The H + ‐ATPase purified from maize root plasma membranes retains fusicoccin in vivo activation

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The H؉ -ATPase is a key enzyme for the establishment and maintenance of plasma membrane potential and energization of secondary active transport in the plant cell. The phytotoxin fusicoccin induces H ؉ -ATPase activation by promoting the association of 14-3-3 proteins. It is still unclear whether 14-3-3 proteins can represent natural regulators of the proton pump, and factors regulating 14-3-3 binding to the H ؉ -ATPase under physiological conditions are unknown as well. In the present study in vivo and in vitro evidence is provided that 14-3-3 proteins can associate with the H ؉ -ATPase from maize roots also in a fusicoccin-independent manner and that the interaction depends on the phosphorylation status of the proton pump. Furthermore, results indicate that phosphorylation of H ؉ -ATPase influences also the fusicoccin-dependent interaction of 14-3-3 proteins. Finally, a protein phosphatase 2A able to impair the interaction between H ؉

Section: Methodsmentioning

confidence: 99%

Phosphorylation-dependent Interaction between Plant Plasma Membrane H+-ATPase and 14-3-3 Proteins

Camoni¹,

Iori²,

Marra³

et al. 2000

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“…In fact, it has been demonstrated that FC in vivo treatment of plant tissues, which produces a strong stimulation of H ϩ -ATPase activity, concomitantly brings about a marked increase in plasma membraneassociated 14-3-3 proteins (4,11). Also, the amount of FC bound to plasma membrane appears to be strictly correlated with stimulation of H ϩ -ATPase activity (12).…”

mentioning

confidence: 99%

“…Purification of H ϩ -ATPase-H ϩ -ATPase from maize roots was solubilized from two-phase partitioned plasma membranes and purified by anion-exchange HPLC as previously described (11). To obtain C-terminally truncated H ϩ -ATPase, before purification, plasma membranes were treated with trypsin under the conditions described by RasiCaldogno et al (19).…”

mentioning

confidence: 99%

Fusicoccin Effect on the in Vitro Interaction between Plant 14-3-3 Proteins and Plasma Membrane H+-ATPase

Fullone¹,

Visconti²,

Marra³

et al. 1998

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A 17-amino acid peptide was selectively cleaved from the highly variant C terminus of the 33-kDa 14-3-3 isoform occurring in fusicoccin receptor preparations from maize and was sequenced. The determined C-terminal sequence was identical to that of the already known maize 14-3-3 homolog GF14-6, thus prompting the use of recombinant GF14-6 in an in vitro protein-protein interaction study. The cDNA of GF14-6 was expressed in Escherichia coli as a 32 P-phosphorylatable glutathione S-transferase fusion protein and was used as a probe in overlay experiments with H ؉ -ATPase partially purified from maize roots. The results demonstrated that the recombinant protein specifically bound to H ؉ -ATPase. The binding was dependent on Mg 2؉ and was strongly increased by fusicoccin. Controlled trypsin digestion of H ؉ -ATPase abolished the association with GF14-6, a finding that was suggestive of an interaction with the C terminus of the enzyme. To confirm this result, the Cterminal domain of H ؉ -ATPase was expressed as a glutathione S-transferase fusion peptide and was used in overlay experiments. GF14-6 was also able to bind to the isolated C terminus, but only in the presence of fusicoccin.

“…Santos) from Dekalb (Mestre, Italy) were germinated and grown in the dark for 6 days as already described (18).…”

Section: Methodsmentioning

confidence: 99%

“…Isolation of Maize Plasma Membranes-Two-phase partitioned plasma membranes from maize roots were obtained as described previously (18).…”

Section: Methodsmentioning

confidence: 99%

Adenosine 5′-Monophosphate Inhibits the Association of 14-3-3 Proteins with the Plant Plasma Membrane H+-ATPase

Camoni

Visconti

Marra

et al. 2001

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Although a well ascertained evidence proves that the activity of the plant plasma membrane H ؉ -ATPase is regulated by 14-3-3 proteins, information about physiological factors modulating the phosphorylation-dependent association between 14-3-3 proteins and the proton pump is largely incomplete. In this paper we show that the 5-AMPmimetic, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), inhibits the fusicoccin-promoted proton extrusion in maize roots. We also demonstrate that 5-AMP inhibits the association of 14-3-3 proteins with the C-terminal domain of the H ؉