron transport from PSII to PSI , at 10 mM severely inhibited phosphorylation , suggesting that light induced phosphorylation of the H ATPase is regulated by photosynthesis.