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ateThr of HATPase and that phosphorylationof the penultimate Thr is really a main widespread regulatorymechanism of HATPase in vascular plants. It shouldbe noted that the pT HATPase has been reportedto be phosphorylated at many sites furthermore tothe penultimate Thr CX-4945 in vascular plants.The C terminus of nonpT HATPase is also thoughtto be important for the regulation of its activity. In theyeast CX-4945 Saccharomyces cerevisiae, it has been reported thatphosphorylation of two tandemly positioned residuesin the C terminusactivatesthe HATPase in response to Glc, indicating that the fungal HATPase is regulatedin a different manner from the pT HATPase. Posttranslationalregulation in the HATPases in red andgreen algae remains unresolved.
In this study, we performed molecular characterizationof plasma membrane HATPase within the liverwortMarchantia polymorpha as a nonvascular plantbryophyte, which represents one of the most basal lineage ofextant land plants. We identified that M. polymorphaexpresses both pT HATPase and nonpT HATPase.We further supply evidence that the pT HATPase inM. polymorpha axitinib is regulated by phosphorylation of itspenultimate Thr in response to physiological signals,including light, Suc, and osmotic shock.RESULTSIdentification of cDNA Sequences of Plasma MembraneHATPase in M. polymorphaWe carried out a BLAST search against M. polymorphaESTs to find sequences with similarity to thetypical plasma membrane HATPase in Arabidopsis,AHA2. Individual ESTs had been derived from thalliand protonemata of M. polymorpha, male accessionTakaragaike1. We identified eight HATPasehomologs, designated MpHA1 to MpHA8.
Allisoforms very conserve a characteristic sequence,GDGVNDAPALKKA, within the catalytic domain of thePtype ATPaseand show high sequence identitywith AHA2,supplying strong assistance to our claim that these isoformsare functional homologs as plasma membrane HATPases.Of these, four isoformspossess NSCLC a penultimate Thr and conserveregion I and region II, which are important for autoinhibitoryeffects on the HATPase, within the Cterminalregion. In contrast, the remainingisoforms lack such a penultimate Thr within the C terminusand have various Cterminal lengths. Phylogeneticanalysis utilizing fulllength amino acid sequencesindicated that MpHA2, MpHA3, and MpHA4are clustered with Arabidopsis HATPase and thatMpHA6, MpHA7, and MpHA8 are close to the nonpTHATPase of Chlamydomonas reinhardtii, which has nopenultimate Thr.
In accordance with the classificationof gene families axitinib within the pT HATPase, MpHA2,MpHA3, and MpHA4 localize in between subfamilies Iand IV. These outcomes suggestthat the M. polymorpha genome encodes both pT HATPase and nonpT HATPase genes. Note thatMpHA5 has high sequence identity with AHA2 aswell as MpHA1 to MpHA4 but no conserved penultimateThr and that MpHA6 has insertions of over 40residues within the Cterminal region and a Cterminalextension of 39 residues.To examine the expression of MpHAs, reverse transcriptionPCR analysis utilizing total RNA fromthalli was performed. The results showed that the HATPase isoforms, except for MpHA7, had been expressedin thalli. All MpHAs showed identical expressionproperties in both maleand femalethalli.
Fusicoccin Induces Phosphorylation in the PenultimateThr of pT HATPasesWe initial performed immunoblot analysis utilizing antibodiesraised against the conserved catalytic domainof AHA2andfound that only an apparent 95kD protein in thalliwas recognized. This suggests that the 95kDprotein CX-4945 is most likely involved in MpHA1 to MpHA5,simply because these isoforms show high identity with AHA2and have really equivalent molecularmasses to AHA2.To analyze regardless of whether the pT HATPase in M. polymorphais regulated by phosphorylationof the penultimate Thr, we treated thalli withthe fungal toxin fusicoccin, which is an activatorof HATPase and accumulates phosphorylated HATPase by means of inhibition of dephosphorylation ofthe phosphorylated penultimate Thr in vascular plants.Phosphorylation in the penultimate Thr was detectedusing antibodies raised against the phosphorylatedpenultimate Thr947 of AHA2.
The results showed that FC at 10 mM inducedphosphorylation in the 95kD protein in thalliwithout altering the level of HATPase present inthe cells. Moreover, proteinblotanalysis utilizing 1433 proteinas a probe axitinib revealed that phosphorylated HATPasebound to the 1433 protein. These outcomes indicate thatthe phosphorylated penultimate Thr creates a bindingmotif for the 1433 protein, as also seen in vascularplants, and that the 95kD protein containsthe pT HATPase in M. polymorpha.As illustrated in Figure 2A, the HATPases in bothmaleand femalethalli showed anidentical response to FC. We performed further experimentsusing Tak1.Mp1433a Binds to the Phosphorylated HATPaseWe detected endogenous 1433 proteins in thallihaving molecular masses of 31 and 32 kD utilizing antibodiesraised against Arabidopsis GF14phi. We then performed aBLAST search against M. polymorpha ESTs and founda common 1433 protein, designated M. polymorpha1433a.Expression of Mp1433a in thalli was confirmed by RTPCR. Moreover, protei